The Research Of Expression And Molecular Mechanism Of KCTD11 In Hepatocellular Carcinoma

Background:Globally,primary liver cancer(PLC)has the sixth highest incidence of all cancers in 2015 and the death rate is the fourth.After decades of effort,the mortality of primary liver cancer patients in China has decreased.However,the number of patients with liver cancer is still numerous,and it is necessary to continuously improve the diagnostic and therapeutic level of liver cancer in China.Hepatocellular carcinoma(HCC)accounted for the most of primary liver cancer.Currently,the main method on hepatocellular carcinoma therapy is comprehensive treatment based on surgery.But the current treatment is still difficult to achieve satisfactory results.In recent years,molecular targeted therapy promoted the treatment effect of some malignant tumors,including breast cancer and lung cancer.However,the molecular target therapy for HCC is still not much improved.Therefore,it is important to research the molecular pathological features and therapeutic targets of hepatocellular carcinoma.Objectives:Through comparing the different expression of KCTD11 in liver cancers and their adjacent tissue,analyzing correlation between KCTD11 expression and the clinical pathological characteristics of HCC patients,and researching the correlation between KCTD11 expression and long-term prognosis in HCC patients,we try to illuminate the potential value of KCTD11 as an HCC biomarker.We also performed a series of cellular function experiments and animal experiments to explore the function of KCTD11 in HCC.We use an mRNA expression chip to search the potential genes and signaling pathways that regulated by KCTD11.Thus,we could expound the function and corresponding mechanism of KCTD11 in HCC progression.Methods:1.Real-time fluorescent quantitative PCR was used to detect KCTD11 mRNA expression in HCC tissues and their adjacent tissues.Immunohistochemical was performed to detect the KCTD11 protein expression in these tissues.KCTD11 expression levels were compare between HCC tissues and their adjacent tissues.2.Twenty cases of HCC tissues and their corresponding adjacent tissues were used to detect the loss of heterozygosity(LOH)in the chromosome location of KCTD11.3.According to the immunohistochemical results of 51 HCC tissues,the correlation between the expression level of KCTD11 protein and the clinicopathological characteristics of patients was analyzed.4.The mRNA and protein expression levels of KCTD11 in liver cancer cell lines and normal liver cell line QSG-7701 were detected.5.Respectively construction KCTD11 stable transfection cell lines in HCCLM3(exogenous expression)and Huh7(interference).Cell Counting Kit-8(CCK 8)assay,clone formation assay,EDU assay,cell cycle assay were performed to study the function of KCTD11 in HCC cells proliferation in vitro.Subcutaneous tumor gowth experiment was performed to research the function of KCTD11 in vivo.Then,we explore the mechanism of KCTD11 in affecting HCC cell proliferation by western blot.6.The genes,signaling pathways and biological processes that regulated by KCTD11 were explored through the analysis of an mRNA expression chip and Gene Ontology(GO)analysis.The function and mechanism of KCTD11 in regulating HCC cells adhesion were studied by performing cell adhesion assay and co-immunoprecipitation(CoIP).7.Migration assay,invasion assay and orthotopic liver tumor and pulmonary metastasis model were performed to study the role of KCTD11 in HCC migration,invasion and distant metastasis.Western blot was performed to explore its corresponding mechanism.8.The regulation effect of KCTD11 on Hippo pathway and the the multiple mechanisms in affecting p21 expression was further research in HCC through western blot.Results:1.The mRNA expression and protein expression of KCTD11 in HCC tissues were both lower than their corresponding adjacent tissues.2.Loss of heterozygosity is one of the main causes of KCTD11 dysregulation in HCC.3.The different expression levels of KCTD11 in HCC tissues were not significantly correlated with the tumor size,distant metastasis,TNM stage and other clinical pathological indicators.However,the low expression of KCTD11 was significantly associated with adverse long-term prognosis for patients with HCC.4.The expression of KCTD11 protein in liver cancer cell lines was lower than that in normal liver cells.The expression of KCTD11 protein in high-invasive liver cancer cell lines was lower than that of low-invasive cell lines.5.KCTD11 inhibits HCC cell proliferation and tumor growth by promoting the expression of p21 protein.6.KCTD11 inhibits HCC cell adhesion by regulating the expression of CTGF and CLDN1.Protein-protein interaction is comfurmed between CTGF and COL3A1.Knock-down of CTGF results in promoting COL3A1 expression.7.KCTD11 inhibits HCC migration ability and distant metastasis by regulating MMP proteins and epithelial-mesenchymal transition(EMT)related proteins.8.KCTD11 activates the Hippo pathway in HCC.KCTD11 promotes p21 expression by stabilizing p53 protein expression or activating MST1/GSK3 beta/p21 signal.Conclusion:1.KCTD11 is low expressed in HCC tissues.Loss of heterozygosity is one of the causes of KCTD11 low expression.The low expression of KCTD11 indicates poor long-term prognosis in patients.2.KCTD11 activates the Hippo pathway,and promote p21 protein expression in HCC,thereby inhibits cell proliferation,migration,cell adhesion,tumor growth and distant metastases.