| Objective:Esophageal cancer(EC)is one of the most common malignant tumors in the world.At present,there are few reports on the mechanism of lncRNAs in multistage progression of EC by regulating genes methylation.The purpose of this study was to use lncRNA microarray detection of EC for screening and analysis,and to identify the significantly different ADAMTS9-AS2.This study was conducted concerning the expression and prognosis,biological functions,molecular mechanisms of ADAMTS9-AS2 in EC.This research will provide the theoretical and experimental basis on the EC to screen,prevent and the evaluation of the prognosis.Materials and Methods:1.The expression and potential prognostic value of ADAMTS9-AS2 in ECThe significant difference of lncRNA ADAMTS9-AS2 in ESCC patients was screened by lncRNAs microarray detection of EC,and the expression of ADAMTS9-AS2 was verified in ESCC cell lines and tissues.We detected ADAMTS9-AS2 expression in 83 patients with ESCC tissues and matched adjacent normal tissues by q RT-PCR method.The relationship between ADAMTS9-AS2 expression and clinicopathological characteristics and survival prognostic value of EC were evaluated by Chi-square test,Kaplan-Meier and Cox regression analysis.Six human ESCC cell lines were cultured to detect the expression of ADAMTS9-AS2 by q RT-PCR method,and the cell lines with small interfering RNA(si RNA) ADAMTS9-AS2 were screened.The expression of ADAMTS9-AS2 in ESCC cell lines was knocked down by si RNA and verified by q RT-PCR method.2.The main biological functions of ADAMTS9-AS2 in ECIn EC-1 and TE-1 cell lines,the expression of ADAMTS9-AS2 was knocked down by si RNA,and the proliferation of ESCC cell lines were detected by CCK8 assay.Si RNA was used to knock down the expression of ADAMTS9-AS2 in ESCC cell lines,and the changes of apoptosis and cell cycle were detected by flow cytometry.Transwell assay and scratch assay were used to detect the changes of ESCC cell invasion and migration ability.3.ADAMTS9-AS2 may inhibit the progression of EC through CADM2Differential methylation sites of CADM2 were screened by genome-wide methylation microarray detection in 5 EC patients.The Agena methylation technique was used to validate differential methylation sites in 24 patients with ESCC tissues and matched adjacent normal tissues.The possible binding of ADAMTS9-AS2 to downstream target genes were predicted by Starbase database.We detected CADM2 expression in 83 patients with ESCC tissues and matched adjacent normal tissues by q RT-PCR method.The relationship between CADM2 expression and clinicopathological characteristics and survival prognostic value of EC were evaluated by Chi-square test,Kaplan-Meier and Cox regression analysis.The m RNA levels and protein expression levels of CADM2 in si-ADAMTS9-AS2 cell lines after were detected by q RT-PCR method and Western Blot,respectively.4.ADAMTS9-AS2 inhibits the progression of EC by regulating CADM2 methylationThe regulation of ADAMTS9-AS2 and DNMT3 B on the methylation level of CADM2 was detected by pyrosequencing technology.The relationship between DNMT family genes and ADAMTS9-AS2 and CADM2 were analyzed by EC m RNA microarray detection and Spearman’s correlation.RIP(RNA Binding Protein Immunoprecipitation)method was used to verify the binding and action between ADAMTS9-AS2 and DNMT3 B.Ch IP assay was used to analyze the effect of ADAMTS9-AS2 expression on binding DNMT3 B and CADM2 gene.The mechanism rescue experiments were performed by co-transfection of si-ADAMTS9-AS2 and DNMT3 B overexpression plasmid in TE-1 cell line.After co-transfection of si-ADAMTS9-AS2 and pc DNA3.1-CADM2 in TE-1 cells,the effects on proliferation,cycle,apoptosis,invasion and migration of ESCC cells were analyzed by CCK8 assay,flow cytometry,Transwell assay and scratch assay.WB method was performed to detect the effect on the protein expression levels of EMT markers after co-transfection si-ADAMTS9-AS2 and pc DNA3.1-CAM2 in TE-1 cells.Results:1.The expression and potential prognostic value of ADAMTS9-AS2 in EC(1)By lncRNAs microarray detection in 5 ESCC patients,it was found that the expression levels of ADAMTS9-AS2 was downregulated in ESCC tissues,and then ADAMTS9-AS2 was identified as a lncRNA related to the progression of ESCC.(2)q RT-PCR method was performed to detect the expression of ADAMTS9-AS2 in 83 ESCC patients,and it was found that the expression level of ADAMTS9-AS2 in ESCC tissues was significantly lower than that in matched adjacent normal tissues,which was consistent with the results of GEPIA database.This result is the validation of the lncRNAs microarray detection in EC.(3)In ESCC patients,the expression levels of ADAMTS9-AS2 was negatively correlated with ESCC tumor location(P=0.004).(4)The 5-year overall survival(OS)of ESCC patients with low ADAMTS9-AS2 expression was shorter than that of ESCC patients with high ADAMTS9-AS2 expression(P=0.031),which is of prognostic value in estimating ESCC patients.(5)The expression of ADAMTS9-AS2 is an independent prognostic maker in ESCC patients.(6)The results of q RT-PCR showed that the expression level of ADAMTS9-AS2 in human ESCC cell lines(9706,KYSE 150,EC-1,KYSE 30,KYSE 70,TE-1)was significantly lower than that of human esophageal mucosal epithelial cell lines HET-1A.EC-1 and TE-1 cells were selected for further study.By using si RNA to knock down the expression of ADAMTS9-AS2 in EC-1 and TE-1 cells,the interference efficiency reached more than 50%.2.ADAMTS9-AS2 can inhibit the proliferation and invasion of ESCC cells(1)The results of CCK8 assay showed that the proliferation ability of ESCC cells was significantly increased after ADAMTS9-AS2 was knocked down in EC-1 and TE-1 cell lines.(2)The results of flow cytometry showed that the cells apoptosis was inhibited and the cells cycle transformation were promoted in the G1/S phase after ADAMTS9-AS2 was knocked down in EC-1 and TE-1 cell lines.(3)The ability of invasion and migration were promoted after ADAMTS9-AS2 was knocked down in EC-1 and TE-1 cell lines.3.ADAMTS9-AS2 may inhibit the progression of EC through CADM2(1)Differential methylation sites cg03455765 and cg12541174 in CADM2 gene were screened by EC genome-wide methylation microarray.(2)The differential methylation site cg03455765 of CADM2 was verified in ESCC tissues and matched adjacent normal tissues by Agena methylation technique(P< 0.05).(3)q RT-PCR method was performed to detect the expression of CADM2 in 83 ESCC patients,and it was found that the expression level of CADM2 in ESCC tissues was significantly lower than that in matched adjacent normal tissues(P<0.0001).(4)The Starbase database predicted that CADM2 was a downstream target gene for ADAMTS9-AS2.ADAMTS9-AS2 expression was positively correlated with CADM2 expression by Spearman’s correlation analysis(r=0.4473,P<0.0001).The m RNA levels(P< 0.05)and protein levels(P< 0.05)of CADM2 significantly reduced after ADAMTS9-AS2 was knocked down in EC-1 cells.(5)In ESCC patients,the expression levels of CADM2 were positive correlated with ESCC patients family history(P=0.007).(6)The 5-year OS of ESCC patients with high CADM2 expression was significantly longer than that of ESCC patients with low CADM2 expression(P=0.002),which is of prognostic value in estimating ESCC patients.4.ADAMTS9-AS2 inhibits the progression of EC by regulating CADM2 methylation(1)Pyrosequencing results showed that the methylation level of CADM2 was significantly higher than that in si-NC group after ADAMTS9-AS2 was knocked down in TE-1 cells(10.98% vs.8.65%,P<0.01).In EC-1 cells,the methylation level of CADM2 in pc DNA3.1-ADAMTS9-AS2 group was significantly lower than that in PCDNA3.1-NC group(30.77% vs.27.44%,P<0.05),suggesting that ADAMTS9-AS2 could regulate the methylation level of CADM2.(2)The results of EC m RNA microarray detection showed that the expression levels of DNMT1(P=0.0073)and DNMT3B(P=0.0047)were significantly different in ESCC tissues and matched adjacent normal tissues,while the expression levels of DNMT3 A was not significantly different.Spearman’s correlation analysis showed that DNMT3 B was correlated with ADAMTS9-AS2(P=0.0376)and CADM2(P=0.0251),while DNMT1 was only correlated with ADAMTS9-AS2(P=0.0186).In TE-1 cells,the methylation level of CADM2 in pc DNA3.1-DNMT3 B group was significantly higher than that in pc DNA3.1-NC group(13.76% vs.11.21%).We speculated that DNMT3 B may act as a protein between ADAMTS9-AS2 and CADM2,and regulate the methylation of CADM2.(3)The binding between DNMT3 B and ADAMTS9-AS2 was confirmed by RIP assay(P<0.05).The results of Ch IP assay showed that DNMT3 B can enrich the CADM2 methylation region(P<0.001),and si-ADAMTS9-AS2 expression could increase the enrichment of DNMT3 B in CADM2 methylation region(P<0.01).The mechanism rescue assay showed that DNMT3 B affected the regulation of ADAMTS9-AS2 target gene CADM2.Functional rescue assay showed that co-transfection of PCDNA3.1-CADM2 could reverse the effects of si-ADAMTS9-AS2 on proliferation,cycle,apoptosis,invasion and migration of ESCC cells.CADM2 is a target gene of ADAMTS9-AS2 to inhibit the progression of ESCC cells.After ADAMTS9-AS2 was knocked down in TE-1 cells,the protein expression levels of epithelial marker E-cadherin were significantly decreased,and the protein expression levels of mesenchymal markers N-cadherin and Vimentin were significantly increased.The effect of si-ADAMTS9-AS2 on the expression of EMT markers were reversed after co-transfection of PCDNA3.1-CADM2 in TE-1 cells.Conclusion:1.The expression levels of ADAMTS9-AS2 in ESCC tissues were significantly decreased,and its expression levels were related to the prognosis of ESCC patients.It was an independent and favorable prognosis marker in ESCC patients.2.ADAMTS9-AS2 is a tumor suppressor that inhibits the progression of EC by promoting tumor cells apoptosis,inhibiting tumor cells proliferation,migration,and invasion in EC.3.CADM2 is the target gene of ADAMTS9-AS2.The expression levels of CADM2 in ESCC tissues were significantly decreased,and its expression levels were related to the family history and prognosis of ESCC patients,which is of prognostic value in estimating ESCC patients.4.ADAMTS9-AS2 can inhibit the progression of EC by regulating the CADM2 gene methylation and thereby affecting the expression of CADM2,and its mechanism of action is realized by DNMT3B. |
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