CMTM6 Expression Promotes Lung Cancer Proliferation And Invasion And Its Mechanism

Objectives:Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world,and is one of the fastest growing malignant tumors.It is the leading cause of cancer deaths in men and women worldwide.Pathological types of lung cancer include non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).More than 85%of cases are non-small cell lung cancer.Non-small cell lung cancer has genetic and cellular heterogeneity.Although great progress has been made in the diagnosis and treatment of lung cancer,the overall cure rate and survival rate of NSCLC are still very low,especially in metastatic disease.The specific molecular mechanism of lung cancer occurrence and metastasis is not yet fully understood.The occurrence and invasion of lung cancer is a complex and dynamic process,involving the inherent genetic abnormalities of tumor tissue and its interaction with the immune system.In view of this,in-depth study of the mechanism of the occurrence,development and metastasis of lung cancer has important clinical value and social benefits for the development of effective treatment measures and improving the survival rate of lung cancer.In recent years,it has been discovered that the CMTM6 gene(chemokine-like MARVEL transmembrane domain containing family member 6)is related to tumor immune escape.This gene is also known as the chemokine-like factor superfamily 6.This gene is located in the short arm of chromosome 3,region 2,zone 2,subband 3,and the encoded protein product has a four-pass transmembrane protein superfamily(TM4SF)and MARVEL domain,so it is closely related to a variety of tumors.Studies have shown that CMTM6 and PD-L1 are co-localized on the surface of tumor cells and are positively correlated.CMTM6 stabilizes PD-L1 molecules on the surface of tumor cells,thereby inhibiting T cells and allowing tumor cells to escape T cell killing.However,the expression of CMTM6 and PD-L1 is not completely positively correlated,indicating that CMTM6 is not only related to PD-L1,there may be other important molecules interacting with CMTM6 to affect tumor prognosis.At the same time,other studies have shown that CMTM6 is expressed in a variety of tumor tissues and is closely related to the poor prognosis of tumors.However,its expression in primary lung tumors and its relationship with prognosis are not yet clear,and the function and molecular mechanism of CMTM6 in the occurrence and development of lung cancer are still poorly understood.At the same time,as a new biomarker,CMTM6 is far from enough to study its function.To this end,this project intends to detect the expression of CMTM6 in non-small cell lung cancer,analyze the relationship between its expression and relevant clinicopathological parameters;through in vivo and in vitro experiments,study the role of lung cancer in the occurrence,development and metastasis;explore CMTM6 regulate the mechanism of lung cancer cell proliferation and invasion.Methods:1.Use immunohistochemistry to detect the expression of CMTM6 in paraffin specimens of non-small cell lung cancer tissues and normal lung tissues,and analyze the relationship between CMTM6 expression and tumor differentiation,staging,metastasis,and clinical prognosis.2.Establish a stable lung cancer cell line that interferes with CMTM6,use in vitro MTT experiment,plate clone formation experiment,flow cytometry to detect apoptosis to detect tumor cell proliferation;use scratch test to detect tumor cell migration ability;Transwell test to detect tumor Invasive ability of cells.3.In vivo experiment:use the stable lung cancer cell line that interferes with CMTM6 to establish a nude mouse subcutaneous lung cancer xenograft model,to detect the effect of CMTM6 on the proliferation of lung cancer cells in nude mice;to detect the heart,liver,spleen,lung,kidney,and brain of nude mice,Whether the large intestine and small intestine have tumor invasion.4.Use transcriptome sequencing to screen differentially expressed genes of CMTM6,and find out differentially expressed genes by counting the mRNA expression of lung cancer cells that knock down CMTM6 and control lung cancer cells;perform GO function enrichment and KEGG signaling pathway for differential genes Enrichment;construct a protein interaction network and visualize it with cytoscape software to extract core genes that interact with CMTM6;construct a differential gene-signal pathway network to extract related proteins that mainly act on signal pathways.5.Verification of differentially expressed genes:Detect interference with CMTM6 lung cancer cell cycle distribution by flow cytometry;detect interference with CMTM6 after cell cycle related proteins P53,P21,P27,Cyclin A,Cyclin D,Cyclin E,CDK2,CDK4 and The expression of ERK,pERK,AKT,and pAKT.Results:1.The expression of CMTM6 in lung cancer tissues1).The results of immunohistochemistry showed that the positive expression of CMTM6 was mainly located in the cell membrane and cytoplasm,which was brown or brown.2).The expression level of CMTM6 protein in lung cancer tissue(161.04±86.60)was higher than that of adjacent normal lung tissue(71.20 ± 45.10)(P<0.05):3).The expression of CMTM6 is related to tumor T stage,tumor histological type and tumor infiltrating lymphocytes(P<0.05),but has nothing to do with patient age,gender,tumor differentiation degree,and lymph node invasion.4).Kaplan-Meier survival analysis results showed that the 5-year overall survival of patients with high CMTM6 expression was significantly shorter than that of patients with low expression(P<0.01).2.CMTM6 promotes lung cancer cell proliferation,migration and invasion1).In vitro experiments:MTT results showed that the growth rate of stable lung cancer strains interfered by CMTM6 was significantly slower than that of the control group(P<0.01).Plate clone formation experiments showed that the number of clones of stable lung cancer strains interfered by CMTM6 was significantly less than that of the control group(P<0.05).Flow cytometry detection of apoptosis results showed that the rate of CMTM6 interference with stable lung cancer cell apoptosis was higher than that of the control group(P<0.05).The results of the scratch test showed that the migration speed of stable lung cancer cells interfered by CMTM6 was significantly slowed down(P<0.05).Transwell experiment results showed that the number of invasive cells of CMTM6 interfered with stable lung cancer strain was significantly less than that of the control group(P<0.05).2).In vivo experiment:The subcutaneous tumor formation experiment in nude mice showed that compared with the control group,the tumor growth rate of the stable lung cancer strain interfered by CMTM6 was slower and the tumor volume was smaller(P<0.05).The results of immunohistochemistry showed that the Ki-67 positive rate(39.6%)of the CMTM6 interference group was significantly lower than that of the control group(76.4%)(P<0.05).There were no metastatic tumors in the HE test of heart,liver,spleen,lung,kidney,brain,large intestine,and small intestine in nude mice.3.The mechanism of CMTM6 regulating lung cancer cell proliferation and invasion1).Transcriptome sequencing revealed 4993 differentially expressed genes of CMTM6,2466 up-regulated genes,and 2527 down-regulated genes.2).The GO function enrichment and KEGG signal pathway enrichment of these genes showed that CMTM6 differentially expressed genes were mainly enriched in the biological process of mitotic cell cycle phase transition,DNA replication and chromosome separation.The cell composition is mainly enriched in chromosomes,centromere regions,chromosomal regions and kinetochore.The molecular functions mainly focus on catalytic activity,acting on DNA,structural constituent of nuclear pore and the tubulin binding.3).The KEGG signaling pathway is mainly enriched in DNA replication,cell cycle and base excision repair.4).The protein interaction network extracts 10 core genes that interact with CMTM6,namely UBC,TP53,PRDM10,HSP90AA1,TSPO,UBB,CDK1,TOP2A,CAD,CDK2.5).Differential gene-signaling pathway network obtains the relevant proteins of the main signal pathways.The main role of P53 signal pathway is TP53,CDK1,CDK2,CCND1,CCND3 and so on.The main effects on cell cycle signaling pathways are TP53,CDK1,CDK2,CDK6,CCNB1 and so on.MCM2,MCM5,MCM7,POLE2,LIG1,etc.act on the DNA replication signal pathway.6).The differentially expressed genes of CMTM6 are mainly enriched in the cell cycle.7).The results of cell cycle detection by flow cytometry showed that interference with CMTM6 blocked the cell cycle in the G0/G1 phase,the cells could not enter the G2/M phase,and inhibited cell proliferation.8).Western blot detects the expression of cell cycle related proteins P53,P21,P27,Cyclin A,Cyclin D,Cyclin E,CDK2,CDK4.Interfering with CMTM6 can activate P53 and further induce the expression of P27 gene,thereby inhibiting the activity of Cyclin A.The cell cycle is arrested in G0/G1 phase.9).Western blot detected the protein expression of ERK,pERK,AKT,and pAKT.The results showed that CMTM6 interfered with stable lung cancer strains to down-regulate the expression of pERK and inhibit the phosphorylation of ERK,but it had no effect on AKT.Conclusions:1.The expression level of CMTM6 in non-small cell lung cancer tissues is significantly up-regulated.The expression of CMTM6 is related to tumor T stage,tumor histological type and tumor infiltrating lymphocytes.2.CMTM6 expression is negatively correlated with lung cancer prognosis.3.Interfere with CMTM6 to inhibit the proliferation,invasion and migration of lung cancer cells.4.CMTM6 promotes the proliferation,invasion and migration of lung cancer cells,and its regulatory mechanism may be related to P5 3-mediated cell cycle arrest.CMTM6 mediates the proliferation and migration of lung cancer cells through the MAPK/ERK signaling pathway.5.CMTM6 can be used as an independent risk factor for assessing the prognosis of NSCLC,providing a basis for clinical development of individualized NSCLC treatment plans.