Research On The Regulation Mechanism Of Irs-1and Its Signal Pathway By MiR-126in Colorectal Cancer

Background:Colorectal cancer (CRC) is one of the common human malignant tumors and the fourth common cause of death from cancer in China. But its pathogenesis has not yet very clear. Numerous studies showed that kinds of miRNAs differential expression in the CRC tissues which may be involved in the pathogenesis of CRC. Few studies found that miR-126was down-expression in CRC. Insulin receptor substrate-1(IRS-1) took part in the development of many cancers including CRC. What is the relationship between miR-126and IRS-1? Could miR-126regulate the expression of IRS-1? And how does miR-126play roles in CRC by regulating IRS-1? However, is it relative with the signal pathway? It needs to discuss the problem in this study. We focus on the relationship between miR-126and IRS-1, discussing the mechanism in regulating IRS-1and its signaling pathway by miR-126in colorectal cancer cells. It is helpful for clarifying the pathogenesis of CRC in order to applying microRNAs for diagnosis and treatment in CRC or other diseases in the future.Part1Research on the relationship between expression of microRNA-126(miR-126) and IRS-1and their clinicopathological parameters in colorectal cancerObjective:Our study aim to detecting the expression of miR-126and IRS-1mRNA and protein in the CRC tissues, analyzing the relationship between the expression of miR-126and IRS-1and discussing their association with clinicopathological parameters in CRC. At last, we want to investigate the possible mechanism of miR-126and IRS-1involved in the occurrence and development process of CRC. Our study may provide a theoretical and technological basis for further exploration in CRC gene therapy.Methods:Twenty normal colorectal tissue samples,40CRC tissue samples,28adjacent CRC cancer tissue samples and26colorectal adenomas tissue samples were selected in our study. Expression of miR-126and IRS-1mRNA were analyzed by Real-time quantitative PCR (qRT-PCR), IRS-1protein expression and the location in CRC tissues were detected by western blotting and immunohistochemical. Moreover, we analyz the relationship between the expression of miR-126and IRS-1and discuss their association with clinicopathological parameters in CRC.Results:The real time qRT-PCR results indicated that the relative expression of miR-126in the CRC tissues were deceased significantly when compared with normal tissues, adjacent CRC tissues and colorectal adenomas, and was0.39times as more as the normal control group, and the difference was statistically significant(P<0.05). However, the miR-126expression of the other three groups had no difference from each other (P>0.05). There is no differential expression of IRS-1mRNA in the normal tissues, CRC tissues, adjacent CRC tissues and colorectal adenomas groups (P>0.05). The western blotting results showed that compared with normal tissues, adjacent CRC tissues and colorectal adenomas, the expression of IRS-1protein in the CRC tissues was decreased significantly (P<0.05). However, there is no difference among the other three groups (P>0.05). Moreover, the IRS-1protein was mainly located in CRC cells cytoplasm, the positive rate of IRS-1protein was87.5%(35/40) and was higher than the other three groups(/’<0.05).Correlation analysis displayed that miR-126and IRS-1protein were negative correlation in CRC tissues(r=-0.420, P<0.05), but there was not obvious correlation between miR-126and IRS-1mKNA(r=0.028, P>0.05). The expression of miR-126and IRS-1was related with TNM stage and Dukes’stage in the CRC, and the higher of TNM or Dukes’stage, the lower expression of miR-126and higher expression of IRS-1(P<0.05).Conclusion:The miR-126and IRS-1were differential expression in the CRC tissues, and they may have negative correlation with each other. MiR-126may be involved in the occurrence and development process of CRC through IRS-1. Part2The effect of the biological behavior and mechanism in regulating IRS-1and its signaling pathway by miR-126in colorectal cancer cellsObjective:Colorectal cancer is one of the leading causes of cancer related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogen esis. MiR-126has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate1(IRS-1) and its possible signaling pathway by miR-126.Methods:After transfected with miR-126mimic or inhibitor to increase or decrease miR-126expression in the CRC HT-29or HCT-116cell lines, the expression of IRS-1mRNA was detected by qRT-PCR.The expression of IRS-1, AKT and ERK1/2protein were analyzed by western blotting. The location of IRS-1in HCT-116cell line was detected by immunofluorescence assay. The cell cycle and cell apotosis were assayed with flow cytometry. CCK-8proliferation assay and transwell chamber migration, invasion assay were used to explore the effect of miR-126on the biological behavior of CRC cells. The3’-untranslated region (3r-UTR) of IRS-1regulated by miR-126was analyzed by using a dual-luciferase reporter assay.Results:(1) The expression of miR-126was different in HT-29, HCT-116, SW480, SW620CRC cell lines, and was higher in HCT-116cells than in the other three cell lines.(2) In the study of transfection with the plasmid vector containing the IRS-1mRNA3’-UTR, the relative luciferase activity of the PmiR-IRS-1-wt construct showed reduction when compared to the PmiR-IRS-1-mut construct or psicheck-2vector, separately, there was significant difference between the two groups (P<0.05). In the study of co-transfected with the above luciferase reporter constructs and miR-126mimic, the luciferase activity of the PmiR-IRS-1-wt construct was significantly inhibited in miR-126mimic group compared with the NC group (P<0.05). There was no difference between miR-126mimic and NC group when co-transfected with the PmiR-IRS-1-mut construct or psicheck-2vector (P>0.05).(3) IRS-1was expressed in the cytoplasm of HCT-116cells. Compared to NC group, the expression of miR-126was significantly increased and the IRS-1, p-AKT and p-ERK1/2protein expression were down-regulated in miR-126mimic group (P<0.05), but there were no differential expression in IRS-1mRNA, AKT and ERK1/2between the two groups (P>0.05). Compared to NC group, the expression of miR-126was dramatically decreased and the IRS-1, p-AKT and p-ERK1/2protein expression were up-regulated in miR-126inhibitor group (P<0.05), but there were no differential expression in IRS-1mRNA, AKT and ERK1/2between the two groups (P>0.05).(4) Compared to NC group, the number of CRC cells in the G0/G1phase was increased in miR-126mimic group (.P<0.05), but there was no difference about the percentage of apoptotic cells between the two groups (P>0.05).(5) Compared to NC group, transfection with miR-126mimic inhibited HT-29cells proliferation, migration and invasion, there was remarkable difference between the two groups (P<0.05).However, compared to NC group, transfection with miR-126inhibitor promoted HCT-116cells migration and invasion, there was remarkable difference between the two groups (P<0.05).Conclusions:miR-126may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1via AKT and ERK1/2signaling pathways.