Preliminary Study On The Expression,Biological Function And Molecular Mechanism Of CPA4 In Hepatocellular Carcinoma

Hepatocellular carcinoma is one of the common malignant tumors in clinical practice.It has the characteristics of high morbidity,high mortality,and poor prognosis.It is the fourth most common cause of cancer death worldwide.Carboxypeptidase A4(Carboxypeptidase A4,CPA4)belongs to the metal carboxypeptidase family,which can cleave the C-terminal residues of peptides and proteins with the participation of zinc ions.CPA4 lacks a transmembrane domain and is a soluble extracellular protein that performs biological functions in the extracellular environment.Studies have found that CPA4 is highly expressed in a variety of tumor tissues and is associated with poor prognosis of patients,and has the potential to be used as a cancer diagnostic marker and therapeutic target.In the early stage of our laboratory,it was found through immunohistochemistry that the expression of CPA4 in liver cancer tissues was higher than that in normal tissues.Through analysis,it is found that the abnormally high expression of CPA4 is significantly related to the tumor grade,depth of invasion,and clinical stage of liver cancer patients;it is also an independent factor affecting the poor prognosis of patients.This study is based on the results of previous research,through cytological experiments,to explore the biological role of CPA4 in hepatoma cells and its molecular mechanism.It aims to provide a basis for the molecule to become a diagnostic marker and therapeutic target for liver cancer.First,we analyzed the expression of CPA4 mRNA in normal tissues and liver cancer tumor tissues in the TCGA database through the UALCAN website.The results showed that the expression level of CPA4 mRNA in LIHC tissues increased compared with normal tissues(P<0.05).It is consistent with the previous research results of our laboratory.Then,this study used real-time fluorescent quantitative PCR(qRT-PCR)and western blot to detect the expression of CPA4 in hepatoma cell lines,and found that the expression of CPA4 in MHCC97L cells was lower,while the expression of CPA4 in MHCC97H and Bel7402 cells was higher.high.Therefore,we chose hepatoma cells MHCC97L to overexpress CPA4,and hepatoma cells MHCC97H and Bel-7402 to knock down CPA4.Overexpression and knockdown of CPA4 are achieved by constructing lentiviral vectors.We tested the in vitro proliferation ability of each group of cells through the CCK-8 test and clone formation experiment.The results showed that overexpression of CPA4 can promote the in vitro proliferation ability of hepatoma cells MHCC97L(P<0.01);knocking down CPA4 can inhibit hepatoma cells MHCC97H and MHCC97H.The proliferation ability of Bel-7402 in vitro(P<0.01).Transwell experiment was used to detect the migration and invasion ability of each group of cells.The results showed that overexpression of CPA4 can promote the migration and invasion ability of hepatoma cells MHCC97L(P<0.01);knocking down CPA4 can inhibit the migration of hepatoma cells MHCC97H and Bel-7402,Invasion ability(P<0.01).The self-renewal ability of each group of cells was tested by serum-free spheroidization experiment.The results showed that overexpression of CPA4 can promote the self-renewal ability of hepatoma cells MHCC97L;knocking down CPA4 inhibits the self-renewal ability of MHCC97H and Bel7402 cells.According to the results of western blot experiments,we found that overexpression of CPA4 increased the expression of p-STAT3(Tyr705)and c-myc protein in hepatoma cells MHCC97L;while knocking down CPA4,p-STAT3(Tyr705)in hepatoma cells MHCC97H and Bel-7402,the expression of c-myc protein decreased.In summary,compared with normal tissues,CPA4 is abnormally highly expressed in hepatocellular carcinoma tissues,and is related to tumor grade,depth of invasion,clinical stage and poor prognosis.Research results suggest that CPA4 may increase the expression of c-myc protein by activating the STAT3 signaling pathway,thereby promoting the proliferation,migration,invasion and self-renewal of hepatoma cells.We speculate that CPA4 is promising as a diagnostic marker and therapeutic target for hepatocellular carcinoma,providing new ideas for the diagnosis and treatment of hepatocellular carcinoma.