| Background:As one of the malignant tumors with the highest incidence and fatality rate,liver cancer seriously endangers human life and health.There are 900,000 new cases of liver cancer every year around the world,and about 830,000 people die from liver cancer each year,which makes liver cancer an increasingly serious global health care problem.The lack of suitable early detection biomarkers and effective treatments is one of the important reasons for the high mortality of liver cancer.Therefore,there is an urgent need to find effective diagnostic markers and therapeutic targets for liver cancer.p27 protein is an atypical tumor suppressor,which regulates the transition from G0 to S phase of cells by binding and regulating the activity of cyclin-dependent kinase(CDK).In many cancers,the decrease in p27 protein expression and the increase in degradation rate will cause cell cycle disturbance and abnormal proliferation,and are closely related to the poor prognosis of patients.Therefore,studying the degradation pathway of p27 protein and finding out how to regulate the molecular mechanism of p27 protein will provide a new theoretical basis and research strategy for the treatment of liver cancer.The DNAJC5 protein studied in this project is widely found in all tissues,but it is rich in nerve tissue and is specifically located on synaptic and grid protein-coated vesicles.It is of great significance for maintaining the normal functioning of nerve tissues,but whether DNAJC5 is meaningful in the process of tumorigenesis and development has not been discussed so far.In the early stage,we found that DNAJC5 is highly expressed in liver cancer tissues through bioinformatics and literature research,and there is a possible interaction with the SKP2 protein,a member of the E3 ligase complex,and confirmed that DNAJC5 can promote the degradation of SKP2’s target protein-p27.However,the effect of DNAJC5 on the occurrence and development of liver cancer,and the specific mechanism of how DNAJC5 regulates the degradation of p27 protein need to be further studied.Therefore,studying the role of DNAJC5 in liver cancer and its intrinsic molecular mechanism provide new targets and new ideas for the diagnosis and treatment of liver cancer.Purpose:1.Detect the expression level of DNAJC5 in liver cancer tissues.2.To study the effect of DNAJC5 on the proliferation of hepatocellular carcinoma cells.3.Verify whether there is an interaction between DNAJC5 and SKP2.4.To study the specific molecular mechanism of DNAJC5 regulating p27 through SKP2.5.To study whether DNAJC5 regulation of p27 to promote the proliferation of hepatocellular carcinoma cells depends on SKP2.Methods:1.Analyze the expression of DNAJC5 in liver cancer tissues and the correlation between the expression level of DNAJC5 and the prognosis of liver cancer patients through public databases.Collect liver cancer clinical tissue samples,and use western blot and immunohistochemistry to detect the protein expression level of DNAJC5 in liver cancer tissues and corresponding adjacent tissues.2.Establish liver cancer cell lines that stably overexpress or interfere with DNAJC5 through the lentiviral infection system.Use Ed U,growth curve experiment,cell plate clone formation experiment and cell cycle experiment to study the effect of DNAJC5 on the proliferation of hepatocellular carcinoma cells.3.The protein immunoprecipitation method was used to verify the interaction between DNAJC5 and SKP2,and the DNAJC5 truncation mutant was constructed to detect the key structural domains that interact with SKP2.4.DNAJC5 was overexpressed in hepatocellular carcinoma cells to detect changes in the expression levels of p27 and SKP2,and DNAJC5 and p27 were simultaneously overexpressed in HEK293 T cells,and changes in p27 expression levels were detected to determine that DNAJC5 could regulate the protein expression levels of p27.Then the cells were treated with protein translation inhibitor(CHX)and proteasome inhibitor(MG132)to test the effect of DNAJC5 on the stability of p27.5.Overexpression or interference with DNAJC5 in HEK293 T cells,and overexpression of SKP2 and p27 at the same time,using protein immunoprecipitation method to verify the interaction of SKP2 and p27.Overexpress or interfere with DNAJC5 in hepatocellular carcinoma cells,and use protein immunoprecipitation to verify the interaction between endogenous SKP2 and p27.6.In the stable cell line overexpressing DNAJC5,the expression of SKP2 was disturbed at the same time.Ed U,growth curve experiment,cell plate clone formation experiment and cell cycle experiment were used to study whether the effect of DNAJC5 on the proliferation of hepatocellular carcinoma cells depends on the expression of SKP2.Results:1.Using public database analysis results showed that in liver cancer tissues,the expression level of DNAJC5 is higher than that of normal tissues,and the high expression of DNAJC5 is related to the poor prognosis of patients.The test results of clinical tissue specimens also proved that in liver cancer tissues,the expression level of DNAJC5 was higher than the corresponding adjacent tissues.2.Overexpression of DNAJC5 can promote the malignant proliferation of hepatocellular carcinoma cells.Conversely,interference with DNAJC5 will inhibit the malignant proliferation of hepatocellular carcinoma cells.3.There is a real interaction between DNAJC5 and SKP2.4.DNAJC5 promotes the degradation of p27,the target protein of SKP2.5.DNAJC5 accelerates the degradation of p27 protein by enhancing the interaction between SKP2 and p27.6.Interfering with SKP2 can inhibit the ability of DNAJC5 to promote the proliferation of hepatocellular carcinoma cells.Conclusion:1.In liver cancer tissues,the expression level of DNAJC5 is higher,and it is related to the prognosis of patients.2.DNAJC5 can promote the proliferation of hepatocellular carcinoma cells and is dependent on SKP2.3.DNAJC5 interacts with SKP2 and can enhance the binding ability of SKP2 and p27.4.DNAJC5 promotes the degradation of p27 protein. |
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