| Hepatocellular carcinoma(HCC)is one of the main causes of malignant tumor-related death in China.Although there are surgery,vascular thrombosis,radiotherapy and other adjuvant therapis,its long-term efficacy is still not optimistic.Due to concealed onset,no obvious symptoms in early stage,most of the initial diagnosed patients lost the opportunity for surgical treatment because of local advances or metastasis.Related research of HCC targeted therapy is still in the initial stage,there is no specific targeted drug for HCC.As a key between DNA and protein,the biological role of RNA attracted more and more researchers' attention,and DDX protein family of RNA helicase involved in the whole process of RNA metabolism.The expression and biological function of DDX 10 in solid tumors has been limited reported,and DDX 10 is only reported to be low in ovarian cancer and to inhibit cell proliferation.In view of the important position of DDX helicase in cell life activities,the function of the family members and its further research on the relationship with tumor,to explore its upstream and downstream effector molecules,to clarify its mechanism of action and regulation mechanism,will provide new avenues to the diagnosis and bio-targeted therapy.This study will explore the expression of DDX 10 in hepatocellular carcinoma,its biological function and the possible mechanism.Part 1 The expression of DDX10 in HCC and its clinical significanceObjective:To study the expression of DDX10,a member of DDX protein family,in hepatocellular carcinoma and cell lines.And the relationship between DDX10 expression and clinical prognosis..Methods:The data of HCC gene expression profiles(GSE36376 and GSE14520)were downloaded from GEO.After the data is downloaded,the RMA algorithm is used to preprocess the chip data.Screening of differentially expressed genes using the gene chip significance analysis(SAM)algorithm of R speech platform.Then DDX10 was analyzed.Immunohistochemical staining was performed on 53 specimens of HCC and adjacent normal tissues.The expression of DDX10 protein in hepatocellular carcinoma tissues and adjacent tissues was analyzed statistically.The expression of DDX10 mRNA in normal liver cell line(L02)and hepatocellular carcinoma cell line(HepG2,Hep3B,Huh7)was analyzed by qPCR.The expression of DDX10 protein in cell line was verified by western blot.The relationship between the expression of DDX10 and clinical prognosis was analyzed by online TCGA database analysis tool GEPIA.Results:1.GEO data showed that DDX10 was highly expressed in HCC.2.Immunohistochemistry of HCC tissues showed that the expression of DDX10 was significantly higher in most hepatocellular carcinoma(79.25%)than in adjacent tissues.3.DDX10 was downregulated in L02 and HepG2 cell line,while was highly expressed in Hep3B and Huh7 cell lines.4.Clinical prognostic analysis showed that DDX10 overexpression group achieved shorter disease-free survival and overall survival.Conclusion:DDX10 gene is highly expressed in HCC tissues and cell lines and predicts poor prognosis.Part 2 The Biological Function of DDX10 in HepatocellularCarcinomaObjective:To explore the biological function of DDX10 in hepatocellular carcinoma(HCC).Methods:The silence of DDX10 gene was performed by shRNA technique in Hep3B and Huh7 cell lines.CCK-8 and cloning assays were performed to detect the effect of DDX10 on proliferation ration of sh-DDX10-lv Hep3B and Huh7.Results:1.The DDX10 gene was silenced by DDX10-shRNA-LV in Hep3B and Huh7.2.The results of CCK-8 showed that the proliferation ability of downregulated DDX10 group was significantly lower than that of control group.3.The clone formation ability of DDX10 gene knockout group was significantly lower than that of control group.Conclusion:shRNA lentivirus-mediated RNAi can effectively inhibit the expression of DDX10 gene.Silencing DDX10 gene can inhibit the growth and proliferation of HCC and inhibit the cloning of HCC.Part 3 Study on the mechanism of silencing DDX10 gene inhibits the proliferation of HCCObjective:To investigate the mechanism of silencing DDX10 gene in inhibiting the proliferation of HCC.Methods:The expression and distribution of ?-catenin before and after DDX10 silencing was verified by western blot.Construction of FOP,TOP plasmids,dual luciferase reporter gene detection was taken.The effect of DDX10 on cell proliferation was examined by wnt/?-catenin signaling pathway inhibitor IWR-endo.Results:1.In Hep3B and Huh7 liver cancer cell lines,the expression of ?-catenin in silencing DDX10 group was significantly lower than that in control group.2.Dual luciferase reporter assay showed a decrease in ?-catenin activity after silencing DDX10.3.The cell proliferation ration of wnt/?-catenin signaling pathway IWR-endo group was significantly lower than that of the control group(P<0.05).All theses results indictes that wnt/?-catenin signaling pathway is the downstream target of DXX10 in HCC.Conclusion:DDX10 regulates cell proliferation by modulating the wnt/p-catenin signaling pathway.. |
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