| BackgroundColorectal cancer（CRC）is currently the third most commom cancer in men and the second in women.More than one million new CRC cases are reported and more than half a million patients died of CRC worldwide every year.Although CRC is one of the most common malignant tumors in the world,but up to now,the lack of an effective biomarker for the diagnosis of CRC and treatment for CRC.microRNA（miRNA）is a small single-stranded RNA that repress the mRNA translation and triggere its drgradaton.miRNA is aberrantly expressed in CRC,and it is related to the initiation,progression and drug resistance of CRC.Many studies have shown that miRNA could influence the cell cycle,differentiation,apoptosis,and migration of CRC cells through regulating target gene expression at the psot-transcriptional level.miR-31-5p plays a oncogene role in mmultiple malignant tumors,but the role miR-31-5p in the development and progression of CRC has rarely been reported.The aim of this study was to investigate the role and specific molecular mechanism of miR-31-5 in CRC,and to privide a new target for CRC treatment.ObjectiveGrowing evidence suggests that aberrant miRNA expressioncontributes to CRC pathogenesis and development.miR-31-5p plays a cruical role in the progression of several cancers.However,the potential role and mechanism of miR-31-5p in the progression of CRC remains largely unknown.This study aimed to investigate the effect on the cell proliferation,migration,invasion,cell cycle and apoptosis and specific mechanism of miR-31-5p in CRC cancer cells.MethodsPart one Expression of in human colonrectal cancer tissues and cell lines and influence of miR-31-5p on biological behavior of colonrectal cancer cell in vivo and vitro1.The relative expression of miR-31-5p in colorectal cancer tissues,paired normal tissues and cell lines was detected by qRT-PCR and the relationship between expression of miR-31-5p and clinical features of CRC patients was analysised.2.To knock down the expression of miR-31-5p,the transfection of miR-31-5p inhibitor was performed.The relative expression of miR-31-5p in colorectal cancer cell lines HT29 and HCT116 was detected by qRT-PCR,and cell proliferation was analyzed by CCK-8 assay.The cell apoptosis and cell cycle was evaluated with flow cytometry.Cell migration and invasion capacity were examined by Transwell assay.3.Xenograft tumours were subcutaneously injection of CRC cells transfected with the miR-31-5p inhibitor into the 4-6 week-old BALB/c mice,the animal model transplantation tumor in nude mice was constructed,tumor volume and mass was calculated and growth curve were drawed.Part two Research on the relationship between miR-31-5p and NUMB1.We predicted the target gene of miR-31-5p using miRBase（http://www.mirbase.org/）and TargetScanHuman（http://www.targetscan.org/vert71/）,Luciferase reporter assay was used to confirm that NUMB was a direct target of miR-31-5p.2.qRT-PCR and Western blotting ware used to analyze NUMB expression in colorectal cancer tissues and adjacent normal tissues,also we analysed the correlation between the expression of miR-31-5p and NUMB.To increase the expression of miR-31-5p,the transfection of miR-31-5p mimic was performed and NUMB mRNA and protein expression in colorectal cancer cells were detected.3.NUMB gene expression was upregulated in colorectal cancer cells transfected with pcDNA3.0-UNMB plasmid,NUMB mRNA expression was detected by qRT-PCR,and cell proliferation was analyzed by CCK-8assay.The cell apoptosis and cell cycle was evaluated with flow cytometry.Cell migration and invasion capacity were examined by Transwell assay.4.To confirm that miR-31-5p regulated migration,invasion,and proliferation of colorectal cancer cells through targeting NUMB,HT29 and HCT116 cells were transfected with miR-31-5p inhibitor and miR-31-5p inhibitor+NUMB siRNA,NUMB mRNA expression was detected by qRT-PCR,CCK-8 assay was used to analyze the colorectal cancer cell proliferation of Blank group,NC group,miR-31-5p inhibitor group and miR-31-5p inhibitor+NUMB siRNA group.The cell apoptosis and cell cycle of each group was evaluated with flow cytometry.Cell migration and invasion capacity of each group were examined by Transwell assay.and cell proliferation was analyzed by CCK-8 assay.Part three MiR-31-5p regulated the Notch signaling pathway through targeting NUMB1.To explore the mechanism by which the miR-31-5p-NUMB axis regulated the proliferation,migration,and invasion of colorectal cancer,we predicted NUMB-interacting protein using database of protein and genetic interaction（BioGRID,http://thebiogrid.org/）,and found Notch1 protein could interact with NUMB.2.HT29 and HCT116 cells were transfected with miR-31-5p inhibitor,NUMB and Notch1 protein expression in colorectal cancer cells were detected.3.NUMB gene expression was upregulated in colorectal cancer cells transfected with pcDNA3.0-UNMB plasmid,NUMB and Notch1 protein expression was detected by Western blotting.4.To confirm that miR-31-5p regulated Notch1 expression through targeting NUMB,HT29 and HCT116 cells were transfected with miR-31-5p inhibitor and miR-31-5p inhibitor+NUMB siRNA,NUMB and Notch1 protein expression was detected by Western blotting.5.HT29 and HCT116 cells were divided into six groups: Blank group,miR-31-5p inhibitor NC group,NUMB siRNA NC group,miR-31-5p inhibitor group,miR-31-5p inhibitor+NUMB siRNA group,miR-31-5p inhibitor+NUMB siRNA+Notch1 siRNA group,NUMB and Notch1 protein expression was detected by Western blotting.Statistical analysisGraphPad Prism 5.0（Graphpad Software Inc.）was used for the statistical analyses.To determine significance between two groups,unpaired t-test was used.Kruskal–Wallis test was used to evaluate the differencesbetween more than two groups.P values < 0.05 were considered statistically significant.ResultsPart one1.We examined the expression of miR-31-5p in colorectal cancer tissues by qRT-PCR and confirmed that miR-31-5p is higher expressed in colorectal cancer tissues than paired normal tissues.The expression of miR-31-5p in colorectal cancer cells was assessed,and the HT29 cell line showed the highest level of miR-31-5p expression.Tumor size（P=0.046）,tumor distance metastasis（P=0.0084）,and tumor node metastasis（TNM）stage（P=0.0387）exhibited positive correlation to miR-31-5p expression,but miR-31-5p expression was not significant correlation with age（P=0.7746）and sex（P=0.3121）.2.The knockdown of miR-31-5p by antisense miR-31-5p inhibitor significantly decreased expression levels of miR-31-5p in HT29 and HCT116 cell lines and suppressed the proliferation of HT29 and HCT116 cells compared with Nnegative control and Blank control group.The knockdown of miR-31-5p induced apoptosis and G1 phase arrest in HT29 and HCT116 cells,with an increase in the percentage of cells in G1 phase and a decrease in the percentage of cells in S phase,and the enhanced cell apoptosis by miR-31-5p knockdown was further confirmed by Hoechst33258 staining assay.Moreover,the migration and invasion of HT29 and HCT116 cells were inhibited by knockdown of miR-31-5p.3.To confirm the role of miR-31-5p in vivo,we used nude mice tumorigenesis assays to confirm the effect of miR-31-5p on tumorigenicity in vivo.Compared with the control group,the miR-31-5p inhibitor groupshowed a significant reduction in tumor quality and volume,and a slower tumor growth rate.Part two1.We predicted the target of miR-31-5p using miRBase and TargetScanHuman and found a potential binding site for miR-31-5p in the3′-UTR region of NUMB.To confirm our prediction,we constructed luciferase reporter plasmids containing either wild-type or mutant NUMB and performed the luciferase activity assay,the results showed miR-31-5p inhibited the activity of luciferase reporter harboring wild-type NUMB but not the mutant NUMB,these data suggested NUMB was a direct target of miR-31-5p.2.Compared to adjacent normal tissues,the mRNA and protein levels of NUMB were significantly lower in colorectal cancer tissues,Further correlation analysis indicated that miR-31-5p and NUMB expression level were negatively correlated.Also the mRNA and protein levels of NUMB was decreased by miR-31-5p overexpression（Mimic）.3.To investigate the role of NUMB in colorectal cancer,we overexpressed NUMB in HT29 and HCT116 cells by transfecting the pcDNA3.0-NUMB plasmid,NUMB expression was upregulated in colorectal cancer cells transfected with pcDNA3.0-UNMB plasmid.The overexpression of NUMB significantly inhibited the proliferation of HT29 and HCT116 cells.G1 phase arrest in HT29 and HCT116 cells was induced by NUMB overexpression,resulting in an increased percentage of cells in G1 phase and a decreased percentage of cells in S phase.Also we found that both the migration and invasion potency of HT29 and HCT116 cells was markedly suppressed by NUMB overexpression.Furthermore,induction of colorectal cancer cells apoptosis was increased by NUMB overexpression.4.To further investigate the role of miR-31-5p and NUMB in colorectal cancer,the expression of miR-31-5p and NUMB in HT29 and HCT116 cells was knocked down alone or together by miR-31-5p inhibitor and NUMB specific siRNA.NUMB was elevated in HT29 and HCT116 cells transfected with miR-31-5p inhibitor,and its expression was then reversed by siRNA-NUMB.The cell viability of HT29 and HCT116 cells was decreased by miR-31-5p inhibitor compared with Negative control group and Blank group,however,the knockdown of NUMB significantly enhanced the cell viability of the HT29 and HCT116 cells transfected with miR-31-5p inhibitor.Cell cycle analysis showed the transfection of miR-31-5p inhibitor induced G1-phase arrest of HT29 and HCT116 cells,and the effect of miR-31-5p inhibitor transfection was reversed by NUMB knockdown.In addition,miR-31-5p inhibitor transfection in HT29 and HCT116 cells significantly inhibited cell migration and invasion,and the inhibition was reversed by NUMB knockdown.Moreover,NUMB knockdown reversed the induction of cell apoptosis by miR-31-5p inhibitor transfection in HT29 and HCT116 cells.Our results supported the findings that NUMB was a functional mediator of miR-31-5p and played a key role in colorectal cancer proliferation,migration,and invasion.Part three1.We found Notch1 protein could interact with NUMB using database of protein and genetic interaction（BioGRID）.Notch1 protein is the critical protein in Notch signaling pathways.Therefore,we speculated that the miR-31-5p-NUMB induced promotion of colorectal cancer cellproliferation,migration,and invasion was associated with the Notch pathway.2.Western blotting showed that the knockdown of miR-31-5p in HT29 and HCT116 cells decreased expression levels of Notch1,Conversely,downregulation of miR-31-5p resulted in increased levels of NUMB.3.The expression of Notch1 protein was inhibited by NUMB overexpression in HT29 and HCT116 cells.4.NUMB protein expression was elevated in HT29 and HCT116 cells transfected with miR-31-5p inhibitor,and its expression was then reversed by siRNA-NUMB.Notch1 protein expression of HT29 and HCT116 cells was inhibited by miR-31-5p inhibitor compared with Negative control group and Blank group,and the inhibition was reversed by NUMB knockdown.5.There were no significant difference in NUMB and Notch1 protein expression between Blank group,miR-31-5p inhibitor NC group and NUMB siRNA NC group.Compared with the three groups,miR-31-5p silencing increased expression of NUMB and decreased expression of Notch1,but NUMB siRNA reversed upregulation of NUMB and downreglation of Notch1.Furthermore,Notch1 siRNA reversed upreglation of Notch1 again caused by NUMB silencing at protein levels.These results suggested that miR-31-5p regulated the NOTCH signaling pathway through targeting NUMB.Conclusion1.miR-31-5p is higher expressed in colorectal cancer tissues than paired normal tissues,and tumor size,tumor distance metastasis and tumor node metastasis（TNM）stage exhibited positive correlation to miR-31-5p expression.2.The proliferation,migration and invasion of colorectal cancer cells were inhibited by knockdown of miR-31-5p.Also knockdown of miR-31-5p could induce cells apoptosis and G1 phase arrest.3.Compared with the control group,the miR-31-5p inhibitor group showed a significant reduction in tumor transplanted in nude mice quality and volume,and a slower tumor growth rate.4.NUMB was a direct target of miR-31-5p.miR-31-5p suppresses NUMB expression to promote colorectal cancer cell proliferation,migration and invasion.5.miR-31-5p promotes colorectal cancer cell proliferation,migration and invasion of colorectal cells by targeting NUMB through the Notch signaling pathway. |
