The Study Of CDK8 As A Target For Inhibition Of Colorectal Cancer Liver Metastasis

Colorectal cancer is one of the common malignant tumor.Liver metastasis is the most common form of distant metastasis of colorectal cancer.Liver metastasis occures in approximately 25% of the patients when they are diagnosed as colorectal cancer.Prognosis of colorectal cancer closely relates to distant metastasis especially for liver metastasis.In recent years,integrated surgical treatment improves the curative effect of liver metastasis of colorectal cancer.But the prognosis is still not good.It is very important to explore a new therapy to inhibit the liver metastasis of colorectal cancer.Gene therapy is expected to become a new therapy for liver metastasis of colorectal cancer.Cyclin dependent kinase 8(CDK8)is a proto oncogene of colorectal cancer,which plays an important role in the regulation of Wnt/β-catenin pathway in the development of colorectal liver metastasis.Therefore,CDK8 is expected to be a therapeutic target for liver metastasis of colorectal cancer.Part one:The expression of CDK8 in colorectal cancer.Objective: To detect the expression of CDK8 in colorectal cancer tissues and to analyze the relationship between CDK8 expression and clinicopathological features of colorectal cancer.Method:Paraffin sections of 70 cases of colorectal cancer were selected,and 20 sections of normal colorectal tissues were selected as control group.The expression of CDK8 in colorectal cancer tissues and normal colorectal tissues was detected by immunohistochemistry,and the relationship between CDK8 expression and clinicopathological features was analyzed.Result:The positive rate of CDK8 expression in colorectal cancer group was(71.43%)higher than that in normal colorectal(30%)(P<0.05).The increasing expression of CDK8 related to clinical stage progressing,lymph node metastasis and distant metastasis of colorectal cancer(P<0.05);and did not relate to sex and age of patients,tumor differentiation,the serum level of CEA(P >0.05).Conclusion:1 The expression of CDK8 increased in colorectal cancer tissues.2 High expression of CDK8 related to clinical stage progressing and metastasis of colorectal cancer.Part two: CDK8 as a target for the inhibition of proliferation,migration and invasion of colorectal cancer cells.Objective:To observe the effect of silencing CDK8 gene on the proliferation,migration and invasion of colorectal cancer cells by cell experiments.Method:1 Built recombinant lentivirus of silent CDK8 gene(LV-CDK8-sh RNA)and control empty virus(LV-NC).Infected HCT116 cell with the lentiviruses.2 Detected CDK8 m RNA level by fluorescent quantitative PCR and detected CDK8 protein expression level by western blot.3 Assessed cell proliferation ability by MTT,assessed cell migration ability by wound healing assays,and assessed cell invasion ability by transwell invasion assays.Result:1 HCT116 cells with silenced CDK8 gene was built successfully.(The control group cell was called HCT116-luc-GFP-NC and the experience group cell was called HCT116-luc-GFP-sh CDK8).2 The level of CDK8 m RNA and protein expression in HCT116-luc-GFP-sh CDK8 cell was lower than that in HCT116-luc-GFP-NC cell(P<0.05).3 The proliferation ability,migration ability and invasion ability of HCT116-luc-GFP-sh CDK8 cell were lower than those of HCT116-luc-GFP-NC cell(P<0.05).Conclusion:1 LV-CDK8-sh RNA could effectively silence the expression of CDK8 gene in colon cancer cell line HCT116.2 The proliferation ability,migration ability and invasion ability of HCT116 cell were inhibited after the CDK8 gene being silenced.Part three: Mechanisms of the inhibition of colorectal cancer liver metastasis by downregulating CDK8.Objective: To observe the effect of silencing CDK8 gene on liver metastasis of colorectal cancer by animal experiments.Method:1 Built BLAB/c nu/nu nude mouse model of colorectal cancer liver metastasis.2 Observed the tumor growth and metastasis in nude mice by in vivo imaging.3 The expressions of CDK8,β-catenin,E2F1 and MMP-7 protein in the liver metastases of nude mice were detected by Western blot.Result:1 BLAB/c nu/nu nude mouse model of colorectal cancer liver metastasis was built successfully.2 Silencing CDK8 gene expression could effectively inhibit the development of colorectal cancer liver metastasis in nude mice.3 The expression of CDK8,β-catenin and MMP-7 in colorectal cancer liver metastases in nude mice decreased,while the expression of E2F1 increased after silencing the expression of CDK8 gene.Conclusion:1 Silencing CDK8 gene expression could inhibit the development of colorectal cancer liver metastasis in nude mice.2 Silencing the expression of CDK8 gene could inhibit the colorectal cancer liver metastasis in nude mice by inhibiting the Wnt/β-catenin signaling pathway.All Conclusions Silencing CDK8 gene expression could effectively inhibit colorectal cancer liver metastasis.CDK8 could be a target for inhibiting liver metastasis of colorectal cancer.