Study On The Role And Mechanism Of CMTM6 In Mediating Immune Regulation Of Hepatocellular Carcinoma

Objectives: We aim to clarify the expression of CMTM6 and PDL1/CD4/CD8 in hepatocellular carcinoma(HCC)tissues,their relationships with clinicopathological features of HCC patients,and the effect of CMTM6 expression,co-expression of CMTM6/PD-L1 with T cell infiltration on the prognosis of HCC patients.We also try to investigate the impact of abnormal expression of CMTM6 on the biological functions of HCC cells,such as proliferation,apoptosis,cell cycle,invasion,and migration.The CMTM6 related immune signal pathways,target genes and immune cell infiltration,are preliminarily explored to elucidate the role and molecular mechanism of CMTM6 in the HCC tumor microenvironment via mediating immune regulation.Our study will provide new strategies and ideas to find potential immune therapy biomarkers and clinical prognosis markers for HCC patients.Methods: We collected the epidemiological data and clinicopathological features of HCC patients who survived the surgery and followed up prospectively(n=90).The immunohistochemical method was used to detect the expression of CMTM6 and PD-L1/CD4/CD8 in these 90 cases of HCC and its corresponding adjacent non-tumor liver tissues.The relationship between CMTM6/PD-L1/CD4/CD8 expression and the clinicopathological features of HCC patients was also analyzed.The role of CMTM6 expression and the co-expression effect of CMTM6/PD-L1 with T cell infiltration were clarified for the survival and prognosis of HCC patients,and TCGA-LIHC database was verified for these results.The change of biological functions,such as proliferation,cell cycle,invasion,apoptosis,and metastasis,was detected in vitro and vivo after CMTM6 knocked-down in HCC cells,including CCK8,plate cloning experiment,Transwell chamber invasion and migration experiment,cell scratch,subcutaneous tumorigenesis and metastasis in nude mice.Based on Affymetrix’s whole gene expression profile chip,we screened and verified the CMTM6-related immune signaling pathways,and target genes.Combined with bioinformatics,we analyzed the correlation between CMTM6 and immune cell infiltration,predicted the related immune pathways and target genes when CMTM6 and PD-L1 were co-expressed,and verified them through GSEA pathway enrichment analysis.Results:(1)In the 90 cases(pairs)of HCC and its corresponding adjacent non-tumor liver tissues,the positive rate of CMTM6 expression in HCC tissues was 55.56%(50/90),and the positive rate of CMTM6 expression in adjacent tissues was 36.67%(33/90).Compared with adjacent tissues,CMTM6 was highly expressed in HCC tissues(P<0.05).The clinical characteristic analysis showed that CMTM6 was significantly correlated with tumor size,and lymph node metastasis(P<0.05).Survival analysis showed that the expression of CMTM6 affected the clinical prognosis of HCC patients,and the prognosis of HCC patients with high expression of CMTM6 was poor.(2)We also analyzed the relationship between the clinical characteristics of HCC patients with PD-L1/CD4/CD8 expression.The results showed that PD-L1 protein expression was related to tumor differentiation and CD8 protein expression(P<0.05).The expression of CD8 was correlated with the tumor size of HCC patients(P<0.05).Pearson correlation analysis showed that CMTM6 was positively correlated with the expression of PD-L1(R=0.300,P=0.026),and CD4(R=0.209,P=0.047),but not with the expression of CD8(P>0.05).(3)Multivariate COX regression analysis showed that CMTM6(β=0.583)and co-expression of CMTM6/PD-L1/CD4(β=-0.351)were related to the overall survival of HCC patients(P<0.05),and the co-expression of CMTM6/PD-L1/CD4 was also related to the progression-free survival of HCC patients(β=-1.236;P<0.05).Kaplan-Meier analysis showed that HCC patients with high CMTM6+PD-L1+CD4 had better overall survival and progression free survival than those with low CMTM6+PD-L1+CD4(P<0.05).At the same time,when CMTM6/PD-L1 was highly expressed,we found that the overall survival and progression free survival of HCC patients with high CD4 expression were better than those with low CD4 expression(P<0.05).In addition,we validated the above results by bioinformatics analysis of TCGA-LIHC data,and the results were consistent with this analysis.(4)By constructing a silencing CMTM6 HCC cell line,we found that knocking-down of CMTM6 inhibited the proliferation,cell cycle,invasion,and migration of HCC cells,and affected the EMT process of HCC cells by regulating the expression of EMT related factors.(5)Using the whole gene expression profile chip of Affymetrix,we found that CMTM6 regulated IL-17 F,Rho GDI,and axon trigger signaling pathways.29 related target genes were screened in those pathways for verification,and the expression of 28 genes was consistent with the results of the microarray.In the IL-17 F signal pathway,five members of CXCL family(CXCL1,CXCL10,CXCL5,CXCL6,and CXCL8)were significantly increased.Western blot results that verified CXCL8 expression were consistent with q PCR results.(6)Based on TCGA-LIHC transcriptome chip and the whole gene expression profile chip,we selected CMTM6 related 20 immune genes,including IL18R1,CREB1,and CRLF3(R>3.0).GSEA/PPI/Venn analysis showed the co-expression of CMTM6 and PD-L1 participated in T cell-related pathways,regulated T cell activation,activated T cell immunity,and promoted the occurrence and development of HCC.(7)In the microenvironment of HCC,we used TCGA-LIHC data to find that CMTM6 regulated T cells and NK cells.CMTM6 was negatively related to NK cells(R=-0.37,P<0.05).PD-L1 was positively related with CD8 T cell(R=0.40,P<0.05)and active memory CD4 T cell(R=0.55,P<0.05),which suggested PD-L1 regulated CD8/CD4 T cells.All these results suggested CMTM6 combined with PD-L1 to regulate immune cell infiltration in HCC,promoted HCC immune microenvironment,and affected the prognosis of HCC patients.Conclusions:(1)In HCC tissues,high expressed CMTM6 was associated with poor prognosis of HCC patients.(2)knocking-down of CMTM6 expression inhibited the proliferation,invasion,and migration of HCC cells and regulated the EMT process in vitro.(3)HCC patients having high CMTM6/PD-L1 expression combined with high CD4 T cell infiltration showed favourable prognosis.(4)In HCC,CMTM6 activated IL-17 F signaling pathway,and then increased the expression of CXCLs,which resulted in the malignant progression of HCC.It provided new clues for finding potential diagnostic and therapeutic targets for HCC patients.(5)In the microenvironment of HCC,CMTM6 cooperated with PD-L1 to participate in T cell-related pathways,regulated T cell activation,activated T cell immunity,and promoted the occurrence and development of HCC.At the same time,CMTM6 cooperated with PD-L1 to regulate immune cell infiltration in HCC,played the role of jointly regulating CD4/CD8 T cells,participated in the regulation of immune microenvironment of HCC,and then affected the clinical prognosis of patients with HCC.It is expected to become a new target of specifically targeted immunotherapy.