Expression And Clinical Significance Of CKLF-like MARVEL Transmembrane Domain-containing Member 4 In Hepatocelluar Carcinoma

BackgroundAccording to the latest statistic data,liver cancer is one of the most common malignancies and the fourth leading cause of cancer-related death in the world,with a poor prognosis and a five-year survival rate of about 18%.Hepatocellular carcinoma(HCC)constitutes most pathological types of liver cancer,and HCC refers to liver cancer in our study.In the past 20 years,although some risk factors such as hepatitis C virus(HCV),hepatitis B virus(HBV)infection and aflatoxin exposure have been controlled.The advent of HCV direct acting anti-viral(DAA)drugs have cured HCV hepatitis successfully and HBV antiviral drugs nucleoside analogs have also achieved clinical cure of HBV hepatitis.However,the persistence of factors such as hepatitis viruses,alcohol consumption,obesity,and non-alcoholic fatty liver disease has made the incidence of HCC remains high,which causes certain health losses and economic burdens on the people and their country.As an aggressive solid tumor,HCC has the characteristics of early metastasis,rapid growth and infiltration,poor prognosis and high malignancy.The routine treatments of HCC are mainly surgical treatment,local radiofrequency ablation of the liver,and conservative medical treatment.However,as a malignant tumor,HCC has a high rate of metastasis and recurrence,which leads to poor clinical treatment effect and high risk of tumor-related death.In the process of tumorigenesis and development,the malignant transformation of somatic cells is often accompanied by genetic and epigenetic changes,for example,inactivation of tumor suppressor genes,activation of oncogenes,methylation and methylation of genes,etc.These events may induce the production of a variety of tumor-related antigens,new antigens or targets of anti-tumor immune responses.Therefore,the investigation on abnormal expression of genes in the process of tumorigenesis and development can deepen our understanding of HCC and may provide some theoretical basis for the improvement of tumor diagnosis methods and therapeutic effects.Increasing number of reports indicated that the expression of genes and the growth and division of cells are precisely regulated by genetic and epigenetic inheritance.Abnormal genetic changes(such as genetic mutations,gene deletions,gene amplifications,or chromosomal translocations)and/or epigenetic abnormalities(such as histone modifications)may cause tumors to develop or progress.CKLF-like MARVEL transmembrane domain-containing member(CMTM),originally known as chemokine like factor superfamily(CKLFSF),is a new gene family discovered and named by the Human Disease Research Center of Peking University in China.The new family was later renamed the CMTM family due to the structural characteristics of the MARVEL transmembrane domain in its member-encoded products,including nine members,CLSF and CMTM 1-8.The structural and functional characteristics of CMTM family members are between chemokines and the Transmembrane 4 Super Family(TM4SF).In recent years,the research on this family has been continuously improved.Studies have shown that the coding products of this family are closely related to the male reproductive system,immune system,hematopoietic system,cardiovascular system,and tumor progression.The relationship between the CMTM family and tumors has not yet reached a unified conclusion.Many studies believe that the CMTM family is a tumor suppressor gene family,members of which is lowly expressed in tumors and related to the malignant progression of tumors.However,some studies have demonstrated that CMTM1 is highly expressed in breast cancer,renal cell carcinoma,lung cancer,and ovarian cancer,and that breast cancer patients with low expression in the CMTM1 high expression group have lower survival rates.CMTM4 and CMTM8 were down-expression in many solid tumors,while CMTM2,CMTM3,and CMTM6 are highly expressed in gliomas and HCC.Among them,at the transcriptome level,the mRNA level of CMTM6 was upregulated in glioma,while at the protein level,the CMTM6 protein was overexpressed in HCC and the head and neck squamous cell carcinoma(HNSCC).Even the same member may have different roles in different pathological types of the same tumor.The high expression of CMTM7 has a positive correlation with the prognosis of patients with lung adenocarcinoma,and a negative correlation with the prognosis of patients with lung squamous cell carcinoma.Therefore,members of the CMTM family have different functions in different malignancies,so it is of great significance to further study their role and mechanism in tumorigenesis,development and metastasis.Researches are aimed to find new molecular targets for tumor detection and gene therapy and bring new hope for cancer patients.Bei et al.performed immunohistochemical staining(IHC)on 75 HCC clinical samples and found that CMTM4 protein is under-expressed in HCC,which is an independent risk factor for the prognosis of patients with HCC.However,CMTM4 mRNA levels have not been explored.Considering the complex relationship between CMTM family members and tumors,this study aims to validate the relationship between CMTM4 expression and HCC from transcriptomes and proteomes,from tissues to cells,through network public databases and clinical samples,and explores its possible mechanism through bioinformatics technology.Provide a theoretical basis for deepening the understanding of CMTM4 molecules and promote the transformation of CMTM4 as a clinical diagnostic and therapeutic target.ObjectiveOur study explores the diagnostic and prognostic value of CMTM4 for HCC patients from the transcriptome and proteome comprehensively through the network public database and liver cancer tissue microarray(TMA)data.Combined with bioinformatics technology,we also investigate the possible mechanism of CMTM4 in the occurrence and development of HCC.Methods1.Download the human liver cancer gene mRNA expression profile files from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO).Analyze the expression level of CMTM4 mRNA in tumor tissues,and the relationship between expression level of CMTM4 mRNA and the clinicopathological characteristics and survival prognosis of HCC patients.2.Validate the expression level of CMTM4 protein in HCC tissues and HCC cell lines by TMA-IHC technology western blotting technology;3.Statistical analysis and mapping were implemented with Graph Pad Prism 8.0 and SPSS23.0 software.Gene mRNA expression profile data was used as a continuous variable.Comparisons between groups were performed using two-sided Student’s t-test.Comparisons between disordered classification data were compared using Chi-square test.Comparisons between ordered classification data were compared using group Mann-Whitney’s test.Survival curve analysis and Log Rank test were used to explore the relationship between gene expression and prognosis.Receiver Operating Characteristic(ROC)curves were used to evaluate the diagnostic value of CMTM4 for HCC tissues.α<0.05 was considered statistically significant.4.Realize the installation,loading and operation of Gene Set Variation Analysis(GSVA)package by using R software.Use Hall-Mark gene set as background gene set and analyze the enriched functional sets and paths of CMTM4-related genes in tumor.Result1.The expression level of CMTM4 mRNA was significantly higher in HCC tissues than in adjacent tissues,and the difference was statistically significant.Univariate analysis suggested that the expression of CMTM4 mRNA was different in patients with different TNM staging(P=0.007)and gender(P=0.019).However,there was no significant correlation between the expression level of CMTM4 mRNA and age(P=0.384),race(P=0.065),histological type(P=0.592).Multivariate analysis suggested that the expression level of CMTM4 mRNA(HR 1.434,P=0.013)and TNM stage(HR 1.904,P=0.000)were independent risk factors for prognosis of HCC patients.Kaplan-Meier analysis suggested that patients with higher CMTM4 mRNA expression had shorter overall survival(OS)time(P=0.005)and progression-free survival(PFS)time(P=0.015)than patients with lower CMTM4 mRNA expression.2.CMTM4 mRNA is of promising diagnostic value in patients with HCC.The AUCs of different cohorts were as follows:the TCGA cohort,AUC=0.704(P<0.001);GSE6764,AUC=0.816(P<0.001);GSE45436,AUC=0.921(P<0.001);GSE5423 8,AUC=0.710(P=0.007);GSE62232,AUC=0.854(P<0.001);GSE76297,AUC=0.887(P<0.001);GSE77314,AUC=0.758(P<0.001);and GSE102083,AUC=0.745(P<0.001).3.The expression level of CMTM4 protein is reduced in HCC cell lines and tissues(P=0.0008).4.The CMTM4 mRNA high expression group was related to immune system response and has negative correlation with immune cells in HCC tissues.Conclusion1.The high expression of CMTM4 mRNA is of great diagnostic value and associated with poor prognosis and malignant clinical phenotype in patients with HCC,which proves that CMTM4 mRNA may serve as a prognostic and diagnostic marker for HCC patients.2.The CMTM4 mRNA high expression group was enriched in immune system response and has negative correlation with immune cells,which proves that CMTM4 may related to immune function inhibition in HCC and provided research direction for the further investigation.