The Function And Mechanism Study Of Lgr5 In Hepatocellular Carcinoma

PartⅠThe expression of Lgr5 in hepatocellular carcinoma and its effect on cellular biological behavior of tumor cell Object In order to investigate the expression of leucine-rich repeat-containing Gprotein-coupled receptor 5 in hepatocellular carcinoma.To study the relationship between Lgr5 and clinical pathological data and prognosis of patients.To observe the effect of Lgr5 on tumor biological behavior of HCC cell lines.Method The expression of Lgr5 in HCC tissue and cell lines was detected by Real time Quantitative PCR and Western blot.To determine the relationship between the expression of Lgr5 in HCC tissue detected by Immunohistochemistry(ICH)and the clinical pathological data.Kaplan–Meier test analysis the relationship between expression of Lgr5 and prognosis of HCC patient.The Cox regression model is used to determine if Lgr5 is an independent risk factor for HCC prognosis.We use tanswell assay and wound-healing migration assay to analyse the migratory and invasive ability of hepatocellular carcinoma cell lines(SK-Hep1 and HepG2)in which Lgr5 was overexpressed or koncked-down.We also use CCK8 assay to identify the amplification capacity of those cells.The EMT associated protein were detected by western blot.We establish a cancer model using a nude mice to detected the ability of HCC cells transfected with Lgr5 in lung metastasis.The resistance to Doxorubicin(Dox)in HCC cells were analyzed by CCK8 assay.Result TheLgr5 expression was significantly upregulated in tumor tissue compared with adjacent nontumor tissue.An analysis of the clinical characteristics of HCC revealed that Lgr5 expression was significantly correlated with tumor size(P <0.01),tumor number(P <0.01),BCLC stage(P=0.0005)and PVTT(P <0.01).Kaplan-Meier survival curves revealed an obvious correlation between high Lgr5 expression and poor prognosis in patients with HCC.The Cox regression model and multivariate analysis showed that high Lgr5 expression was an independent predictor of poor prognosis in patients with HCC.Lgr5 could enhance the ability of migratory and invasive and amplification in HCC cells and induce the resistance to Dox.Conclusion Lgr5 is an oncoprotein associated with tumorigenesis and cancer progression and it is an important factor in HCC metastasis.Lgr5 is expected to be used to predict the prognosis of patients with HCC and is meaningful for the potential tumor therapy.PartⅡIdentification of the Lgr5 interacting with the programmed cell death protein 5(PDCD5)Object To screen and identify the interactional protein for Lgr5.To study the molecular mechanism of resistance to chemotherapy mediated by Lgr5.Method We constructed the c DNA library from the HCC tissue collected clinically.Lgr5 c DNA was cloned into p GBKT7 as bait and used to screen a human HCC c DNA library constructed in the prey plasmid by yeast two hybrid system.The Flag-Lgr5 and MYC-PDCD5 fusion protein expression vector were co-transfected into HEK 293 T cell and the interaction between the those protein were analyzed by CoImmunocoprecipitation(Co-IP)and immunofluorescence staining assay(IF).To confirm the interaction between endogenous Lgr5 and PDCD5 in the Hep G2 cells that has a high expression of Lgr5 by Co-IP.Finally,to identify the interaction between Lgr5 and PDCD5 by GST-pull down.Additionally,to detect the m RNA level of PDCD5 of 100 pairs of HCC specimens.To analyze the expression of PDCD5 in HCC specimens with western blot and immunohistochemistry(ICH).To analyze the relationship between the expression of PDCD5 and the corresponding clinical pathological characteristics of HCC patients.Kaplan–Meier curve was used to analysis the relationship between expression of PDCD5 and overall survival rates of HCC patient.The Cox regression model is used to evaluate the value of PDCD5 in prognosis of HCC patient.To overexpress and knock down the PDCD5 in HCC cell lines(SK-Hep1 and Hep G2)and to detect the resistance to Dox with CCK8 assay and to analyze the proliferation ability with cell colony formation assay in those cell.Result We screened 30 different proteins from HCC c DNA library with Lgr5 as a bait by yeast two hybrid.We tested these proteins and focused our attention on the programmed cell death protein 5(PDCD5).The Co-IP assay in HEK 293 T cell cotransfected with Flag-Lgr5 and MYC-PDCD5 overexpression vector showed that the anti-MYC antibody could detect MYC-PDCD5 in the immune complex precipitated by anti-Flag and the anti-Flag antibody could detect Flag-Lgr5 in the immune complex precipitated by anti-MYC.Moreover,immunofluorescence analysis showed that Lgr5 and PDCD5 have the same location in HEK 293 T cells transfected with both Flag-Lgr5 and Myc-PDCD5.The anti-Lgr5 could identify the Lgr5 in the immune complex precipitated by anti-PDCD5 in the nondenaturing lysis of Hep G2 and vice versa.The RT-q PCR and western blot assay showed that the expression of PDCD5 was significantly downregulated in HCC tissue compared with the adjacent nontumor tissue.The expression of PDCD5 has a remarkable negative correlation with tumor size(P =0.03),BCLC stage(P =0.01)and PVTT(P =0.01).Kaplan-Meier curve showed that the high expression of PDCD5 predict a favorable prognosis in HCC patients.The Cox regression and multivariate analysis suggested that PDCD5 is an independent risk factor of prognosis in HCC.PDCD5 could enhance the chemosensitivity of HCC cells to Dox.PDCD5 could inhibit the resistance to Dox induced by Lgr5 in HCC cells overexpressed with Lgr5.Conclusion There is an interaction between Lgr5 and PDCD5.PDCD5 take participated in the progress that Lgr5 induced the resistance to chemotherapy.Part Ⅲ Lgr5 promotes chemotherapeutic resistance by competitively binding with PDCD5Object To clarify how Lgr5 interacted with PDCD5 and regulated the chemotherapeutic resistance of HCC.Method RT-q PCR and western blot assay were used to find out if the expression of Lgr5 and PDCD5 influence each other.The location of PDCD5 in those cells overexpressed or knocked down with Lgr5 were observe under the microscope by immunofluorescence.The cytoplasmic and nuclear extracts of protein from the cells were analyzed by western blot to clarify the distribution of PDCD5 in cells and the expression of Lgr5 and p53.To analyze the level of p53 that interact with PDCD5 in the HCC cell overexpressing Lgr5.To determinate the relationship between the expression of Lgr5 and p53 in HCC tissue by western blot and ICH.Flow cytometric analysis was used to detect apoptosis in cells stably transfected with Lgr5 and sh RNA-Lgr5 and treated with Dox(0 μg/ml or 1 μg/ml).Western blot was used to analyze the expression of apoptosis-related proteins in cells stably transfected with Lgr5 and sh RNA-Lgr5 and treated with Dox(0 μg/ml or 1 μg/ml).Result Lgr5 and PDCD5 could not influence the expression each other.The knock down of Lgr5 could not influence the expression of PDCD5.Lgr5 could competitively binding with PDCD5 and block its nuclear translocation.The protein level of p53 that interacted with PDCD5 was significantly decreased when overexpression of Lgr5.There was a conspicuous negative correlation between the level of Lgr5 and p53 in HCC tissue(P <0.01).Doxorubicin could induce the cell apotosis while Lgr5 could inhibit the apotosis induced by Dox.Dox could enhance the level of cleaved PARP1 and p53 and change the expression of several apoptosis-related targets of p53,for example,Bax protein expression was increased and Bcl2 protein expression was decreased.Lgr5 could decrease the level of p53 and Bax and increase the level of Bcl2.