Role Of MiR-377 In The Proliferation Or Apoptosis Of Pancreatic Cancer And The Regulation Mechanisms

Background:Pancreatic ductal adenocarcinoma(PDAC)is a serious lethal malignancy and remains the fourth leading cause of cancer-related death,with a 5-year survival rate of 6 % globally.Due to its early aggressive nature,more than 80% of PDAC patients have invasive disease at the time of diagnosis,the curative efficacy of surgical and medical interventions is manifestly unsatisfactory.And the survival rate of PDAC remains extremely lower and the prognosis remains poor,compared with other cancer types.Investigate its reason we can from following aspects thinking,firstly,lacking of early diagnostic and prognostic markers,most of PDAC patients often have an advanced stage with local invasion and distant metastasis at the presence of symptoms,who have lost the best opportunity for surgical treatment.Secondly,PDAC is a highly heterogeneous disease,and various pathogenic genes are involved in its tumorigenesis progress,which revealed its complexity and clinical variability.As a consequence,it is urgent to understand the molecular mechanism/pathways governing oncogenesis,tumor progression and metastasis,and to scan out novel molecular markers with more sensitivity and specificity.Progress in fundamental research may provide novel potential therapeutic targets for better management of this lethal disease.Recent reports highlighted the important role of microRNAs(miRNAs)in tumor cell growth,development,differentiation,proliferation,and apoptosis.MiRNAs regulate the post-transcriptional level of key gene mRNAs by binding to 3'-UTR directly.Because of highly evolutionary conservation,thermodynamic stability,tissue specificity and ease of availability,these deregulated miRNAs may may function as novel biomarkers for PDAC.But,the accurate molecular mechanism of the deregulated miRNA expression remains largely unknown.Objective:This study aimed to investigate the miR-377 expression features of PDAC tissue and cell lines.And it may provide new insight into the role of miR-377 in the proliferation or apoptosis of pancreatic ductal adenocarcinoma cells and the regulation mechanisms.Materials and Methods:1.In this study,we identified the miRNAs expression feature of PDAC in miRNA profiling of tumor data from The Cancer Genome Atlas(TCGA)database and miRNA array of GEO database by using bioinformatics.We scanned out that miR-377 is significantly down-regulated in PDAC tumor and associated with overall survival.2.A total of 30 patients with PDAC were recruited prior to any therapy,and 30 paired tumor tissues and adjacent non-tumor tissues were obtained and we performed quantitative real-time polymerase chain reaction(QT-PCR)to validate the expression and significance of miR-377 in a cohort of 30 pairs of frozen PDAC tumors and adjacent matched NT tissues and cell lines.Statistical analysis was carried out to analyze the association between miR-377 level and the clinical parameter of the patients.3.To explore the potential role of miR-377 in PDAC growth,we generated stable ectopic miR-377 expression cell lines by transfection with lentiviral miR-377 overexpression vector,while aberrant miR-377 knock-down was conducted by lentiviral miR-377 inhibition vector respectively.CCK-8 assay and colony formation assay were performed to study the proliferation and viability.Transwell for Cell migration assay was used to investigate the migration ability.Flow cytometric analyses were performed to test the cell cycle distribution.Flow cytometric analyses were adopted to quantify the apoptotic cell proportion and TUNEL assays were performed to detect the apoptosis respectively.4.Binding-site predictions by bioinformatics showed that Pim-3 might be a potential target of miR-377.QT-PCR assay and Western Blotting(WB)assay was used to quantify the expression levels of Pim-3 mRNA and protein in PDAC cell lines with ectopic miR-377 expression respectively.And we analysised the correlation between miR-377 and Pim-3 mRNA expression in human pancreatic cancer tissues.Pim-3 protein expression was observed in four primary PDAC tissues(T)compared to adjacent non-tumorous tissues(AN)and in six PDAC cell lines and HPDE cell by Western blot analysis.And luciferase reporter assay was performed to confirm that Pim-3 is a direct target of miR-377.5.The changes of the target protein PIM-3 and downstream protein levels in PDAC cell lines were observed by WB assay.6.To determine the impact of Pim-3 alone on PDAC cell proliferation,colony formation,migration,cell cycle and survival,we knock-downed its expression by specific siRNA in SW1990 cell.Statistical analysis:All statistical analyses were performed adopting SPSS 23.0 software for Windows while graphs were generated using the GraphPad Prism 5.0.Comparison within groups was made by repeated measures ANOVA(or Student t test when only two groups were compared).The relationship between miR-377 and PIM3 mRNA was determined by Pearson's correlation analysis.Log rank Kaplan-Meier survival analysis was used to test for significant differences.Chi-square tests were used to identify the association between the level of PIM-3 mRNA and clinical parameters.All data are presented as mean ± S.D.;p< 0.05 was identified to be statistically significant.Results:1.miRNA expression profiling data from GEO and TCGA were scanned and we found that miR-377 is down-regulated in PDAC tissues and inversely correlates with the overall survival(OS)of PDAC.2.MiR-377 expression was specifically down-regulated in PDAC tumors and cell lines compared with paired AN tissues and human pancreatic ductal epithelium(HPDE)cell respectively.In addition,data indicated that the levels of miR-377 were significantly lower in PDAC patients with lymph node metastasis or larger tumor size.3.Ectopic stable expression of miR-377 can suppress PDAC cell proliferation,migration,and colony formation efficiency;it also can block the G1/S transition and induce cell apoptosis in vitro,and the resistance to chemotherapeutics is attenuated synchronously.4.Luciferase report assay identified that PIM-3 is a target gene of miR-377 and miR-377 suppresses PIM-3 expression by directly targeting its 3'-UTR.The negative correlation between miR-377 and Pim-3 mRNA expression was assessed by QT-PCR.WB assay data indicated,PIM-3 and Bad constitutively phosphorylated at Ser112 were simultaneously down-regulated in PDAC cell line with ectopic miR-377 expression.All data illustrates that miR-377 and PIM-3 regulates the proliferation and apoptosis of pancreatic cancer cells by inhibition of Bad-Bcl-XL anti-apoptotic pathway.5.Knock-down of Pim-3 by siRNA prevents cell proliferation,colony formation and migration,and induces cell cycle arrest,promotes cell death in SW1990 cell line.Taken together,these results indicated that Pim-3 is critical for miR-377-induced tumor suppressive effect in PDAC.Conclusions:Thus,this finding suggests that miR-377 is strongly down-regulated in PDAC,and inversely correlates with the overall survival of PDAC.MiR-377 level in PDAC tissues is associated with lymph node metastasis or larger tumor size.And miR-377 can inhibit the proliferation or colony formation of PDAC cells,induce apoptosis and block the G1/S transition and enhance the sensitivity to chemotherapeutics.Pim-3-pBad112-Bcl-XL regulation pathway is the downstream process of miR-377.Thus,the miR-377-Pim-3-pBAD112 as a novel regulatory pathway of apoptosis may provide potential therapeutic sites in PDAC and other malignancies.