Expression Of YAP1 And β-catenin In Human Colorectal Cancer And Influence On Biological Characteristics Of RKO Cells By SiRNA-mediated Silencing YAP1 Gene

Object:1) To investigate the expression and relationship of YES associated protein 1(YAP1) and β-catenin in colorectal cancer (CRC) and their association with the clinicopathological features.2) To investigate the influence on biological characteristics of RKO cells by siRNA-mediated silencing YAP1 gene.Methods:1) The expression of YAP 1 and P-catenin in human colorectal cancer and normal colorectal mucosa (99 cases of human colorectal cancer,30 cases of normal colorectal mucosa)were detected by immunohistochemistry Envision two step method.2) To knockdown YAP1 expression,a small interfering RNA targeting against YAP1 was synthesized and transfected into RKO cells with lipofectamine 2000. The expression of YAP1 mRNA was detected by RT-PCR. The expression of YAP1 protein was detected by Western Blot analysis.The effect on proliferation of cells was detected by CCK8 assay. The effect on apoptosis of cells was detected by flow cytometry(FCM).Results:1) The positive rate of YAP1 in CRC (56.57%) were higher than that in normal mucosa (16.67%) (p<0.01).The abnormal positive rate of β-catenin in CRC (51.52%) were higher than that in normal mucosa (0%) (p<0.01).2) The expression of YAP1 showed no significant correlation with sex, age,lymph node metastasis,invasion depth, differentiation and Duke’s stage (p>0.05). The nuclear P-catenin expression was correlated with the invasion depth and differentiation degree (p<0.05). The cytoplasmic β-catenin expression was correlated with the differentiation degree (p<0.05).The membranal β-catenin expression showed no significant correlation with clinicopathological features (p>0.05),3) The cytoplasmic β-catenin expression and nuclear β-catenin expression was postively correlated with the nuclear YAP1 expression and cytoplasmic/nuclear YAP1 expression(p<0.05). The nuclear β-catenin expression was postively correlated with the cytoplasmic YAP1 expression(p<0.05).4) RT-PCR shows that the brightness of DNA bands in YAP1-siRNA group were significantly lower than negative control group. Western Blot shows that the brightness of protein bands in YAP1-siRNA group were significantly lower than negative control group.5) CCK8 test results show that the YAP1-siRNA cell growth inhibition was significantly higher, compared with the negative control group (p<0.05).6)Flow cytomrtry results show that the apotosis rate of YAP1-siRNA group was significantly higher, compared with the negative control group (p<0.05).Conclusion:YAP1 and β-catenin proteins play an important role in the carcinogenesis of CRC. siRNA silencing YAP1 gene can effectively inhibit the expression of YAP1 mRNA and protein of colorectal cancer cells,and suppress the proliferation of colorectal cancer cells and induce apoptosis to a certain degree. YAP1 may be a effective gene therapy target of colorectal cancer by the application of RNA interference technology.