The Effect And Mechanism Of Charged Multivesicular Body Protein 3 On The Proliferation,Migration And Invasion Of Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide and it is not only a serious challenge but also a major topic in healthcare.In most cases,HCC is associated with underlying cirrhosis and chronic liver inflammation.Patients with hepatocellular carcinoma have several treatment options,including liver transplantation,surgical resection,transarterial embolisation,radiofrequency ablation and systemic therapy that can extend the patient’s life expectancy.Patients with hepatocellular carcinoma are asymptomatic in the early stages,which greatly delays the optimal time for treatment.Treatment for patients with advanced stages is limited to systemic therapy.Although new treatments have emerged in recent years,such as the use of immunosuppressive drugs to treat hepatocellular carcinoma,it remains one of the causes of death from tumours worldwide.Charged Multivesicular Body Protein 3(CHMP3)is a charged multivesicular body protein that is an essential component of the Endosomal Sorting Complexes Required for Transport(ESCRT-III).Previous studies have shown that ESCRT-III plays different roles in various benign and malignant diseases,and CHMP3 also plays a biological role in breast cancer,but no studies have been conducted on whether CHMP3 has a biological role in hepatocellular carcinoma.Therefore,this project proposes to further investigate the effect of CHMP3 on hepatocellular carcinoma.The endosomal sorting complex,essential for transport(ESCRT)III,is comprised of CHMP3,a charged multivesicular body protein.No studies have been published to determine if CHMP3 has a biological role in hepatocellular carcinoma,despite ESCRT-III’s various roles in benign and malignant diseases,as well as its biological role in breast cancer,being demonstrated by prior research.Therefore,this study is intended to further investigate the effect of CHMP3 in hepatocellular carcinoma.Methods: Firstly,by examining The Cancer Genome Atlas Program(TCGA)database,we derived different expression levels of 52 scorch-related proteins in HCC and normal liver tissue.Based on these Differentially Expressed Genes(DEGs),all HCC cases will be classified into different subtypes and the prognostic value of survival for each scorch-related gene will be assessed.A gene signature containing seven genes was constructed by Cox regression and combined with the literature to identify CHMP3 as the final focus of this study.Gene Expression Profiling Interactive Analysis(GEPIA)and The Human Protein Atlas(HPA)databases were used to verify the relationship between CHMP3 expression levels and survival time and poor prognosis in HCC patients.Secondly,we used in vitro and in vivo experiments to verify the biological role of CHMP3 in hepatocellular carcinoma.The expression levels of CHMP3 in HCC and paraneoplastic tissues were measured by immunohistochemical staining and Western blot,as well as the expression of the scorch death-related genes caspase-1,IL-1β,IL-18 and gasdermin D(GSDMD).The proliferation of HCC cells after knockdown and overexpression of CHMP3 was measured by colony formation assay,and the intracellular expression of scorch death-related proteins was examined by Western blot.After the addition of caspase-1 inhibitor Ac-YVAD-CMK(AYC),the proliferation ability of HCC cells in vitro was confirmed by colony formation assay and CCK-8 assay.The expression levels of key proteins in the caspase-1 pathway after knockdown of CHMP3 and application of AYC were examined by Western blot.The ability of CHMP3 to regulate the proliferation of hepatocellular carcinoma cells in vivo was verified using tumour formation assays in nude mice.Finally,the migration and invasion abilities of HCC cells after knockdown and overexpression of CHMP3 were examined by cell scratch assay and Transwell invasion assay,and the changes in the expression levels of migration and invasion-related proteins were detected by Western blot.Results: We screened for 39 differentially expressed genes by comparing the expression levels of scorch death-related genes in normal liver tissue with those in HCC tissue in the TCGA database.By co-clustering analysis,all HCC cases were classified into two subtypes and a significant difference in overall survival time was found between the two subtypes.The Cox regression method eventually constructed a pool containing seven genes and we selected CHMP3 as the focus of our study.In the GEPIA and HPA databases,CHMP3 was found to be highly expressed in hepatocellular carcinoma compared to normal liver tissue,and survival times were shorter in patients with high expression.Immunohistochemical staining and Western blot showed that CHMP3 was highly expressed in HCC patients’ tissues.5 In HCC and paraneoplastic tissues,the expression levels of focal death-associated proteins were significantly downregulated in cancer tissues.Knockdown or overexpression of CHMP3 in HCC cell lines significantly inhibited or enhanced cell proliferation.Western blot results of the knockdown group showed that the expression levels of key proteins of the scorch death pathway were significantly lower than those of the control group.The functional reversion assay of colony formation assay and CCK8 assay demonstrated that CHMP3 affects the proliferation ability of HCC by influencing the caspase-1 signalling pathway.The tumour formation assay using nude mice further demonstrated that knockdown of CHMP3 significantly inhibited the development of HCC in vivo,and the inhibitory ability was diminished by the addition of AYC.The cell scratch assay and Transwell assay confirmed that the reduction or increase of CHMP3 expression level could affect the migration and invasion of hepatocellular carcinoma cells.The results of Western blot assay in the knockdown group showed significant changes in the expression of e-cadherin,n-cadherin,MMP9 and Vimentin.Conclusion: These results suggest that high CHMP3 expression is associated with poor prognosis in HCC patients.In both in vivo and in vitro experiments,high CHMP3 expression inhibited caspase-1-dependent scorching and thus promoted the proliferation ability of hepatocellular carcinoma.high CHMP3 expression promoted the migration and invasion of hepatocellular carcinoma.