MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer.
Zhao Liuyang,Sun Yan,Hou Yixuan,Peng Qiongle,Wang Liyang,Luo Haojun,Tang Xi,Zeng Zongyue,Liu Manran
The international journal of biochemistry & cell biology
Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.
10.1016/j.biocel.2012.08.005
Effects of TNFα on cell viability, proliferation and apoptosis of glioma cells U251.
Peng Zhang,Ying Lu
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
PURPOSE:To analyse the relationship between the expression of TNFα and the characteristics of glioma cell line U251. METHODS:U251 cell line was transfected with overexpressed TNFα or depleted TNFα constructs. The cell viability, proliferation and apoptosis were determined with MTT assay and flow cytometry analysis. The expressions of TNFα, caspase or microRNA (miR) were detected by Western blot analysis or real-time PCR (RT-PCR). The activity of caspase3 was examined by the absorbance of p-nitroaniline (pNA) production. RESULTS:Overexpression of TNFα could decrease cell viability, suppress cell proliferation and promote cell apoptosis, whereas knockdown of TNFα displayed the opposite effects. In addition, the effects of TNFα on the expression of caspase3, miR-9-3p, miR-9-5p, miR-17-3p, miR-17-5p, miR-31-3p and miR-31-5p were also probed. Overexpression of TNFα could raise the expression of caspase3 and miR-9 and knockdown of TNFα had an opposite effect. Our results also showed the overexpression of miR-9 could increase the expression of caspase3, while the inhibition of miR-9 had the opposite effect. CONCLUSION:These results suggested that TNFα might be involved in the attenuation of growth, proliferation and promotion of apoptosis in U-251 cells by regulating the expression of miR-9 and caspase3 and that miR-17 and miR- 31 perhaps were not correlated with the apoptosis of U251 cells or not acted as important factors in mediating the apoptosis of U251 cells induced by TNFα. Furthermore, the caspase3 could be regulated by miR-9 in an indirect way.
[Dectection and analysis of miRNA expression in breast cancer-associated fibroblasts].
Zeng Zongyue,Hu Ping,Tang Xi,Zhang Hailong,Du Yane,Wen Siyang,Liu Manran
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
OBJECTIVE:To investigate the difference of miRNA expression levels of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) in human breast cancer microenvironment and its effect on the biological features of CAFs. METHODS:Collagenase-1 was used to digest the cancer and adjacent tissues to isolate CAFs and NFs. The isolated cells were cultured and characterized in purity and biological features. The expression of fibroblast secretory protein (FSP) in CAFs and NFs was detected by immunofluorescence staining and Western blotting. Transwell(TM) assay was adopted to compare the invasion ability of CAFs and NFs. The different expressions of miRNAs in CAFs versus NFs were detected by miRNA microarray and analyzed by Significance Analysis of Microarrays (SAM). The differences in miR-205 and miR-221 expressions were verified by real-time quantitative PCR (qRT-PCR). The common target genes of the miRNAs were predicted using multi-bioinformatics tools. The pathway analysis was conducted through the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. The secreting products of TGF-β or IL-6 signaling pathway, matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 were analyzed by ELISA. RESULTS:The primary CAFs and NFs were isolated from breast cancer patients with a purity of over 95%. Compared with NFs, the expression of FSP was obviously elevated in CAFs, and the invasion ability of CAFs was enhanced. The miRNA microarray results showed that there were 10 miRNA genes dysregulated in CAFs, including 3 up-regulated (miR-221-5p, miR-31-3p, miR-221-3p) and 7 down-regulated genes (miR-205, miR-200b , miR-200c, miR-141, miR-101, miR-342-3p, let-7g). The common targets genes of the dysregulated miRNAs were mainly focused on HGF, chemokine signaling, insulin signaling, MAPK signaling, tight junction signaling, adherence junction signaling, EGF1 signaling, androgen-receptor signaling, Wnt and IL-7 signaling. In addition, dysregulated miR-200b/c and miR-141 et al. affect TGF-β and IL-6 signaling through inhibiting their target genes in CAFs, thus promoting invasion and migration of CAFs. CONCLUSION:The miRNA expression profile was markedly dysregulated in CAFs. Those dysregulated miRNAs may take part in the transformation from NFs to CAFs, and also have a close relationship with adhesion, migration, proliferation, secretion and cell-cell interaction of CAFs.
MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis.
Fang Kai,Law Ivy Ka Man,Padua David,Sideri Aristea,Huang Vanessa,Kevil Christopher G,Iliopoulos Dimitrios,Pothoulakis Charalabos
The American journal of pathology
Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460-NK-1R cells on SP stimulation. Our results reveal a novel SP-miR-31-3p-RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment.
10.1016/j.ajpath.2017.10.023
Two GEO MicroRNA Expression Profile Based High-Throughput Screen to Identify MicroRNA-31-3p Regulating Growth of Medullary Thyroid Carcinoma Cell by Targeting RASA2.
Jiang Mei,Shi Xin,Zhu Hua,Wei Wu,Li Jinyan
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND Medullary thyroid carcinoma (MTC), a rare type of thyroid cancer, is a big challenge in clinical treatment. However, the pathogenesis of MTC remains poorly understand. MicroRNAs (miRNAs) were previously demonstrated to be involved in the pathogenesis of MTC, however, the roles of majority of miRNAs in MTC are still undetermined. MATERIAL AND METHODS Two GEO miRNA expression profiles (GSE40807, GSE97070) were downloaded, and the differentially expressed miRNAs (DEmiRNAs) of GSE40807 and GSE97070 were analyzed by bioinformatics methods. Expressions of miRNAs were detected by quantitative real-time polymerase chain reaction; cell proliferation was examined through Cell Counting Kit-8, colony formation and in vivo tumor growth assays; the interaction between miRNA and mRNA was verified by dual-luciferase reporter assay; functional analysis of target genes was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, www.david.ncifcrf.gov) software. RESULTS Ten miRNAs were identified to be dysregulated in both GSE40807 and GSE97070 datasets, and miR-31-3p showed the highest change fold (Log fold change=-3.460625 in GSE40807 and Log fold change=-0.07084374 in GSE97070). MiR-31-3p expression was significantly downregulated in MTC, and low miR-31-3p expression showed a poor prognosis relative to high miR-31-3p expression (P<0.05). Functionally, miR-31-3p inhibited MTC cell proliferation in vitro and in vivo. Functional analysis also showed that the target genes of miR-31-3p were involved in numerous of biochemical processes and pathways, of which Ras signaling pathway was selected for further study. RASA2, overexpressed in MTC, were negatively regulated by miR-31-3p. In addition, we found that knockdown of RASA2 inhibited MTC cell proliferation. CONCLUSIONS Reduced expression level of miR-31-3p might play a key role in the tumorigenesis of MTC by targeting critical pathways, especially Ras signaling pathway.
10.12659/MSM.916815
Circular RNA ANKRD36 attends to lipopolysaccharide-aroused MRC-5 cell injury via regulating microRNA-31-3p.
BioFactors (Oxford, England)
BACKGROUND:Some circular RNAs (circRNAs) are reported to attend to the pathogenesis of pneumonia. This study tested the impact of circRNA ankyrin repeat domain 36 (circANKRD36) on human embryonic lung fibroblast MRC-5 cell injury irritated by lipopolysaccharide (LPS). METHODS:After LPS irritation, viability, apoptosis, ROS, protein, and cytokines, along with circANKRD36 were tested by CCK-8, Annexin V-FITC, DCFH-DA, and ELISA or Western blot. si-circANKRD36 and microRNA-31-3p/5p (miR-31-3p/5p) inhibitor were applied to silence circANKRD36 and miR-31-3p/5p. miR-31-3p/5p mimic was utilized to upregulate miR-31-3p/5p. RT-qPCR was used to detect miRNAs. The relationship between miRNAs and MyD88 or IL-34 was analyzed by luciferase activity reporter assay. RESULTS:LPS aroused a decrease in viability, increases in apoptosis, ROS, and IL-6, IL-8, and TNF-α, along with circANKRD36, and activation of NF-κB pathway. Silencing circANKRD36 weakened the above-mentioned influences of LPS. Moreover, silencing circANKRD36 hoisted miR-31-3p expression. Silencing miR-31-3p mitigated the impacts of circANKRD36 silence on LPS-irritated MRC-5 cells. Besides, MyD88 was a downstream target of miR-31-3p, and 3'UTR of IL-34 mRNA was targeted by miR-31-5p. LPS induced the accumulation of MyD88. Silencing MyD88 was constructive to maintain cell viability, retard apoptosis and inhibit adverse oxidation and inflammation. CONCLUSION:This research verified that silencing circANKRD36 could weaken LPS-irritated MRC-5 cell injury via regulating miR-31/MyD88-mediated repression of NF-κB pathway.
10.1002/biof.1592
Hsa-miR-31-3p expression is linked to progression-free survival in patients with KRAS wild-type metastatic colorectal cancer treated with anti-EGFR therapy.
Manceau Gilles,Imbeaud Sandrine,Thiébaut Raphaële,Liébaert François,Fontaine Karine,Rousseau Francis,Génin Bérengère,Le Corre Delphine,Didelot Audrey,Vincent Marc,Bachet Jean-Baptiste,Chibaudel Benoist,Bouché Olivier,Landi Bruno,Bibeau Frédéric,Leroy Karen,Penault-Llorca Frédérique,Van Laethem Jean-Luc,Demetter Pieter,Tejpar Sabine,Rossi Simona,Mosakhani Neda,Osterlund Pia,Ristamäki Raija,Sarhadi Virinder,Knuutila Sakari,Boige Valérie,André Thierry,Laurent-Puig Pierre
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:To identify microRNAs (miRNA) that predict response to anti-EGFR antibodies in patients with wild-type KRAS metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN:miRNA profiling was performed in a training set of 87 patients with mCRC refractory to chemotherapy treated with anti-EGFR antibodies. This included 33 fresh-frozen (FF) and 35 formalin-fixed paraffin-embedded (FFPE) samples retrospectively collected and 19 prospectively collected FF samples. An independent validation cohort consisting of 19 FF and 26 FFPE prospectively collected samples from patients with mCRC treated with anti-EGFR antibodies was used to confirm our findings. RESULTS:After screening the expression of 1,145 miRNAs in FF samples from the training set, we identified that hsa-miR-31-3p expression level was significantly associated with progression-free survival (PFS). Statistical models based on miRNA expression discriminated between high and low risk of progression for both FF and FFPE samples. These models were confirmed in the validation cohort for both FF [HR, 4.1; 95% confidence interval (CI), 1.1-15.3; P < 0.04] and FFPE samples (HR, 2.44; 95% CI, 1.1-5.4; P = 0.028). The percentage of variation of RECIST criteria in the validation series was significantly associated with the expression level of hsa-miR-31-3p (r(2) = 0.49; P = 0.0035) and risk status determined by hsa-miR-31-3p expression level (P = 0.02, Kruskal-Wallis rank test). Nomograms were built and validated to predict PFS-depending on hsa-miR-31-3p expression level. Following in vitro studies, we identified 47 genes regulated by hsa-miR-31-3p. CONCLUSION:Hsa-miR-31-3p seems to be a new mCRC biomarker whose expression level allows for the identification of patients with wild-type KRAS mCRC who are more likely to respond to anti-EGFR therapy.
10.1158/1078-0432.CCR-13-2750
Association between miR-31-3p expression and cetuximab efficacy in patients with KRAS wild-type metastatic colorectal cancer: a post-hoc analysis of the New EPOC trial.
Oncotarget
BACKGROUND:High miR-31-3p expression is associated with inferior outcomes in KRAS wild-type (WT) advanced colorectal cancer patients treated with anti-EGFR therapy. This study evaluated miR-31-3p expression in patients with operable colorectal liver metastases (LM) enrolled in the New EPOC study. METHODS:MiR-31-3p expression was measured in primary tumors (PT) from 149 KRAS WT patients including 71 receiving chemotherapy alone (CT) and 78 receiving chemotherapy plus cetuximab (CTX). Each treatment arm was split into tertiles based on miR-31-3p expression levels. MiR-31-3p expression was also measured in LM from 94 patients with tumor tissue available. RESULTS:The median progression-free survival for the combined populations with mid or high miR-31-3p expression was shorter in the CTX versus the CT arm (26.7 months versus 12.3 months, HR=2.28 95%CI 1.27; 4.09 p=0.006). Low miR-31-3p expressers had similar outcomes irrespective of treatment (HR=1.06 95%CI 0.43; 2.61 p=0.9). MiR-31-3p expression was correlated between paired PT and LM samples in the CT group but not in the CTX group. CONCLUSIONS:Patients with low miR-31-3p expression in the New EPOC study were not harmed by the addition of cetuximab. This supports miR-31-3p as a promising predictive biomarker for anti-EGFR therapy in KRAS WT advanced colorectal cancer.
10.18632/oncotarget.21291
Validation of miR-31-3p Expression to Predict Cetuximab Efficacy When Used as First-Line Treatment in Wild-Type Metastatic Colorectal Cancer.
Laurent-Puig Pierre,Grisoni Marie-Lise,Heinemann Volker,Liebaert François,Neureiter Daniel,Jung Andreas,Montestruc François,Gaston-Mathe Yann,Thiébaut Raphaële,Stintzing Sebastian
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:MiR-31-3p expression has been shown to be associated with response to anti-EGFR therapy. We investigated the predictive role of this biomarker in the FIRE-3 study population, including its ability to differentiate outcomes between patients receiving anti-EGFR and anti-VEGF therapy. EXPERIMENTAL DESIGN:MiR-31-3p expression was measured in primary tumors obtained from 340 patients with WT mCRC enrolled in the FIRE-3 Trial. This included 164 patients randomized to receive FOLFIRI plus cetuximab (FOLFIRI+Cetux) and 176 to FOLFIRI plus bevacizumab (FOLFIRI+Beva). Patients were divided into subgroups defined by low or high miR-31-3p expression using a prespecified cut-off and by treatment arm. Analyses were performed to assess treatment efficacy by subgroup. Overall survival (OS) and progression-free survival (PFS) were analyzed using Kaplan-Meier curves and Cox regression models. Investigator-assessed objective response (iOR), early tumor shrinkage at 6 weeks (ETS), and centrally reviewed objective response (cOR) were analyzed using logistic regression models. The predictive value of miR-31-3p expression level was assessed through a treatment interaction test using multivariate models adjusted for potential confounding factors. RESULTS:Low miR-31-3p expressers benefited from cetuximab compared with bevacizumab for PFS [HR, 0.74; 95% confidence interval (CI), 0.55-1.00; = 0.05], OS (HR, 0.61; 95% CI, 0.41-0.88; < 0.01), iOR (OR, 4.0; 95% CI, 1.9-8.2; < 0.01), ETS (OR, 4.0; 95% CI, 2.1-7.7; < 0.01 and cOR (OR, 4.9; 95% CI, 2.3-10.5; < 0.01) in multivariate analyses. There was no difference in outcomes for high expressers between treatment arms. MiR-31-3p expression level was predictive of treatment effect for PFS ( = 0.03), OS ( = 0.05), iOR ( = 0.02), ETS ( = 0.04), and cOR ( < 0.01). CONCLUSIONS:MiR-31-3p expression level was validated as a predictive biomarker of cetuximab therapy efficacy for patients with WT mCRC.
10.1158/1078-0432.CCR-18-1324
Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens.
Ramon Lucas,David Catherine,Fontaine Karine,Lallet Elodie,Marcaillou Charles,Martin-Lannerée Séverine,Decaulne Virginie,Vazart Céline,Gélibert Anne-Héloise,Abdelali Raouf Ben,Costa Jean-Marc,Rousseau Francis,Thiébaut Raphaële,Yost Larry,Gaston-Mathé Yann
Biomarker insights
MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.
10.1177/1177271918763357
miR-31-3p Expression and Benefit from Anti-EGFR Inhibitors in Metastatic Colorectal Cancer Patients Enrolled in the Prospective Phase II PROSPECT-C Trial.
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Anti-EGFR mAbs are effective in the treatment of metastatic colorectal cancer (mCRC) patients. status and tumor location (sidedness) are predictive markers of patients' response to anti-EGFR mAbs. Recently, low miR-31-3p expression levels have been correlated with clinical benefit from the anti-EGFR mAb cetuximab. Here, we aimed to validate the predictive power of miR-31-3p in a prospective cohort of chemorefractory mCRC patients treated with single-agent anti-EGFR mAbs. EXPERIMENTAL DESIGN:miR-31-3p was tested by hybridization (ISH) in 91 pretreatment core biopsies from metastatic deposits of 45 patients with mCRC. Sequential tissue biopsies obtained before treatment, at the time of partial response, and at disease progression were tested to monitor changes in miR-31-3p expression overtreatment. miR-31-3p expression, sidedness, and status in pretreatment cell-free DNA were combined in multivariable regression models to assess the predictive value of each variable alone or in combination. RESULTS:Patients with low miR-31-3p expression in pretreatment biopsies showed better overall response rate, as well as better progression-free survival and overall survival, compared to those with high miR-31-3p expression. The prognostic effect of miR-31-3p was independent from age, gender, and sidedness. No significant changes in the expression of miR-31-3p were observed when sequential tissue biopsies were tested in long-term or poor responders to anti-EGFR mAbs. miR-31-3p scores were similar when pretreatment biopsies were compared with treatment-naïve archival tissues (often primary colorectal cancer). CONCLUSIONS:Our study validates the role of miR-31-3p as potential predictive biomarker of selection for anti-EGFR mAbs.
10.1158/1078-0432.CCR-18-3769
Sema4C mediates EMT inducing chemotherapeutic resistance of miR-31-3p in cervical cancer cells.
Jing Li,Bo Wang,Yourong Feng,Tian Wang,Shixuan Wang,Mingfu Wu
Scientific reports
Sema4C, the target of many miRNAs, is involved in EMT-mediated chemotherapeutic resistance of many malignant tumors. However, the underlying upstream regulatory mechanisms of Sema4C-induced EMT and Sema4C-mediated drug resistance are still unclear. The aim of this study was to explore the potential role of miR-31-3p/Sema4C in regulating EMT in cisplatin-resistant (CR) cervical cancer cells. High expression levels of Sema4C were more frequently found in cervical cancer tissues and were associated with poor prognosis, whereas miR-31-3p was significantly downregulated in cervical cancer tissues, which was associated with shorter disease-free and overall survival. Overexpression of miR-31-3p inhibited malignant behaviors and EMT of cervical cancer cells in vitro. Furthermore, miR-31-3p was identified to directly target Sema4C, and upregulation of miR-31-3p reversed EMT-mediated biological functions, including cisplatin resistance of Sema4C in cervical cancer cells. These results suggest that Sema4C promoted EMT-mediated cisplatin resistance in cervical cancer cells and that this effect was inhibited by overexpression of miR-31-3p. Thus, silencing Sema4C or overexpression of miR-31-3p could be a novel approach to treat drug resistance to chemotherapy in cervical cancers.
10.1038/s41598-019-54177-z
Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells.
Pfizenmaier Jennifer,Junghans Lisa,Teleki Attila,Takors Ralf
Biotechnology journal
Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.
10.1002/biot.201500606
Identification of CNS Injury-Related microRNAs as Novel Toll-Like Receptor 7/8 Signaling Activators by Small RNA Sequencing.
Wallach Thomas,Wetzel Max,Dembny Paul,Staszewski Ori,Krüger Christina,Buonfiglioli Alice,Prinz Marco,Lehnardt Seija
Cells
Toll-like receptors (TLRs) belong to pattern recognition receptors, which respond to danger signals such as pathogen-associated molecular patterns or damage-associated molecular patterns. Upon TLR activation in microglia, the major immune cells in the brain, distinct signaling cascades trigger the production of inflammatory molecules, being a critical feature in neuroinflammation and neurodegenerative processes. Recently, individual microRNAs (miRNAs) were shown to act as endogenous TLR ligands. Here, we conducted systematic screening for miRNAs as potential TLR7/8 ligands by small RNA sequencing of apoptotic neurons and their corresponding supernatants. Several miRNA species were identified in both supernatants and injured neurons, and 83.3% of the media-enriched miRNAs activated murine and/or human TLR7/8 expressed in HEK293-derived TLR reporter cells. Among the detected extracellular miRNAs, distinct miRNAs such as miR-340-3p and miR-132-5p induced cytokine and chemokine release from microglia and triggered neurotoxicity in vitro. Taken together, our systematic study establishes miRNAs released from injured neurons as new TLR7/8 activators, which contribute to inflammatory and neurodegenerative responses in the central nervous system (CNS).
10.3390/cells9010186
LINC01551 promotes metastasis of nasopharyngeal carcinoma through targeting microRNA-132-5p.
Xue M-Y,Cao H-X
European review for medical and pharmacological sciences
OBJECTIVE:Previous studies have shown that long intergenic non-coding RNA01551 (LINC01551) is a cancer-promoting gene. However, the role of LINC01551 in nasopharyngeal carcinoma (NPC) has not been reported. Therefore, the aim of this study was to investigate the expression characteristics of LINC01551 in NPC, and to further explore its mechanism in promoting the metastasis. PATIENTS AND METHODS:Quantitative real time-polymerase chain reaction (qRT-PCR) was used to detect the expression levels of LINC01551 in tumor tissue samples and paracancerous normal ones of 36 patients with NPC; meanwhile, the expression of LINC01551 in NPC cell lines was also verified using the qRT-PCR assay. In addition, the LINC01551 knockdown model was constructed in NPC cell lines (CNE2 and 6-10B) using lentivirus, and the influence of LINC01551 on the function of NPC cells was analyzed by cell counting kit-8 (CCK-8) and transwell invasion assays. Finally, the interaction between LINC01551 and microRNA-132-5p was examined by Luciferase reporter gene assay, while the potential mechanism was further explored by cell reverse experiments. RESULTS:The results of qRT-PCR indicated that the expression level of LINC01551 in tumor tissue specimens of these patients was remarkably higher than that in adjacent tissues, and the difference was statistically significant. Meanwhile, LINC01551 expression was also found remarkably higher in cell lines than that in normal ones. In addition, compared with blank or control group, the proliferation, invasion and metastasis ability of NPC cells in LINC01551 knockdown group (si-LINC01551) was significantly reduced. Subsequently, the result of Luciferase reporting assay demonstrated that overexpression of microRNA-132-5p attenuated the Luciferase activity of the wild-type LINC01551 vector without attenuating that of the mutant vector, further demonstrating that LINC01551 can be combined with miR-132-5p. Additionally, the result of cell reverse experiment revealed that knockdown of microRNA-132-5p could reverse the effect of LINC01551 silencing on proliferation rate and metastasis of NPC cells, thus further demonstrating the mutual regulation between LINC01551 and microRNA-132-5p. CONCLUSIONS:The above studies indicated that LINC01551 was remarkably up-regulated in NPC tissues, as well as in cell lines. In addition, studies have shown that LINC01551 may promote the metastatic ability by regulating microRNA-132-5p.
10.26355/eurrev_202004_20836
Plasma miR-22-5p, miR-132-5p, and miR-150-3p Are Associated with Acute Myocardial Infarction.
Li Huixian,Zhang Pengxiang,Li Fangjiang,Yuan Guili,Wang Xiaoyuan,Zhang Aiai,Li Feixing
BioMed research international
Circulating microRNAs (miRNAs) are potential biomarkers for cardiovascular diseases. Our study aimed to determine whether miR-22-5p, miR-132-5p, and miR-150-3p represent novel biomarkers for acute myocardial infarction (AMI). Plasma samples were isolated from 35 AMI patients and 55 matched controls. Total RNA was extracted, and quantitative real-time PCR and ELISA were performed to investigate the expressions of miRNAs and cardiac troponin I (cTnI), respectively. We found that plasma levels of miR-22-5p and miR-150-3p were significantly higher during the early stage of AMI and their expression levels peaked earlier than cTnI. Conversely, circulating miR-132-5p was sustained at a low level during the early phase of AMI. All three circulating miRNAs were correlated with plasma cTnI levels. A receiver operating characteristic (ROC) analysis suggested that each single miRNA had considerable diagnostic efficacy for AMI. Moreover, combining the three miRNAs improved their diagnostic efficacy. Furthermore, neither heparin nor medications for coronary heart disease (CHD) affected plasma levels of miR-22-5p and miR-132-5p, but circulating miR-150-3p was downregulated by medications for CHD. We concluded that plasma miR-22-5p, miR-132-5p, and miR-150-3p may serve as candidate diagnostic biomarkers for early diagnosis of AMI. Moreover, a panel consisting of these three miRNAs may achieve a higher diagnostic value.
10.1155/2019/5012648
miR-132-5p regulates apoptosis and autophagy in MPTP model of Parkinson's disease by targeting ULK1.
Zhao Jianli,Yang Manyi,Li Qi,Pei Xiaorui,Zhu Xiaodong
Neuroreport
Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by a loss of dopaminergic neurons in the substantia nigra of the brain. Numerous investigations have focused on the underlying mechanism involved in the progression of PD in recent decades. miR-132 is abnormal expression in many diseases including PD. However, the functional role and molecular mechanism of miR-132-5p in PD pathogenesis are still not elucidated. In our study, we found miR-132-5p was upregulated in 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP) model of PD. MTT assay and flow cytometric analysis revealed that inhibition of miR-132-5p increased cell survival ability and reduced MPTP-induced apoptosis of SH-SY5Y cells. Furthermore, inhibition of miR-132-5p could significantly suppressed mRNA and protein expression levels of LC3 and Beclin 1, indicating inhibition of miR-132-5p might restrain autophagy in PD. Subsequently, ULK1 was identified as a target of miR-132-5p and positively regulated by miR-132-5p at both mRNA and protein levels. Additionally, ectopic expression of ULK1 was able to reverse the effects of miR-132-5p inhibition. Taken together, our results demonstrated that miR-132-5p inhibition might exert a protective role in MPTP-treated PD models by targeting ULK1, indicating that miR-132-5p may be a prospective therapeutic target for PD.
10.1097/WNR.0000000000001494
Nicotine-induced upregulation of miR-132-5p enhances cell survival in PC12 cells by targeting the anti-apoptotic protein Bcl-2.
Shrestha Tejashwi,Takahashi Tetsuya,Li Chengyu,Matsumoto Masayasu,Maruyama Hirofumi
Neurological research
: Activation of nicotinic acetylcholine receptors (nAChRs) results in neuroprotection via a poorly understood molecular mechanism. In this study, we aimed to investigate the effect of nAChR stimulation with nicotine on the regulation of microRNA (miRNA) expression and identify the molecular pathway involved in neuroprotection.: We conducted miRNA expression profiling using a microarray to identify the miRNAs regulated by nicotine. miR-132-5p expression was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Cells were treated with nicotine, a miR-132-5p mimic or its inhibitor, and the cell viability was assessed. CREB, Bcl-2, Bax, cleaved caspase-3, and α-tubulin protein expression levels were determined by Western blotting analysis.: Using a rodent miRNA microarray, we identified 37 miRNAs regulated by nicotine. The microarray and RT-qPCR results showed 1.67-fold and 1.5-fold increases in miR-132-5p, respectively, upon nicotine treatment. Immunoblotting revealed a >2-fold increase in phosphorylation of CREB with nicotine, peaking at 4 h. Nicotine treatment of cells increased viability from 35% to 54%, and Bcl-2 immunoreactivity increased by 1.4-fold. Overexpression of miR-132-5p increased cell viability from 38% to 70% and increased Bcl-2 expression by 3.9-fold. Inhibition of miR-132-5p decreased cell viability to 25%, whereas no change was observed in Bcl-2. Bax expression remained unchanged following treatment with a miR-132-5p mimic or its inhibitor.: Our results suggest that nAChR activation facilitates cell survival by upregulating miR-132-5p, which upregulates the anti-apoptotic protein Bcl-2. These results present miR-132-5p as a potential novel therapeutic target to achieve neuroprotection via stimulation of nAChRs.: CCK-8: Cell counting kit-8; nAChR: Nicotinic acetylcholine receptor; NGF: Nerve growth factor; WST-8: [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt].
10.1080/01616412.2020.1735817
A P53-related microRNA model for predicting the prognosis of hepatocellular carcinoma patients.
Fang Shuang-Sang,Guo Jin-Cheng,Zhang Jian-Hua,Liu Jin-Na,Hong Shuai,Yu Bo,Gao Yuhan,Hu Su-Pei,Liu Hai-Zhong,Sun Liang,Zhao Yi
Journal of cellular physiology
Studies have shown that microRNAs (miRNAs) play a vital role in tumor progression and patients' prognosis. Therefore, we aimed to construct a miRNA model for forecasting the survival of hepatocellular carcinoma (HCC) patients. The gene expression data of 433 patients with HCC from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus public databases were remined by survival analysis and receptor manipulation characteristic curve (ROC). A prognostic model including six miRNAs (hsa-mir-26a-1-3p, hsa-mir-188-5p, hsa-mir-212-5p, hsa-mir-149-5p, hsa-mir-105-5p, and hsa-mir-132-5p) were constructed in the training dataset (TCGA, n = 333). HCC patients were stratified into a high-risk group and a low-risk group with significantly different survival (median: 2.75 vs. 8.93 years, log-rank test p < .001). Then we proved its performance of stratification in another independent dataset (GSE116182, median: 2.55 vs 6.96 years, log-rank test p = .008). Cox regression analysis showed that the prognostic model was an independent prognostic indicator for HCC patients. Then time-dependent ROC analyses were performed to test the prognostic ability of the model with that of TNM staging, we found the model had a better performance, especially at 5 years (AUC = 0.76). Functional prediction showed that the genes targeted by the six prognostic miRNAs in the prognostic model were highly expressed in the P53-related pathway. In conclusion, we constructed a prognostic miRNA model that could indicate the survival of HCC patients.
10.1002/jcp.29245
DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC).
Sannigrahi M K,Sharma Rajni,Singh Varinder,Panda Naresh K,Rattan Vidya,Khullar Madhu
Molecular and cellular biochemistry
INTRODUCTION:Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma. METHODOLOGY:HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2'-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV -ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV -ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis. RESULTS:Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV -ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV -ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls and both were up-regulated in HPV +ve compared to HPV -ve TCGA tissue samples. In silico analysis revealed hsa-miR-132-5p targeted canonical β-catenin/wnt pathway and modulation of down-stream genes of the pathway was observed on over-expression/inhibition of hsa-miR-132-5p. CONCLUSION:This study suggests the role of epigenetic modifications in regulating expression of miRNAs in HPV +ve HNSCC.
10.1007/s11010-018-3336-6
The expression, induction and pharmacological activity of CYP1A2 are post-transcriptionally regulated by microRNA hsa-miR-132-5p.
Chen Yinting,Zeng Linjuan,Wang Yong,Tolleson William H,Knox Bridgett,Chen Si,Ren Zhen,Guo Lei,Mei Nan,Qian Feng,Huang Kaihong,Liu David,Tong Weida,Yu Dianke,Ning Baitang
Biochemical pharmacology
Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3'-untranslated region (3'-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3'-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.
10.1016/j.bcp.2017.08.012