Effects Of Prdx1 On Proliferation,Migration And Apoptosis Of Hepatocellular Carcinoma

Background:Primary Liver cancer(PLC)is one of the most common cancers in China,with the second highest mortality rate of newly diagnosed malignant tumors in China,accounting for about 400,000 deaths per year.HCC accounts for 80%-90%of PLC,while bile duct carcinoma accounts for 10%-15%.Although the level of diagnosis and treatment of cancer has been improved,the conventional treatment has not achieved good curative effect,and its morbidity and mortality are still very high.At present,surgical excision,interventional therapy,chemotherapy and targeted drug therapy are the main therapeutic methods for PLC,but the therapeutic effect is not ideal due to its high metastasis and recurrence rate.Therefore,it is an urgent task to further study the molecular mechanism of PLC occurrence and development and find more possible therapeutic targets.Peroxiredoxins(Prdxs)are a family of antioxidant proteins that have received much attention recently and play an important role in antioxidant defense and peroxide detoxification.Mammalian PRDXs family contains 6 members and Prdx1-6,which is a multifunctional protein involved in cell growth,differentiation and apoptosis.Prdx1 has been reported to be expressed to a certain extent in different types of cancer,including lung adenocarcinoma,breast cancer,soft tissue sarcoma,colorectal cancer and prostate cancer.In recent years,it has been found that by affecting the cell cycle,it can increase the proportion of G1/S phase and increase the gene mismatch of cells,thus promoting the occurrence of tumors.However,Fang et al.found that Prdx1 expression was low in HCC cells.Prdx1 is up-regulated in cervical cancer and enhances proliferation,migration and invasion by inhibiting cell apoptosis.Analysis of a disease model showed that Prdx1 is expressed at higher levels in the brain,and it is associated with toll-like receptor-4(TLR-4)inflammation and apoptosis.Some studies have reported its expression in liver tumor tissues.However,the cellular role of Prdx1 in hepatocellular carcinoma and its mechanism of action in relation to related proteins remain unclear。Objective:In this study,PRDX1 was detected in different liver cancer cell lines and tissues to explore the correlation between the concentration of PRDX1 and the survival time of HCC patients.Next,in vitro,The effect of PRDX1 on the proliferation and migration of hepatocellular carcinoma cell lines was studied.To explore the mechanism of PRDX1 in the occurrence and development of HCC,and to provide a new therapeutic target and experimental basis for the prevention and treatment of liver cancer patients in the futureMethod:1.Examination of the significance of PRDX1 in different liver cancer cell lines and tissues1)Download HCC data from TCGA(Cancer Genome Atlas)database for bioinformatics analysis to explore the significance of PRDX1 in the occurrence,development and prognosis of HCC.2)Real-time PCR and western blot were used to determine the differences of PRDX1 gene expressions between normal live cell lines(LO2)and HCC cell liens(Hep-3B、Huh-7、HepG2、MHCC97-1).2.Investigation of the significance of PRDX1 expression in HCC cells1)The coding sequence of PRDX1 was cloned into the lentiviral vector p CDH-EGFP in vitro,and p CDH-EGFP-PRDX1 lentiviral vector was constructed,which was transfected into HepG2 cells to construct lentivirus-mediated,stably transduced overexpressing PRDX1 Cell Line.2)HCC cell lines were obtained,with silent expression of PRDX1.3)The impact of PRDX1 on HCC cell line proliferation was evaluated by CCK8,Plate clone formation assays.4)Immunofluorescence assay was conducted to evaluate the effect of PRDX1 on HCC cell line mitochondrial fragmentation.5)Scratch-wound assay was conducted to evaluate the effect of PRDX1 on HCC cell line migration.6)Flow cytometry was subjected to evaluate the effects of PRDX1 on the apoptosis phase distribution of HCC cell line.7)Western blot assay was performed to detect apoptosis-inducing-related proteins and apoptosis-related proteins in HCC cell line.3.Exploring molecular mechanism of PRDX1-mediated HCC cell proliferation and migration1)Following the overexpression of PRDX1 in HCC cell line,the cells were subjected to evaluate the expression of EMT、MAPK-PI3K-AKT-related proteins and p-HSP27、p-P38.2)Following the silencing of PRDX1 in HCC cell line,the cells were subjected to evaluate the expression of EMT,p-HSP27,p-P38,MAPK-PI3K-AKT-related proteins.3)The mechanism of PRDX1 regulating cell fate was analyzed by analyzing the ratio of phosphorylated HSP27 to HSP27 and the ratio of phosphorylated P38 to P38.Result:Chapter 1:1)In the TCGA database,we found the expression level of PRDX1 in the cancer tissue was significantly lower than that of the paraneoplastic tissues[P<0.001].Kaplan-Meier survival analysis showed that the overall survival period of patients with high PRDX1 expression was significantly shorter than that of patients with low PRDX1 expression(P<0.05).2)Compared with normal hepatocyte LO2 cell line,PRDX1 m RNA expression was up-regulated in the Hep-3B、Huh-7、HepG2、MHCC97-1 cells,and HepG2 cell had the highest expression than that of others(P<0.01).Chapter 2:1)The data of the CCK8 Plate clone formation assays demonstrated that downregulation of PRDX1 expression impaired cell proliferation ability in HCC cell line.Otherwise,overexpression of PRDX1 encouraged cell proliferation ability in HCC cell line.2)Scratch-wound assay revealed that downregulation of PRDX1 expression inhibited HCC cell line migration.Otherwise,overexpression of PRDX1 encouraged cell migration in HCC cell line.3)Immunofluorescence assay revealed that downregulation of PRDX1expression encouraged HCC cell line mitochondrial fragmentation.Otherwise,overexpression of PRDX1 inhibited mitochondrial fragmentation in HCC cell line.4)The results of Western Blot showed that downregulation of PRDX1expression,the expressions of apoptosis induction related proteins,apoptosis related proteins and mitochondrial fragmentation related proteins were significantly increased.Otherwise,overexpression of PRDX1 inhibited the expressions of apoptosis induction related proteins,apoptosis related proteins and mitochondrial fragmentation related proteins.5)Flow cytometry revealed that downregulation of PRDX1 expression,encouraged the apoptosis in HCC cell line.Otherwise,overexpression of PRDX1inhibited the apoptosis in HCC cell line.Chapter 3:1)The results of Western Blot showed that downregulation of PRDX1expression,the expressions of E-cardherin,p-HSP27 and p-P38 were significantly increased,when p-ERK,p-AKT,were significantly decreased.2)The results of Western Blot showed that upregulation of PRDX1 expression,the expressions of vimentin,p-ERK and p-AKT were significantly increased,when,p-HSP27 and p-P38 were significantly decreased.3)The results of Western Blot showed that downregulation of PRDX1expression,increased the ratio of p-HSP27 to HSP27 and p-P38 to P38,finally induced the cell death in HCC cell line.Otherwise,overexpression of PRDX1decreased the ratio of p-HSP27 to HSP27 and p-P38 to P38,to inhibited the cell death in HCC cell line.Conclusion1.The expression level of PRDX1 protein in liver cancer tissues is correlated with patient survival period negatively.2.Downregulation of the expression of PRDX1 inhibits the proliferation and migration of liver cancer cell lines.3.PRDX1 can promote the phosphorylation of HSP27 and P38 via reduction of mitochondrial fragmentation,p38MAPK-and PI3K/AKT signaling activation,which leads to ultimately encourage the cell proliferation and tumor growth.