Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis.
Pizzini Silvia,Bisognin Andrea,Mandruzzato Susanna,Biasiolo Marta,Facciolli Arianna,Perilli Lisa,Rossi Elisabetta,Esposito Giovanni,Rugge Massimo,Pilati Pierluigi,Mocellin Simone,Nitti Donato,Bortoluzzi Stefania,Zanovello Paola
BACKGROUND:Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed. RESULTS:Most changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples. CONCLUSIONS:Using a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.
SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b.
Kugel Sita,Sebastián Carlos,Fitamant Julien,Ross Kenneth N,Saha Supriya K,Jain Esha,Gladden Adrianne,Arora Kshitij S,Kato Yasutaka,Rivera Miguel N,Ramaswamy Sridhar,Sadreyev Ruslan I,Goren Alon,Deshpande Vikram,Bardeesy Nabeel,Mostoslavsky Raul
Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD(+)-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%-40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset. PAPERCLIP.
Up-Regulation of hsa-miR-210 Promotes Venous Metastasis and Predicts Poor Prognosis in Hepatocellular Carcinoma.
Ji Jia,Rong Yuan,Luo Chang-Liang,Li Shuo,Jiang Xiang,Weng Hong,Chen Hao,Zhang Wu-Wen,Xie Wen,Wang Fu-Bing
Frontiers in oncology
To investigate the potential biomarkers for venous metastasis of hepatocellular carcinoma (HCC), and briefly discuss their target genes and the signaling pathways they are involved in. The dataset GSE6857 was downloaded from GEO. Significantly differentially expressed miRNAs were identified using the R package "limma," After that, the survival analysis was conducted to discover the significance of these up-regulated miRNAs for the prognosis of HCC patients. Additionally, miRNAs which were up-regulated in venous metastasis positive HCC tissues and were significant for the prognosis of HCC patients were further verified in clinical samples using RT-qPCR. The miRNAs were then analyzed for their correlations with clinical characteristics including survival time, AFP level, pathological grade, TNM stage, tumor stage, lymph-node metastasis, distant metastasis, child-pugh score, vascular invasion, liver fibrosis and race using 375 HCC samples downloaded from the TCGA database. The target genes of these miRNAs were obtained using a miRNA target gene prediction database, and their functions were analyzed using the online tool DAVID. 15 miRNAs were differentially expressed in samples with venous metastasis, among which 7 were up-regulated in venous metastasis positive HCC samples. As one of the up-regulated miRNAs, hsa-miR-210 was identified as an independent prognostic factor for HCC. Using RT-qPCR, it was evident that hsa-miR-210 expression was significantly higher in venous metastasis positive HCC samples ( = 0.0036). Further analysis indicated that hsa-miR-210 was positively associated with AFP level, pathological grade, TNM stage, tumor stage and vascular invasion. A total of 168 hsa-miR-210 target genes, which are mainly related to tumor metastasis and tumor signaling pathways, were also predicted in this study. hsa-miR-210 might promote vascular invasion of HCC cells and could be used as a prognostic biomarker.
Identification of invasion-metastasis-associated microRNAs in hepatocellular carcinoma based on bioinformatic analysis and experimental validation.
Lou Weiyang,Chen Jing,Ding Bisha,Chen Danni,Zheng Huilin,Jiang Donghai,Xu Liang,Bao Chang,Cao Guoqiang,Fan Weimin
Journal of translational medicine
BACKGROUND:Hepatocellular carcinoma (HCC) is one of the most lethal cancer, mainly attributing to its high tendency to metastasis. Vascular invasion provides a direct path for solid tumor metastasis. Mounting evidence has demonstrated that microRNAs (miRNAs) are related to human cancer onset and progression including invasion and metastasis. METHODS:In search of invasion-metastasis-associated miRNAs in HCC, microarray dataset GSE67140 was downloaded from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) were obtained by R software package and the potential target genes were predicted by miRTarBase. The database for annotation, visualization and integrated discovery (DAVID) was introduced to perform functional annotation and pathway enrichment analysis for these potential targets of DE-miRNAs. Protein-protein interaction (PPI) network was established by STRING database and visualized by Cytoscape software. The effects of the miR-494-3p and miR-126-3p on migration and invasion of HCC cell lines were evaluated by conducting wound healing assay and transwell assay. RESULTS:A total of 138 DE-miRNAs were screened out, including 57 upregulated miRNAs and 81 downregulated miRNAs in human HCC tumors with vascular invasion compared with human HCC tumors without vascular invasion. 762 target genes of the top three upregulated and downregulated miRNAs were predicted, and they were involved in HCC-related pathways, such as pathway in cancer, focal adhesion and MAPK signaling pathway. In the PPI network, the top 10 hub nodes with higher degrees were identified as hub genes, such as TP53 and MYC. Through constructing the miRNA-hub gene network, we found that most of hub genes could be potentially modulated by miR-494-3p and miR-126-3p. Of note, miR-494-3p and miR-126-3p was markedly upregulated and downregulated in HCC cell lines and tissues, respectively. In addition, overexpression of miR-494-3p could significantly promote HCC migration and invasion whereas overexpression of miR-126-3p exerted an opposite effect. CONCLUSIONS:Targeting miR-494-3p and miR-126-3p may provide effective and promising approaches to suppress invasion and metastasis of HCC.
MicroRNA-200b is downregulated and suppresses metastasis by targeting LAMA4 in renal cell carcinoma.
Li Yifan,Guan Bao,Liu Jingtao,Zhang Zhongyuan,He Shiming,Zhan Yonghao,Su Boxing,Han Haibo,Zhang Xiaochun,Wang Boqing,Li Xuesong,Zhou Liqun,Zhao Wei
BACKGROUND:Metastasis is the primary cause of tumor death in renal cell carcinoma (RCC). Improved diagnostic markers of metastasis are critically needed for RCC. MicoRNAs are demonstrated to be stable and significant biomarkers for several malignancies. In this study, we aimed to explore the metastasis related microRNAs and its mechanism in RCC. METHODS:The relationship between microRNAs expression and prognosis and metastasis of RCC patients were explored by data mining through expression profiles from The Cancer Genome Atlas (TCGA). A total of 80 RCC tissues and adjacent normal kidney tissues were obtained from Department of Urology, Peking University First Hospital. Expression of microRNA-200b (miR-200b) in RCC tissues and cell lines were determined by bioinformatic data mining and quantitative real-time PCR (qRT-PCR). The effects of miR-200b on cell proliferation, migration and invasion were determined by cell counting kit-8 and colony formation assay, wound healing assay and Boyden chamber assay. Mouse cell-derived xenograft and patient-derived xenograft model were also performed to evaluate the effects of miR-200b on tumor growth and metastasis in vivo. The molecular mechanism of miR-200b function was investigated using bioinformatic target predication and high-throughput cDNA sequencing (RNA-seq) and validated by luciferase reporter assay, qRT-PCR, Western blot and immunostaining in vitro and in vivo. FINDINGS:Our findings indicates that miR-200b is frequently downregulated and have potential utility as a biomarker of metastasis and prognosis in RCC. Interestingly, ectopic expression of miR-200b in the Caki-1 and OSRC-2 cell lines suppresses cell migration and invasion in vitro as well as tumor metastases in vivo. However, miR-200b has no effect on cell proliferation in vitro and tumor growth in vivo. In addition, bioinformatics target predication and RNA-seq results reveals that Laminin subunit alpha 4 (LAMA4) is one target of miR-200b and significantly inhibited by miR-200b in vitro and in vivo. INTERPRETATION:These results demonstrate a previously undescribed role of miR-200b as a suppressor of tumor metastasis in RCC by directly destabilizing LAMA4 mRNA.
Exosomal miR-660-5p promotes tumor growth and metastasis in non-small cell lung cancer.
Qi Yongjian,Zha Wangjian,Zhang Wei
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
PURPOSE:Non-small cell lung cancer (NSCLC) is still the commonest fatal malignancy worldwide. The relationship between miR-660-5p and progress of NSCLC has not been well confirmed in recent studies. This manuscript focused to the function of miR-660-5p during the appearance and progression of NSCLC. METHODS:To identify the expression level of miR-660-5p in NSCLC, patient plasma and exosomes, quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed. Cell proliferation and colony formation abilities were examined by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Then, the influence of miR-660-5p on migration and invasion was analyzed by transwell assay. Bioinformatics and Luciferase report assay were used to find potential target genes. Western blot was chosen to assess the expression level of KLF9. Stably transfected NSCLC cells (A549 and H1299) were injected into nude mice to identify the function of miR-660-5p in tumorigenesis in vivo. RESULTS:Compared with healthy controls, the release of miR-660-5p in plasma and exosomes was increased in patients with NSCLC (n=80). Knockdown of miR-660-5p significantly suppressed proliferation, migration, and invasion, whereas overexpression of miR-660-5p had the opposite effect. KLF9 might be a potential target of miR-660-5p. In addition, up-regulation of miR-660-5p promoted tumorigenesis in vivo, and the protein level of KLF9 also decreased in xenografts. CONCLUSIONS:Our current study suggests that miR-660-5p may control NSCLC proliferation, viability, and metastasis by targeting KLF9, which provides a potential therapeutic target for NSCLC.
microRNA-146a inhibits cancer metastasis by downregulating VEGF through dual pathways in hepatocellular carcinoma.
Zhang Zheng,Zhang Yang,Sun Xiu-Xuan,Ma Xi,Chen Zhi-Nan
UNLABELLED:Growing evidence indicates that miR-146a is involved in carcinogenesis and tumor progression in several human malignancies. However, the molecular details underlying miR-146a mediated regulation of its target genes and its precise biological function in cancer, especially in hepatocellular carcinoma (HCC) remains unclear. METHODS:The expression levels of genes including miR-146a, APC, VEGF and HAb18G were examined in HCC cell lines and patient specimens were compared with control levels using quantitative reverse transcription-PCR. The functions of miR-146a and HAb18G in migration/invasion and liver metastasis formation were determined by transwell and spleen injection assays, respectively. miR-146a related genes were determined by PCR array. The potential regulatory targets of miR-146a were determined by bioinformatics and prediction tools, correlation with target protein expression, and luciferase reporter assay. DNA methylation status of miR-146a promoter were performed by PCR analysis of bisulfite-modified genomic DNA. RESULTS:We demonstrated that miR-146a expression was markedly downregulated in hepatoma cells and hepatoma tissues compared to immortalized normal liver epithelial cells and normal hepatic tissues. DNA methylation of miR-146a promoter correlated with its downexpression and with liver cancer metastasis. The restoration of miR-146a dramatically suppressed HCC cell invasion and metastasis by repressing VEGF expression through upregulating APC, which inhibits β-catenin accumulation in nucleus, and downregulating NF-κB p65 by targeting HAb18G. In human HCC, miR-146a expression was negative correlated with increased HAb18G, VEGF, NF-κB p65 and beneficial prognosis. CONCLUSION:This study identified a novel target of miR-146a and defined miR-146a as a crucial tumor suppressor in human HCC that acts through multiple pathways and mechanisms to suppress HCC invasion or metastasis.
MicroRNA-330-3p promotes cell invasion and metastasis in non-small cell lung cancer through GRIA3 by activating MAPK/ERK signaling pathway.
Wei Chun-Hua,Wu Gang,Cai Qian,Gao Xi-Can,Tong Fan,Zhou Rui,Zhang Rui-Guang,Dong Ji-Hua,Hu Yu,Dong Xiao-Rong
Journal of hematology & oncology
BACKGROUND:Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). However, the exact parts played by miR-330-3p in BM of NSCLC remain unknown. Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Here, we evaluated the expression and biological effects of miR-330-3p in NSCLC cells and explored the underlying mechanism of miR-330-3p in promoting cell migration and invasion in NSCLC. METHODS:Stable over-expression and knockdown of miR-330-3p in NSCLC cells was constructed with lentivirus. Expression levels of miR-330-3p in NSCLC cells were quantified by quantitive real-time PCR (qRT-PCR). The effects of miR-330-3p on NSCLC cells were investigated using assays of cell viability, migration, invasion, cell cycle, apoptosis, western blotting, immunohistochemical, and immunofluorescence staining. A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis. The potential target of miR-330-3p in NSCLC cells was explored using the luciferase reporter assay, qRT-PCR, and western blotting. The miR-330-3p targets were identified using bioinformatics analysis and verified by luciferase reporter assay. The correlation between GRIA3 and DNA methyltransferase (DNMT) 1 and DNMT3A was tested by RT-PCR, western blotting, and co-immunoprecipitation (IP). RESULTS:miR-330-3p was significantly up-regulated in NSCLC cell lines. MTT assay, transwell migration, and invasion assays showed that miR-330-3p promoted the growth, migration, and invasion of NSCLC cells in vitro and induced tumor growth and metastasis in vivo. Luciferase reporter assays showed that GRIA3 was a target of miR-330-3p. qRT-PCR and western blotting exhibited that miR-330-3p promoted the growth, invasion, and migration of NSCLC cells by activating mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinases (ERK) signaling pathway. Furthermore, miR-330-3p up-regulated the total DNA methylation in NSCLC cells, and co-IP-demonstrated GRIA3 was directly related with DNMT1 and DNMT3A. CONCLUSIONS:miR-330-3p promoted the progression of NSCLC and might be a potential target for the further research of NSCLC brain metastasis.
Inhibition of miR-328-3p Impairs Cancer Stem Cell Function and Prevents Metastasis in Ovarian Cancer.
Srivastava Amit K,Banerjee Ananya,Cui Tiantian,Han Chunhua,Cai Shurui,Liu Lu,Wu Dayong,Cui Ri,Li Zaibo,Zhang Xiaoli,Xie Guozhen,Selvendiran Karuppaiyah,Patnaik Srinivas,Karpf Adam R,Liu Jinsong,Cohn David E,Wang Qi-En
Cancer stem cells (CSC) play a central role in cancer metastasis and development of drug resistance. miRNA are important in regulating CSC properties and are considered potential therapeutic targets. Here we report that miR-328-3p (miR-328) is significantly upregulated in ovarian CSC. High expression of miR-328 maintained CSC properties by directly targeting DNA damage binding protein 2, which has been shown previously to inhibit ovarian CSC. Reduced activity of ERK signaling in ovarian CSC, mainly due to a low level of reactive oxygen species, contributed to the enhanced expression of miR-328 and maintenance of CSC. Inhibition of miR-328 in mouse orthotopic ovarian xenografts impeded tumor growth and prevented tumor metastasis. In summary, our findings provide a novel mechanism underlying maintenance of the CSC population in ovarian cancer and suggest that targeted inhibition of miR-328 could be exploited for the eradication of CSC and aversion of tumor metastasis in ovarian cancer. SIGNIFICANCE: These findings present inhibition of miR-328 as a novel strategy for efficient elimination of CSC to prevent tumor metastasis and recurrence in patients with epithelial ovarian cancer.
The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis.
Ling Hui,Pickard Karen,Ivan Cristina,Isella Claudio,Ikuo Mariko,Mitter Richard,Spizzo Riccardo,Bullock Marc,Braicu Cornelia,Pileczki Valentina,Vincent Kimberly,Pichler Martin,Stiegelbauer Verena,Hoefler Gerald,Almeida Maria I,Hsiao Annie,Zhang Xinna,Primrose John,Packham Graham,Liu Kevin,Bojja Krishna,Gafà Roberta,Xiao Lianchun,Rossi Simona,Song Jian H,Vannini Ivan,Fanini Francesca,Kopetz Scott,Zweidler-McKay Patrick,Wang Xuemei,Ionescu Calin,Irimie Alexandru,Fabbri Muller,Lanza Giovanni,Hamilton Stanley R,Berindan-Neagoe Ioana,Medico Enzo,Mirnezami Alex,Calin George A,Nicoloso Milena S
OBJECTIVE:MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN:We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS:MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS:MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.
MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer.
Asangani I A,Rasheed S A K,Nikolova D A,Leupold J H,Colburn N H,Post S,Allgayer H
Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3'-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3'-UTR at 228-249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3'-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228-249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken-embryo-metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.
MicroRNA-134 prevents the progression of esophageal squamous cell carcinoma via the PLXNA1-mediated MAPK signalling pathway.
Wang Wei-Wei,Zhao Zhi-Hua,Wang Li,Li Pan,Chen Kui-Sheng,Zhang Jian-Ying,Li Wen-Cai,Jiang Guo-Zhong,Li Xiang-Nan
BACKGROUND:MicroRNAs (miRNAs) are involved in oncogenesis of esophageal squamous cell carcinoma (ESCC). miR-134 is reported to have a tumour-suppressive role but its role in ESCC is not known. The present study was designed to examine whether miR-134 inhibits ESCC development and further explored relevant underlying mechanisms. METHODS:Differentially expressed genes related to ESCC were identified from microarray gene expression profiles. Immunohistochemical staining and RT-qRCR assays identified elevated PLXNA1 expression levels and low miR-134. The relationship between miR-134 and PLXNA1 was predicted and further verified by a dual-luciferase reporter assay. The expression levels of miR-134 and PLXNA1 in ESCC cells were modified by miR-134 mimic/inhibitor and siRNA against PLXNA1, respectively. Thereafter, the expression of MAPK signalling pathway-related proteins, as well as the viability, migration, invasion, cell cycle and cell apoptosis of ESCC cells was investigated. FINDINGS:The results showed that miR-134 could block the MAPK signalling pathway by downregulating PLXNA1. When miR-134 was overexpressed or PLXNA1 was silenced, cell apoptosis was enhanced, the cell cycle was retarded, and the cell proliferation, migration and invasion were suppressed. In vivo experiments confirmed that miR-134 overexpression or PLXNA1 silencing restrained tumour growth and lymph node metastasis. INTERPRETATION:These findings demonstrate that cancer cell proliferation, migration, invasion, and tumour metastasis of ESCC can be suppressed by overexpression of miR-134 through downregulating PLXNA1, which subsequently blocks the MAPK signalling pathway. These results provide new potential targets and strategies for the treatment of ESCC.
MiR-33a suppresses breast cancer cell proliferation and metastasis by targeting ADAM9 and ROS1.
Zhang Chuankai,Zhang Yunda,Ding Weiji,Lin Yancheng,Huang Zhengjie,Luo Qi
Protein & cell
MicroRNAs (miRNAs) are small noncoding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression by sequence-specifically targeting multiple mRNAs. Although miR-33a was recently reported to play an important role in lipid homeostasis, atherosclerosis, and hepatic fibrosis, the functions of miR-33a in tumor progression and metastasis are largely unknown. Here, we found that downregulated miR-33a in breast cancer tissues correlates with lymph node metastasis. MiR-33a expression is significantly lower in the highly metastatic breast cancer cell lines than the noncancerous breast epithelial cells and non-metastatic breast cancer cells. Moreover, the overexpression of miR-33a in metastatic breast cancer cells remarkably decreases cell proliferation and invasion in vitro and significantly inhibits tumor growth and lung metastasis in vivo, whereas its knockdown in non-metastatic breast cancer cells significantly enhances cell proliferation and invasion in vitro and promotes tumor growth and lung metastasis in vivo. Combining bioinformatics prediction and biochemical analyses, we showed that ADAM9 and ROS1 are direct downstream targets of miR-33a. These findings identified miR-33a as a negative regulator of breast cancer cell proliferation and metastasis.
MicroRNA-222 Promotes Invasion and Metastasis of Papillary Thyroid Cancer Through Targeting Protein Phosphatase 2 Regulatory Subunit B Alpha Expression.
Huang Yanrui,Yu Shuang,Cao Siting,Yin Yali,Hong Shubin,Guan Hongyu,Li Yanbing,Xiao Haipeng
Thyroid : official journal of the American Thyroid Association
BACKGROUND:Increasing evidence indicates that microRNA dysfunction is involved in the pathogenesis and progression of cancer. MicroRNA-222 (miR-222) is upregulated in papillary thyroid carcinoma (PTC). However, the role of miR-222 in invasion and metastasis of PTC remains unknown. This study investigated the function of miR-222 and its underlying mechanism in the progression of PTC. METHODS:The expression of miR-222 was detected by quantitative reverse transcription polymerase chain reaction, and its correlation with various clinical characteristics was analyzed. The role of miR-222 in PTC cell migration ability was assessed with Transwell assays and wound-healing assays in both TPC-1 and K1 cells. By using bioinformatics analyses and dual-luciferase 3'-UTR reporter assays, the study identified the direct target of miR-222 and the downstream pathways of PTC. Further, the study confirmed the role of miR-222 in promoting PTC distant metastasis in vivo by injecting TPC-1 cells into nude mice. RESULTS:This study confirmed that miR-222 was upregulated in PTC tissues compared to adjacent thyroid tissues and that it correlated with aggressive cancer phenotypes. The results indicate that ectopic miR-222 enhanced cell migration and invasion of thyroid cancer cells in vitro and distant pulmonary metastases in vivo. Protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), a tumor suppressor, was identified as a direct target of miR-222 through the 3'-UTR of PPP2R2A. Restoring PPP2R2A expression led to the attenuation of migration and invasion in miR-222-overexpressing thyroid cancer cells. Moreover, we found that miR-222 promoted invasion and metastasis partly through the AKT signaling pathway. CONCLUSIONS:Taken together, the results suggest that miR-222 promotes tumor invasion and metastasis in thyroid cancer by targeting PPP2R2A. Thus, miR-222 could serve as a potential diagnostic biomarker, as well as an attractive therapeutic tool for thyroid cancer.
Downregulation of miR-142-5p promotes tumor metastasis through directly regulating CYR61 expression in gastric cancer.
Yan Jing,Yang Bing,Lin Shuye,Xing Rui,Lu Youyong
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association
BACKGROUND:Recurrence is a primary cause of gastric cancer (GC)-related deaths. We reported previously that low expression of miR-142-5p could predict recurrence in GC. The present study aimed to investigate the function and mechanism of miR-142-5p in metastasis of GC. METHODS:MiR-142-5p expression was detected in 101 GC samples by qRT-PCR. Its clinical significance was statistically analyzed. The roles of miR-142-5p and its candidate target gene CYR61 in metastasis were determined both in vivo and in vitro. RESULTS:MiR-142-5p downregulation was significantly associated with the recurrence (P = 0.031) and poor prognosis of GC (P = 0.043). MiR-142-5p inhibited cancer cell migration and invasion both in vitro and in vivo. CYR61 was identified as a novel direct target of miR-142-5p by bioinformatics analysis of target prediction and luciferase reporter assay. The re-expression and knockdown of CYR61 could, respectively, rescue the effects induced by miR-142-5p overexpression and knockdown. MiR-142-5p attenuated GC cell migration and invasion, at least partially, by inactivation of the canonical Wnt/β-catenin signaling pathway through CYR61. CONCLUSIONS:The newly identified miR-142-5p-CYR61-Wnt/β-catenin axis partially illustrates the molecular mechanism of GC recurrence and represents a novel prognosis biomarker for GC.
miR-629-3p may serve as a novel biomarker and potential therapeutic target for lung metastases of triple-negative breast cancer.
Wang Jin,Song Cailu,Tang Hailin,Zhang Chao,Tang Jun,Li Xing,Chen Bo,Xie Xiaoming
Breast cancer research : BCR
BACKGROUND:Different breast cancer subtypes show distinct tropisms for sites of metastasis. Notably, the lung is the most common site for the first distant recurrence in triple-negative breast cancer (TNBC). The identification of novel biomarkers for lung metastasis is of great importance to improving the outcome of TNBC. In this study, we sought to identify a microRNA (miRNA)-based biomarker and therapeutic target for lung metastasis of TNBC. METHODS:A total of 669 patients without de novo stage IV TNBC were recruited for this study. miRNA profiling was conducted in the discovery cohort. Diagnostic accuracy and prognostic values of candidate miRNAs were evaluated in the training and validation cohorts, respectively. The biological functions of candidate miRNAs, as well as potential targets, were further evaluated through bioinformatic analysis as well as by performing in vitro and in vivo assays. RESULTS:In the discovery set, we found that miR-629-3p was specifically upregulated in both metastatic foci (fold change 144.16, P < 0.0001) and primary tumors (fold change 74.37, P = 0.004) in patients with lung metastases. In the training set, the ROC curve showed that miR-629-3p yielded high diagnostic accuracy in discriminating patients with lung metastasis from patients without recurrence (AUC 0.865, 95% CI 0.800-0.930, P < 0.0001). Although miR-629-3p predicted poor overall survival and disease-free survival in the validation set, it failed to show significance after multivariate analysis. Notably, logistic regression analyses confirmed that miR-629-3p was an independent risk factor for lung metastasis (OR 4.1, 95% CI 2.5-6.6, P < 0.001). Inhibition of miR-629-3p drastically attenuated the viability and migration of TNBC cells, and it markedly suppressed lung metastasis in vivo. Furthermore, we identified the leukemia inhibitory factor receptor (LIFR), a well-known metastatic suppressive gene, to be a direct target of miR-629-3p. CONCLUSIONS:miR-629-3p may serve as a novel biomarker and potential therapeutic target for lung metastases of TNBC mediated via LIFR.
miR-19b-3p promotes colon cancer proliferation and oxaliplatin-based chemoresistance by targeting SMAD4: validation by bioinformatics and experimental analyses.
Jiang Tao,Ye Ling,Han Zhongbo,Liu Yuan,Yang Yinxue,Peng Zhihai,Fan Junwei
Journal of experimental & clinical cancer research : CR
BACKGROUND:As a disease with extremely complex molecular mechanisms, many deregulated miRNAs have been identified in colon cancer. Few studies have been performed by using Ingenuity Pathways Analysis (IPA) to predict miRNAs specifically expressed in colon cancer. METHODS:A characteristic microRNA-target network of colon cancer was explored using IPA. Then the clinical significance of miR-19b-3p was evaluated in 211 colon cancer patients. The roles of miR-19b-3p and its candidate target gene, SMAD4, in colon cancer progression were examined both in vitro and in vivo. RESULTS:Bioinformatics analysis showed that 15 microRNAs screened by IPA were significantly correlated with malignant biological behaviors of colon cancer. miR-19b-3p was the most significantly upregulated candidate based on the validation experiment using 211 colon cancer samples. High expression of miR-19b-3p was significantly associated with high N stage (P < 0.001), high AJCC stage (P < 0.001), poor histologic grade (P = 0.032), frequent venous and lymphatic invasion (P = 0.027), and liver metastasis (P < 0.001). Survival analysis revealed that miR-19b-3p was an independent prognostic factor associated with colon cancer patient's overall survival (OS) and disease-free survival (DFS). miR-19b-3p promoted proliferation and chemoresistance of colon cancer cells, but had no effect on invasion in vitro, along with tumorigenesis in vivo. In addition, we confirmed that miR-19b-3p mediates resistance to oxaliplatin-based chemotherapy via SMAD4. CONCLUSIONS:Our findings demonstrate the role of miR-19b-3p-SMAD4 axis in colon cancer progression, which may become a potential therapeutic target against chemotherapy resistance.
MicroRNA-29b attenuates non-small cell lung cancer metastasis by targeting matrix metalloproteinase 2 and PTEN.
Wang Hongyan,Guan Xiaoying,Tu Yongsheng,Zheng Shaoqiu,Long Jie,Li Shuhua,Qi Cuiling,Xie Xiaobin,Zhang Huiqiu,Zhang Yajie
Journal of experimental & clinical cancer research : CR
BACKGROUND:Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells. METHODS:Ten human non-small cell lung cancer (NSCLC) cell lines and samples from thirty patients with NSCLC were analyzed for the expression of miR-29b by quantitative RT-PCR. Bioinformatics analysis combined with tumor metastasis PCR array showed the potential target genes for miR-29b. miR-29b lentivirus and inhibitors were transfected into NSCLC cells to investigate its role on regulating cell proliferation which was measured by CCK-8 assay in vitro and nude mice xenograft tumor assay in vivo. Cell motility ability was evaluated by transwell assay. The target genes of miR-29b were determined by luciferase assay, quantitative RT-PCR and western blot. RESULTS:Bioinformatics analysis combined with tumor metastasis PCR array showed that matrix metalloproteinase 2 (MMP2) and PTEN could be important target genes of miR-29b. The expression of miR-29b was down regulated in NSCLC tissues compared to the normal tissues. Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis. The gain-of-function studies revealed that ectopic expression of miR-29b decreased cell proliferation, migration and invasion abilities of NSCLC cells. In contrasts, loss-of-function studies showed that inhibition of miR-29b promoted cell proliferation, migration and invasion of NSCLC cells in vitro. Nude mice xenograft tumor assay confirmed that miR-29b inhibited lung cancer growth in vivo. High-invasion (A549-H) and low-invasion (A549-L) NSCLC cell sublines from A549 cells were created by using the repeated transwell assay aimed to confirm the effect of miR-29b on migration and invasion of NSCLC. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29b inhibited the expression of the luciferase gene containing the 3'-UTRs of MMP2 and PTEN mRNA. Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels. CONCLUSION:Taken together, our results demonstrate that miR-29b serves as a tumor metastasis suppressor, which suppresses NSCLC cell metastasis by directly inhibiting MMP2 expression. The results show that miR-29b may be a novel therapeutic candidate target to slow NSCLC metastasis.
Downregulation of miR-133a-3p promotes prostate cancer bone metastasis via activating PI3K/AKT signaling.
Tang Yubo,Pan Jincheng,Huang Shuai,Peng Xinsheng,Zou Xuenong,Luo Yongxiang,Ren Dong,Zhang Xin,Li Ronggang,He Peiheng,Wa Qingde
Journal of experimental & clinical cancer research : CR
BACKGROUND:Bone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear. METHODS:miR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues. RESULTS:miR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues. CONCLUSION:Our results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.
Role of microRNA-141-3p in the progression and metastasis of hepatocellular carcinoma cell.
Hou Xu,Yang Le,Jiang Xiaohong,Liu Zhiheng,Li Xuehua,Xie Shuli,Li Guangbing,Liu Jun
International journal of biological macromolecules
Hepatocellular carcinoma (HCC) is a leading cause of cancer related death worldwide. However, the mechanisms underlying HCC progression and metastasis are still in obscure. Here, we used bioinformatic analysis to identify miRNAs that regulate GP73, a specific marker for HCC diagnosis and prognosis. The correlations between miR-141-3p and clinic-pathological factors were analyzed in HCC patient samples; proliferation, migration, invasion, and colony formation were studied using established HCC cell lines. Expression levels of target genes (miR-141-3p, GP73, E-cadherin, N-cadherin, occludin, vimentin, and cytokeratin 18) were detected by either Western blot or qRT-PCR analysis. Xenograft models were established to evaluate tumor growth and metastasis. MiR-141-3p was significantly reduced in HCC tumors and cell lines, highly correlated with tumor progression. In contrast, GP73 was negatively correlated with miR-141-3p in HCC tumors. MiR-141-3p overexpression significantly decreased HCC cell proliferation, migration, and invasion by inhibiting epithelial-mesenchymal transition (EMT). GP73 overexpression partially restored the inhibitory effects of miR-141-3p, while miR-141-3p overexpression markedly inhibited tumor growth and pulmonary metastasis, which were partially reversed by GP73 overexpression. Our findings suggest that miR-141-3p targets GP73 to reverse EMT, subsequently inhibiting HCC progression and metastasis. Thus, overexpression of miR-141-3p could serve as a therapeutic strategy to arrest HCC.
miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling.
Shi Dong-Min,Li Li-Xin,Bian Xin-Yu,Shi Xue-Jiang,Lu Li-Li,Zhou Hong-Xin,Pan Ting-Jia,Zhou Jian,Fan Jia,Wu Wei-Zhong
Journal of experimental & clinical cancer research : CR
BACKGROUND:Accumulation of evidence indicates that miRNAs have crucial roles in the regulation of EMT-associated properties, such as proliferation, migration and invasion. However, the underlying molecular mechanisms are not entirely illustrated. Here, we investigated the role of miR-296-5p in hepatocellular carcinoma (HCC) progression. METHODS:In vitro cell morphology, proliferation, migration and invasion were compared between HCC cell lines with up- or down-regulation of miR-296-5p. Immunofluorescence and Western blot immunofluorescence assays were used to detect the expression of EMT markers. Bioinformatics programs, luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-296-5p. Xenograft nude mouse models were established to observe tumor growth and metastasis. Immunohistochemical assays were conducted to study the relationships between miR-296-5p expression and Neuregulin-1 (NRG1)/EMT markers in human HCC samples and mice. RESULTS:miR-296-5p was prominently downregulated in HCC tissues relative to adjacent normal liver tissues and associated with favorable prognosis. Overexpression of miR-296-5p inhibited EMT along with migration and invasion of HCC cells via suppressing NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling in vitro. More importantly, miR-296-5p disrupted intrahepatic and pulmonary metastasis in vivo. NRG1, as a direct target of miR-296-5p, mediates downstream biological responses. In HCC tissues from patients and mice, the levels of miR-296-5p and NRG1 also showed an inverse relationship. CONCLUSIONS:miR-296-5p inhibited EMT-related metastasis of HCC through NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling.
Dual regulatory role of CCNA2 in modulating CDK6 and MET-mediated cell-cycle pathway and EMT progression is blocked by miR-381-3p in bladder cancer.
Li Jiangfeng,Ying Yufan,Xie Haiyun,Jin Ke,Yan Huaqing,Wang Song,Xu Mingjie,Xu Xin,Wang Xiao,Yang Kai,Zheng Xiangyi,Xie Liping
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Emerging evidence has elucidated that microRNAs (miRNAs) transcribed from miRNA cluster at DLK-DIO3 imprinted domain are involved in various cancers. However, as one member of this cluster, the underlying mechanisms and functions of miR-381-3p in bladder cancer (BCa) still remains elusive. Here we demonstrate that the hypermethylated status of upstream maternally expressed gene 3 divergent methylation region reduces the expression of miR-381-3p in BCa by bisulfite-sequencing PCR. In vitro and in vivo experiments indicate that overexpression of miR-381-3p significantly inhibits cell proliferation via inducing G phase arrest and migration via down-regulating MET and CCNA2 induced EMT progression. CDK6/CCNA2/MET are all identified as the direct targets of miR-381-3p by bioinformatics analysis and dual-luciferase reporter assay. Furthermore, inhibition of CCNA2 mediated by miR-381-3p as the crucial biregulator not only participates in the proliferation regulation with CDK6 in cell cycle but also modulates the EMT progression via ROCK/AKT/β-catenin/SNAIL pathway, which establishes an EMT circuit combined with miR-381-3p/MET/AKT/GSK-3β/SNAIL pathway, and SNAIL is the last confocal target to induce EMT progression. To conclude, we propose 2 novel regulatory circuits mediated by miR-381-3p in BCa, which may assist in the development of more effective therapies against BCa in the future.-Li, J., Ying, Y., Xie, H., Jin, K., Yan, H., Wang, S., Xu, M., Xu, X., Wang, X., Yang, K., Zheng, X., Xie, L. Dual regulatory role of CCNA2 in modulating CDK6 and MET-mediated cell-cycle pathway and EMT progression is blocked by miR-381-3p in bladder cancer.
MicroRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44.
Yu Gan,Yao Weimin,Xiao Wei,Li Heng,Xu Hua,Lang Bin
Journal of experimental & clinical cancer research : CR
BACKGROUND:Metastasis have considered as an important clinical obstacle in the treatment of human cancer including bladder cancer. Post-transcriptional regulation has emerged as robust effectors of metastasis. MiRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. In this study, we focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppress angiogenesis in bladder cancer by directly targeting CD44. METHODS:The expression of mir-34a was detected by quantitative real-time PCR. Oligonucleotide and lentivirus were used to overexpress miR-34a. Tube formation assay and transwell assay were used to examine the effect on bladder cancer tube formation, migration and invasion in vitro. Animal models were used to examine the effect on metastasis and angiogenesis in vivo. Luciferase assay was carried out to verify the precise target of miR-34a. RESULTS:We not only proved that mir-34a was significantly downregulated in bladder cancer tissues and cell lines but also that circulating miR-34a levels are reduced in bladder cancer, and their levels were positively relevance. Gain-of-function experiments investigated that increased mir-34a expression suppressed tube formation and reduced cell migration and invasion. In vivo metastasis, assays also demonstrated that overexpression of mir34a markedly inhibited bladder cancer metastasis. CD31, an endothelial cell-specific marker which stained in T24 tumors to evaluate for blood vessel density, the immunohistochemistry results showed that blood vessel quantification reduced dramatically in the T24 tumors over-expressing mir-34a. Combining with our previous studies and bioinformatics analysis, we expected that CD44 gene was a direct target of mir-34a, siRNA-mediated knockdown of CD44 partially phenocopied mir-34a overexpression suggesting that the pro-apoptotic role of mir-34a may be mediated primarily through CD44 regulation, whereas restoring the expression of CD44 attenuated the function of mir-34a in bladder cancer cells. Additionally, we identified that EMT (epithelial-mesenchymal transition) related proteins could be regulated by mir-34a which indicated that mir-34a could partially reserve EMT. CONCLUSION:Our study defines a major metastasis and angiogenesis suppressive role for mir-34a, a microRNA functions as a tumor suppressor in bladder cancer by directly targeting CD44, which would be helpful as a therapeutic approach to block bladder cancer metastasis.
miR-302a-5p/367-3p-HMGA2 axis regulates malignant processes during endometrial cancer development.
Ma Jian,Li Da,Kong Fan-Fei,Yang Di,Yang Hui,Ma Xiao-Xin
Journal of experimental & clinical cancer research : CR
BACKGROUND:Metastasis is one of the main reasons for treatment failure in endometrial cancer. Notably, high mobility group AT-hook 2 (HMGA2) has been recognized as a driving factor of tumour metastasis. microRNAs (miRNAs) are powerful posttranscriptional regulators of HMGA2. METHODS:The binding sites of miR-302a-5p and miR-367-3p on HMGA2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-302a-5p and miR-367-3p were detected using quantitative real-time PCR and in situ hybridization. Western blotting and immunohistochemistry were used to detect the levels of HMGA2 and epithelial-mesenchymal transition pathway-related proteins. Co-immunoprecipitation was used to detect protein interactions. The roles of miR-302a-5p and miR-367-3p in the regulation of HMGA2 during the progression of endometrial cancer were investigated using both in vitro and in vivo assays. RESULTS:In the present study, high HMGA2 expression was correlated with poor clinical outcomes in endometrial cancer. The binding sites of miRNAs on HMGA2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. In the endometrial cancer cell lines Ishikawa and HEC-1A, the overexpression of miR-302a-5p/367-3p significantly inhibited the expression of HMGA2 mRNA. In endometrial cancer tissues, we showed that miR-302a-5p and miR-367-3p were significantly downregulated and thus inversely correlated with HMGA2. The miR-302a-5p and miR-367-3p expression levels were closely correlated with FIGO stage and lymph node metastasis. High expression of miR-302a-5p/367-3p was correlated with high survival rates in endometrial cancer. In addition, miR-302a-5p/367-3p suppressed the malignant behaviour of endometrial carcinoma cells via the inhibition of HMGA2 expression. CONCLUSION:Our findings indicate that miR-302a-5p/367-3p-mediated expression of HMGA2 regulates the malignant behaviour of endometrial carcinoma cells, which suggests that the miR-302a-5p/367-3p-HMGA2 axis may be a predictive biomarker of endometrial cancer metastasis and patient survival and a potential therapeutic target in metastatic endometrial cancer.
MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression.
Cao Qinghua,Liu Fang,Ji Kaiyuan,Liu Ni,He Yuan,Zhang Wenhui,Wang Liantang
Journal of experimental & clinical cancer research : CR
BACKGROUND:MicroRNA-381 (miR-381) has been reported to play suppressive or promoting roles in different malignancies. However, the expression level, biological function, and underlying mechanisms of miR-381 in gastric cancer remain poorly understood. Our previous study indicated that transmembrane protein 16A (TMEM16A) contributed to migration and invasion of gastric cancer and predicted poor prognosis. In this study, we found that miR-381 inhibited the metastasis of gastric cancer through targeting TMEM16A expression. METHODS:MiR-381 expression was analyzed using bioinformatic software on open microarray datasets from the Gene Expression Omnibus (GEO) and confirmed by quantitative RT-PCR (qRT-PCR) in human gastric cancer tissues and cell lines. Cell proliferation was investigated using MTT and cell count assays, and cell migration and invasion abilities were evaluated by transwell assay. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Luciferase reporter assay, western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were employed to explore the mechanisms of the effect of miR-381 on gastric cancer cells. RESULTS:MiR-381 was significantly down-regulated in gastric cancer tissues and cell lines. Low expression of miR-381 was negatively related to lymph node metastasis, advanced tumor stage and poor prognosis. MiR-381 decreased gastric cancer cell proliferation, migration and invasion in vitro and in vivo. TMEM16A was identified as a direct target of miR-381 and the expression of miR-381 was inversely correlated with TMEM16A expression in gastric cancer tissues. Combination analysis of miR-381 and TMEM16A revealed the improved prognostic accuracy for gastric cancer patients. Moreover, miR-381 inhibited TGF-β signaling pathway and down-regulated epithelial-mesenchymal transition (EMT) phenotype partially by mediating TMEM16A. CONCLUSIONS:MiR-381 may function as a tumor suppressor by directly targeting TMEM16A and regulating TGF-β pathway and EMT process in the development of progression of gastric cancer. MiR-381/TMEM16A may be a novel therapeutic candidate target in gastric cancer treatment.
Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer.
Huang Shuai,Wa Qingde,Pan Jincheng,Peng Xinsheng,Ren Dong,Huang Yan,Chen Xiao,Tang Yubo
Journal of experimental & clinical cancer research : CR
BACKGROUND:Clinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. miR-141-3p is an extensively studied miRNA in cancers and downregulation of miR-141-3p has been widely reported to be involved in the progression and metastasis of several human cancer types. However, the clinical significance and biological roles of miR-141-3p in bone metastasis of PCa are still unclear. METHODS:miR-141-3p expression was examined in 89 non-bone metastatic and 52 bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to investigate the clinical correlation between miR-141-3p expression levels and clinicopathological characteristics in PCa patients. The biological roles of miR-141-3p in bone metastasis of PCa were evaluated both in vitro and a mouse intracardial model in vivo. Bioinformatics analysis, Western blot, luciferase reporter and miRNA immunoprecipitation assays were performed to explore and examine the relationship between miR-141-3p and its potential targets. Clinical correlation of miR-141-3p with its targets was examined in clinical PCa tissues. RESULTS:miR-141-3p expression is reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Low expression of miR-141-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Furthermore, upregulating miR-141-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Conversely, silencing miR-141-3p yields an opposite effect. Importantly, upregulating miR-141-3p dramatically reduces bone metastasis of PC-3 cells in vivo. Our results further show that miR-141-3p inhibits the activation of NF-κB signaling via directly targeting tumor necrosis factor receptor-associated factor 5(TRAF5) and 6 (TRAF6), which further suppresses invasion, migration and bone metastasis of PCa cells. The clinical negative correlation of miR-141-3p expression with TRAF5, TRAF6 and NF-κB signaling activity is demonstrated in PCa tissues. CONCLUSION:Our findings unravel a novel mechanism underlying the bone metastasis of PCa, suggesting that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis.
A CREB1/miR-433 reciprocal feedback loop modulates proliferation and metastasis in colorectal cancer.
Yan Li,You Wei-Qiang,Sheng Neng-Quan,Gong Jian-Feng,Hu Lan-Dian,Tan Ge-Wen,Chen Hong-Qi,Wang Zhi-Gang
Increasing evidence has indicated the prognostic value of miR-433 across a series of malignancy types. However, the underlying mechanisms involved in cancer progression haven't been sufficiently elucidated. In the present work, we found that miR-433 was downregulated in CRC tissues and cell lines. Ectopic expression of miR-433 obviously suppressed the proliferation, invasion and metastasis activity of CRC cells in vitro and in vivo. CREB1, CCAR1 and JNK1 were highly expressed and negatively correlated with miR-433 expression in CRC. CRC patients with higher expression of CREB1, CCAR1 or JNK1 presented a worse outcome relative to those with lower expression. CREB1 transactivated the expression of miR-433, and CREB1, CCAR1 and JNK1 simultaneously served as its targets, which in turn composed a feedback loop between CREB1 and miR-433. miR-433 blocked cell cycle progression and abolished EMT. Collectively, our study demonstrated the CREB1/miR-433 reciprocal feedback loop restrained the propagation, invasion and metastasis activities of CRC cells through abrogation of cell cycle progression and constraint of EMT.
miR-665 expression predicts poor survival and promotes tumor metastasis by targeting NR4A3 in breast cancer.
Zhao Xin-Ge,Hu Jing-Ye,Tang Jun,Yi Wei,Zhang Mei-Yin,Deng Rong,Mai Shi-Juan,Weng Nuo-Qing,Wang Rui-Qi,Liu Ji,Zhang Hui-Zhong,He Jie-Hua,Wang Hui-Yun
Cell death & disease
Cancer metastasis is the main cause of death in breast cancer (BC) patients. Therefore, prediction and treatment of metastasis is critical for enhancing the survival of BC patients. In this study, we aimed to identify biomarkers that can predict metastasis of BC and elucidate the underlying mechanism of the functional involvement of such markers in metastasis. miRNA expression profile was analyzed using a custom microarray system in 422 BC tissues. The relationship between the upregulated miR-665, metastasis and survival of BC was analyzed and verified in another set of 161 BC samples. The biological function of miR-665 in BC carcinogenesis was explored with in vitro and in vivo methods. The target gene of miR-665 and its signaling cascade were also analyzed. There are 399 differentially expressed miRNAs between BC and noncancerous tissues, of which miR-665 is the most upregulated miRNA in the BC tissues compared with non-tumor breast tissues (P < 0.001). The expression of miR-665 predicts metastasis and poor survival in 422 BC patients, which is verified in another 161 BC patients and 2323 BC cases from online databases. Ectopic miR-665 expression promotes epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of BC cells, and increases tumor growth and metastasis of BC in mice. Bioinformatics, luciferase assay and other methods showed that nuclear receptor subfamily 4 group A member 3 (NR4A3) is a target of miR-665 in BC. Mechanistically, we demonstrated that miR-665 promotes EMT, invasion and metastasis of BC via inhibiting NR4A3 to activate MAPK/ERK kinase (MEK) signaling pathway. Our study demonstrates that miR-665 upregulation is associated with metastasis and poor survival in BC patients, and mechanistically, miR-665 enhances progression of BC via NR4A3/MEK signaling pathway. This study provides a new potential prognostic biomarker and therapeutic target for BC patients.
MiR-429 increases the metastatic capability of HCC via regulating classic Wnt pathway rather than epithelial-mesenchymal transition.
Tang Jing,Li Liang,Huang Wentao,Sui Chengjun,Yang Yingcheng,Lin Ximeng,Hou Guojun,Chen Xin,Fu Jing,Yuan Shengxian,Li Shao,Wen Wen,Tang Shanhua,Cao Dan,Wu Mengchao,Chen Lei,Wang Hongyang
Epigenetic modification of miR-429 can manipulate liver T-ICs via targeting the RBBP4/E2F1/Oct4 axis, which might be crucial for hepatocarcinogenesis. However, whether miR-429 plays a role in regulating metastasis of hepatocellular carcinoma is still unclear. Using quantitative methylation analysis and real-time PCR, we have identified the hypomethylated status and upregulation of miR-429 in portal vein metastasis samples in comparison with their matched primary tumor. The ectopic expression of miR-429 dramatically induced the expression of MMP2/7/9 and enhanced HCC migration and invasion in vitro and in vivo in an EMT-independent manner. Both bioinformatics and functional studies elucidated the direct regulation of miR-429 on the 3'UTR of the PTEN gene, which leads to the activation of PI3K/AKT signaling and the nuclear translocation of β-catenin, eventually. Conversely, the knockdown of miR-429 efficiently recovered the expression of PTEN and attenuated PI3K/AKT/β-catenin-mediated cell metastasis. Clinically, the higher expression of miR-429 and nucleus relocation of β-catenin were identified as the adverse prognosis factors for recurrence-free survival (RFS) and overall survival (OS). In summary, our results here defined miR-429 as a key inducer for HCC pathogenesis and metastasis with potential utility for tumor intervention.
miR-1236 regulates hypoxia-induced epithelial-mesenchymal transition and cell migration/invasion through repressing SENP1 and HDAC3.
Chen Sung-Yuan,Teng Shu-Chun,Cheng Tzu-Hao,Wu Kou-Juey
Intratumoral hypoxia induces epithelial-mesenchymal transition and promotes cancer metastasis. MicroRNAs (miRNAs) are endogenous, single-strand RNA molecules that regulate gene expression. MiRNAs control cell growth, proliferation, differentiation and cell death and may function as oncogenes or tumor suppressors. HDAC3 and SENP1 are two molecules involved in hypoxia-induced EMT and HIF-1α stability, respectively. In this report, we show that miR-1236 plays a critical role in hypoxia-induced EMT and metastasis. MiRNA prediction programs TargetScan and miRanda show that miR-1236 may target HDAC3 and SENP1. MiR-1236 represses the luciferase activity of reporter constructs containing 3'UTR of HDAC3 and SENP1 as well as the expression levels of HDAC3 and SENP1. MiR-1236 abolishes hypoxia-induced EMT and inhibits migration and invasion activity of tumor cells. Hypoxia represses miR-1236 expression. The promoter region of miR-1236 is identified as the NELFE promoter. Twist1, an EMT regulator activated by hypoxia/HIF-1α, is shown to repress the reporter construct driven by the NELFE promoter. The binding site of Twist1 in the NELFE promoter is identified and chromatin immunoprecipitation assays show the direct binding of Twist1 to this site. Overexpression or knockdown of Twist1 in stable cell lines shows the inverse correlation between Twist1 and miR-1236 expression. These results identify a miRNA that regulates hypoxia-induced EMT and metastasis through repressing HDAC3 and SENP1 expression and present a regulatory network that involves many key players in hypoxia-induced EMT.
Oncogenic miR-210-3p promotes prostate cancer cell EMT and bone metastasis via NF-κB signaling pathway.
Ren Dong,Yang Qing,Dai Yuhu,Guo Wei,Du Hong,Song Libing,Peng Xinsheng
BACKGROUND:The primary issue arising from prostate cancer (PCa) is its high prevalence to metastasize to bone, which severely affects the quality of life and survival time of PCa patients. miR-210-3p is a well-documented oncogenic miRNA implicated in various aspects of cancer development, progression and metastasis. However, the clinical significance and biological roles of miR-210-3p in PCa bone metastasis remain obscure. METHODS:miR-210-3p expression was evaluated by real-time PCR in 68 bone metastatic and 81 non-bone metastatic PCa tissues. The biological roles of miR-210-3p in the bone metastasis of PCa were investigated both in vitro by EMT and Transwell assays, and in vivo using a mouse model of left cardiac ventricle inoculation. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to discern and examine the relationship between miR-210-3p and its potential targets. RT-PCR was performed to identify the underlying mechanism of miR-210-3p overexpression in bone metastasis of PCa. Clinical correlation of miR-210-3p with its targets was examined in human PCa and metastatic bone tissues. RESULTS:miR-210-3p expression is elevated in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Overexpression of miR-210-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Upregulating miR-210-3p enhances, while silencing miR-210-3p represses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Our results further demonstrate that miR-210-3p maintains the sustained activation of NF-κB signaling via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in EMT, invasion, migration and bone metastasis of PCa cells. Moreover, our results further indicate that recurrent gains (amplification) contribute to miR-210-3p overexpression in a small number of PCa patients. The clinical correlation of miR-210-3p with SOCS1, TNIP1 and NF-κB signaling activity is verified in PCa tissues. CONCLUSION:Our findings unravel a novel mechanism for constitutive activation of NF-κB signaling pathway in the bone metastasis of PCa, supporting a functional and clinical significance of epigenetic events in bone metastasis of PCa.
The epithelial-to-mesenchymal transition activator ZEB1 initiates a prometastatic competing endogenous RNA network.
Tan Xiaochao,Banerjee Priyam,Liu Xin,Yu Jiang,Gibbons Don L,Wu Ping,Scott Kenneth L,Diao Lixia,Zheng Xiaofeng,Wang Jing,Jalali Ali,Suraokar Milind,Fujimoto Junya,Behrens Carmen,Liu Xiuping,Liu Chang-Gong,Creighton Chad J,Wistuba Ignacio I,Kurie Jonathan M
The Journal of clinical investigation
Epithelial tumor cells undergo epithelial-to-mesenchymal transition (EMT) to gain metastatic activity. Competing endogenous RNAs (ceRNAs) have binding sites for a common set of microRNAs (miRs) and regulate each other's expression by sponging miRs. Here, we address whether ceRNAs govern metastasis driven by the EMT-activating transcription factor ZEB1. High miR-181b levels were correlated with an improved prognosis in human lung adenocarcinomas, and metastatic tumor cell lines derived from a murine lung adenocarcinoma model in which metastasis is ZEB1-driven were enriched in miR-181b targets. ZEB1 relieved a strong basal repression of α1 integrin (ITGA1) mRNA, which in turn upregulated adenylyl cyclase 9 mRNA (ADCY9) by sponging miR181b. Ectopic expression of the ITGA1 3'-untranslated region reversed miR-181b-mediated metastasis suppression and increased the levels of adenylyl cyclase 9 protein (AC9), which promoted tumor cell migration and metastasis. In human lung adenocarcinomas, ITGA1 and ADCY9 levels were positively correlated, and an AC9-activated transcriptomic signature had poor-prognostic value. Thus, ZEB1 initiates a miR-181b-regulated ceRNA network to drive metastasis.
MicroRNA-409 suppresses tumour cell invasion and metastasis by directly targeting radixin in gastric cancers.
Zheng B,Liang L,Huang S,Zha R,Liu L,Jia D,Tian Q,Wang Q,Wang C,Long Z,Zhou Y,Cao X,Du C,Shi Y,He X
Emerging evidence has shown that aberrantly expressed microRNAs (miRNAs) are highly associated with tumour development and progression. However, little is known about the potential role of miRNAs in gastric cancer (GC) metastasis. In this study, miR-409-3p was found to be downregulated frequently in human GCs, and its expression was significantly associated with tumor-node-metastasis (TNM) stage and lymph node metastasis. Enforced expression of miR-409 in GC cells significantly reduced their migration and invasion in vitro and their capacity to develop distal pulmonary metastases and peritoneal dissemination in vivo. Moreover, we found that miR-409 exerted its function predominantly through the mature miR-409-3p, but not miR-409-5p. Microarray and bioinformatics analysis identified the pro-metastatic gene radixin (RDX) as a potential miR-409-3p target. Further studies confirmed that miR-409-3p suppressed the expression of RDX by directly binding to its 3'-untranslated region. Silencing of RDX by small interfering RNAs phenocopied the effects of miR-409 overexpression, whereas restoration of RDX in miR-409-overexpressed GC cells reversed the suppressive effects of miR-409. Taken together, these results demonstrate that miR-409 suppresses GC cell invasion and metastasis by directly targeting RDX and that patients with downregulated miR-409-3p are prone to lymph node metastasis.