Effect Of Corticotropin Releasing Hormone Binding Protein On The Function Of Hepatocellular Carcinoma Cells

Objective: Hepatocellular carcinoma(HCC)is common malignant tumor in our country,with rapid progression,high malignancy,and poor prognosis,which is the second cause in cancer-related death.In clinical practice,HCC is usually detected at an advanced stage,with extremely limited treatment options.Therefore,the study for the mechanisms of the development and progression of HCC and looking for more treatment target is very important.Corticotropin releasing hormone binding protein(CRHBP)is involved in many physiological processes.The TCGA database showed that the expression of CRHBP was significantly down-regulated in HCC tissues,suggesting that CRHBP may play a role in the development and progression of HCC.This study aims to explore the effect of CRHBP on the function of HCC cells and hope to further provide more theoretical basis for the treatment of HCC.Methods:This experiment mainly includes three parts.1.The first part is to detect the protein expression level of CRHBP in HCC tissues and normal liver tissues by immunohistochemistry.2.The second part is about the effect of CRHBP on the function of HCC cells.(1)Use plasmids to overexpress CRHBP in LM3 cells,and then select monoclonal stable transgenic strains through a 96-well plate using the multiple dilution method.The monoclonal stable transfected strains with high transfection efficiency are screened by qPCR and Western-Blot.(2)Detecting the biological behavior changes of LM3 cells after overexpression of CRHBP,CCK8 proliferation test was used to examine the cell proliferation.Cell scratch test and Transwell test were used to examine cell migration and invasion.3.The third part is about the molecular mechanism of CRHBP inhibiting HCC metastasis.(1)Using Western-Blot technology to detect the overexpression of CRHBP in LM3 cells,detect epithelial mesenchymal transition(EMT)related genes(ZO-1,E-cadherin,N-cadherin,Snail),and study the effects of CRHBP on EMT pathway related genes regulation.Results:1.Part 1: The immunohistochemical result showed that the expression of CRHBP in HCC tissues was significantly lower than that in normal liver tissue(P<0.01).2.Part 2:(1)In LM3 cells overexpressing CRHBP,using q RT-PCR and Western-Blot methods further screened stable transfected monoclonal strains with high transfection efficiency(P<0.05).(2)After LM3 cells overexpressed CRHBP,a stable monoclonal clone was selected from it,and various cell function experiments were performed.CCK-8 result showed that overexpressing CRHBP inhibited the proliferation of LM3 cells(P<0.05).Cell scratch test result showed that overexpressing CRHBP significantly inhibited the migration of LM3 cells(P<0.0001);Transwell test result showed that overexpressing CRHBP significantly inhibited the migration and invasion of LM3 cells(P<0.0001).3.Part 3: In LM3 cells,overexpression of CRHBP inhibited Snail,N-cadherin,and promoted the expression of E-cadherin,ZO-1 and other related EMT indicators(P<0.05).Conclusion: CRHBP can inhibit the proliferation,migration and invasion of HCC cells and promote EMT of HCC cells.The expression of CRHBP in HCC tissues may be a predictor of the clinical prognosis of HCC and a potential therapeutic target.