The Functional And Mechanical Studies Of Chemokine-like Factor Superfamily Protein CMTM7 In Hepatocellular Carcinoma

BackgroundPrimary liver cancer(PHC)mainly includes three pathological types:hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and mixed liver cancer,of which HCC accounts for 75%-85?of PHC.There are more than 0.841 million new cases and 0.782 million deaths of PHC recorded according to the global cancer epidemiological data in 2018.In our country,the incidence of PHC accounts for 55%of that in the world,and the incidence and mortality of PHC rank the fourth and third respectively.The high incidence and mortality of PHC cause great economic burden to the society.Although the level of diagnosis and treatment of PHC have improved,the effect of conventional treatment is still not satisfied.The 5-year survival rate of patients with PHC is only 30%,and the opportunities of surgical treatment is less than 20%.In recent years,with the in-depth studies of cell biology,high stability and specific molecular targeted therapy have become the mainstream of research.It has a wider significance for clarifying the molecular mechanism of PHC,developing new therapeutic targets,and providing a new scheme for the early diagnosis and treatment of PHC.Chemokine-like Factor Super Family(CKLFSF)is a new type of gene familiest,it is discovered by Chinese scholars via suppressing subtractive hybridization technique.The first member of CKLFSF is named CKLF1,and the three variants of CKLF1 are named CKLF2,3,4.CKLFSF1-8 are successfully cloned and verified subsequently.The super family genes contain MARVEL structure which is closely related to gene function,and they are involved in immunity,reproductive,hematopoiesis,cardiac muscle and bone.CMTM1,3,5,7,8 are thought to have a potential role in tumor suppressor genes,including inhibition of cell proliferation,inducing cell cycle arrest and promoting cell apoptosis.CMTM7 acts as a member of the CMTM family,which is expressed widely in the human body tissues.The structure and function of CMTM7 are between four transmembrane proteins and typical chemokines.The four transmembrane proteins play a role by binding other proteins in vivo,and participate in the process of cell differentiation,adhesion,dissolution,signal transduction and other biological characteristics.Some studies have shown that the expression of CMTM7 in non-small cell lung cancer is much lower than that in the paracancerous tissues,and knockdown of CMTM7 inhibited the function of tumor cell apoptosis.In addition,it has been reported that overexpression of CMTM7 could reduce the stability of EGFR,and accelerate its degradation and promote internalization.These results suggest that CMTM7 may play an important role in tumor biotherapy.In this project,we will focus on studying the function and mechanism of CMTM7 in HCC.Objective1.To confirm the expression of CMTM7 in HCC tissues and cell lines,samples of HCC patients and HCC cell lines were collected.At the same time,to explore the correlation between CMTM7 and pathological features of HCC patients,the expression level of CMTM7 in HCC tissue,clinical data and follow-up results were analyzed.2.To explore the influence of CMTM7 on the cell growth,migration and invasion,CMTM7 was overexpressed in HCC cells.3.To explore whether CMTM7 overexpression affected the cell cycle progression of HCC cells.4.To detect whether overexpression of CMTM7 could affect the growth of HCC tumors by establishing nude mice xenografts.5.To detect the effect of CMTM7 on STAT3-driven luciferase activity and STAT3 activation after overexpression of CMTM7 in HCC cells.6.To detect the expression of CMTM7 and the effect of overexpression of CMTM7 on the drug resistance in solafenib-resistant HCC cells.Methods1.The quantitative real-time PCR(qRT-PCR)and western blot were used to detect the expression of CMTM7 in HCC tumor tissues and cell lines.2.Immunohistochemistry(IHC)was used to detect the expression level of CMTM7 in HCC tumor tissues and adjacent normal tissues,and the expression scores of CMTM7 in tumor tissues obtained by IHC was used to analyze the relationship between the scores and the clinicopathological characteristics and prognosis of HCC patients..3.The overexpression plasmids of CMTM7 was transfected into HCC cells by Lipofectamine(?)2000.4.Cell Counting kit-8(CCK-8)was used to determine the cell survival of HCC cells.5.Cell scratch and transwell assays were carried out to detect whether CMTM7 affected cell migration of HCC cells.6.Flow cytometry and western blot were used to detect the effect of overexpression of CMTM7 on HCC cell cycle.7.The effect of CMTM7 on the growth of HCC was detected by using the tumor xenograft model of nude mice.8.Luciferase assay and western blot were used to detect the effect of CMTM7 on STAT3 activation.9.Western blot and CCK-8 were used to detect the expression of CMTM7 and the effect of CMTM7 on the drug sensitivity of sorafenib-resistant HCC cells.Results1.The qRT-PCR analysis showed that CMTM7 was significantly decreased in 78.8%(41/52)HCC paired tumor tissues compared with the adjacent normal tissues.At the same time,the mRNA expression levels of CMTM7 in HCC cell lines were also decreased obviously compared with the normal liver epithelial cell line.2.IHC results showed that there was no significant difference in the scores of CMTM7 among the factors of HCC patients' sex,age,liver fibrosis,hepatitis B surface anti positive and distant metastasis(p>0.05),but there was a significant difference in different pathological stages of tumor(p<0.01).The scores of CMTM7 were negatively correlated with the pathological stages of tumor(R=-0.771).In addition,the 5-year survival rate of HCC patients with low CMTM7 expression was significantly lower than that with high CMTM7 expression(p=0.038).3.CCK-8 assay showed that overexpression of CMTM7 could significantly inhibit the growth of HCC cells.4.The results of cell scratch test showed that there was a significant difference in the mobility of HCC cells between the control and CMTM7-overexpressed groups(p<0.01).In addition,the invasiveness of the cells was measured by transwell chamber method.The results showed that there was no significant difference in the number of cells passing through the membrane between the empty plasmid transfection group and the negative control group(p=0.33),while the CMTM7-overexpressed group was significantly lower than that of the negative control group(p<0.01).5.Flow cytometry showed that overexpression of CMTM7 could block the cell cycle of HCC at G0/G1 phase.In addition,western blot showed that Cyclin D1,CDK4 and CDK6 were down-regulated,while p27 was up-regulated.6.Overexpression of CMTM7 significantly inhibited the tumor growth of HCC in nude mice xenografts.7.Luciferase assay showed that overexpression of CMTM7 could significantly inhibit STAT3-driven luciferase activity.In addition,western blot showed that overexpression of CMTM7 could significantly inhibit STAT3 activation in HCC cells.8.It was found that CMTM7 was downregulated in the sorafenib-resistant HCC cells,and overexpression of CMTM7 could significantly enhance the drug sensitivity of sorafenib-resistant HCC cells to sorafenib.Conclusion1.CMTM7 is downregulated in HCC tumor tissues and cell lines,and the expression of CMTM7 is closely associated with the prognosis of HCC patients.2.Overexpression of CMTM7 obviously inhibits HCC cell growth,migration and invasion,and CMTM7 inhibits HCC cell growth by inducing cell cycle arrest at G0/G1 phase.3.Overexpression of CMTM7 inhibits STAT3 signaling in HCC by upregulating SHP-1 expression.4.Overexpression of CMTM7 enhances the drug sensitivity of sorafenib in sorafenib-resistant HCC cells.5.CMTM7 gene is expected to be a new target for HCC treatment.