| BackgroundEsophageal cancer is a serious malignant tumor with poor prognosis and high mortality.Esophageal cancer is the fifth most common cancer in China and the fourth leading cause of cancer-related death.The incidence of esophageal cancer is as high as 16.7/100000,and the mortality rate is as high as 13.4/100000.Although Esophageal adenocarcinoma(EAC)has become the leading cause of esophageal cancer in the United States and European countries,Esophageal Squamous Cell carcinomas(ESCC)is the predominant histologic type in China.The molecular mechanism of ESCC is not fully understood,but emerging studies suggest that lncRNA may be positively associated with the pathogenesis of ESCC.Lnc RNA is a group of nc RNAs with a length of more than 200 nt,which plays an important regulatory role in various cellular processes and disease progression,but do not participate in protein translation.The studies on lncRNA in ESCC have been conducted for years,multiple evidences suggest that lncRNA plays an important role in disease progression.Firstly,in some cell studies,a few lncRNAs have been reported as oncogenes or tumor suppressors.Secondly,the expression patterns of various lncRNAs are also related to clinical parameters such as therapy,staging and prognosis of ESCC patients.These results indicate that lncRNA-based therapy has great potential in ESCC therapy.Therefore,an in-depth study of the functions of lncRNA is helpful to elucidate the molecular mechanism of ESCC development,so as to provide a new target for clinical treatment of ESCC and a new molecular marker for prognosis of ESCC tumors.ObjectiveIn this study,the expression of lncRNA in ESCC was analyzed to explore the effect of lncRNA in ESCC and the molecular mechanism involved,so as to provide a reliable theoretical basis and new direction for clinical diagnosis,treatment and prognosis evaluation of ESCC.MethodsFirstly,lncRNA GAS8-AS1 in ESCC and its paracancer tissues was analyzed by RNA-seq to confirm the low expression of lncRNA GAS8-AS1 in ESCC.Further analysis by q PCR confirmed the difference in the expression of lncRNA GAS8-AS1 in ESCC and normal tissues,and analyzed the significance of lncRNA GAS8-AS1 expression in clinical ESCC patients.Secondly,the effects of lncRNA GAS8-AS1 expression on the survival,proliferation and apoptosis of ESCC cells were analyzed by means of cell and molecular biology.The effect of lncRNA GAS8-AS1 expression on tumor formation was investigated by subcutaneous tumor transplantation model in nude mice.Finally,the target genes of lncRNA GAS8-AS1 were predicted and analyzed,and the expression of the target genes and relevant signaling pathways involved were detected.Results(1)Expression of lncRNA GAS8-AS1 in ESCC tissuesThrough bioinformatics analysis and statistics,it was found that,compared with paracancer tissues,different gene expressions were up-regulated and down-regulated in ESCC tissues.Lnc RNA GAS8-AS1 was significantly down-regulated in ESCC tissues(p<0.05).We used q PCR to further confirm the expression of lncRNA GAS8-AS1 in ESCC tissues and para-cancer tissues in another 10 pairs,and found that lncRNA GAS8-AS1 was indeed down-regulated in ESCC tissues compared with para-cancer tissues(p<0.05).In addition,we analyzed the relationship between lncRNA GAS8-AS1 expression in TCGA database and survival and prognosis of ESCC patients,and found that patients with high lncRNA GAS8-AS1 expression had better survival and prognosis(p<0.05).(2)The effect of lncRNA GAS8-AS1 expression on the biological characteristics of ESCC cellsCompared with the control group,down-regulation of lncRNA GAS8 AS1 in ESCC cells markedly improved survivability,and the formation of cell proliferation clone size significantly larger,more cells in G1 / S phase,cell apoptosis rate significantly decreased(p<0.05).In ESCC cell line with lower lncRNA GAS8-AS1 expression,EC-9706,using c DNA to overexpress lncRNA GAS8-AS1,the results showed that compared with the control group,lncRNA GAS8-AS1 overexpression decreased cell viability,the formation of cell clone,and the proportion of cells in G1 / S phase significantly,and cell apoptosis rate increased significantly(p<0.05).(3)Overexpression of lncRNA GAS8-AS1 can inhibit the development of ESCC in vivoIn order to prove that in the body lncRNA GAS8-AS1 expression effect on esophageal cancer progression and tumor formation in vivo,we use nude mice subcutaneous tumor model to verify.The results showed that EC-9706 with lncRNA GAS8-AS1 overexpression formed tumor significantly less than the control,the proportion of tumor formation was also significantly lower than the control group(p< 0.05).(4)The molecular mechanism of lncRNA GAS8-AS1 affecting the occurrence and development of ESCCWe compared the gene expression differences between lncRNA GAS8-AS1 knockdown in ESCC cell lines KYSE-510 and KYSE-140 and the control group through RNA-seq analysis,found that 233 genes up-regulated after lncRNA GAS8-AS1 knockdown,such as DNMT3 A and HDAC5,and 47 genes down-regulated after lncRNA GAS8-AS1 knockdown,such as PRMT5 and BAX.Through the phenotypic experiments above,we found that lncRNA GAS8-AS1 knockdown can inhibit cell apoptosis,while lncRNA GAS8-AS1 overexpression can promote cell apoptosis,and BAX is one of the most important protein involved in cell apoptosis.Therefore,we speculated that lncRNA GAS8-AS1 might inhibit cell proliferation and promote cell apoptosis by promoting the expression of apoptotic protein BAX,thereby inhibiting the progression of ESCC.We detected the effect of lncRNA GAS8-AS1 expression on the Bax in ESCC cell lines by qPCR and western-blot.The results showed that when the expression of lncRNA GAS8-AS1 was down-regulated in KYSE-140,an ESCC cell line with high expression of lncRNA GAS8-AS1,the expression of Bax was also decreased(p<0.05).Lnc RNA GAS8-AS1 was overexpressed in EC-9706,an ESCC cell line with low expression of lncRNA GAS8-AS1,and the expression of Bax also increased(p<0.05).Therefore,lncRNA GAS8-AS1 affects the formation of ESCC,at least,partially by regulating the expression of Bax.Then we studied the Bax gene polymorphism and the expression of Bax protein in ESCC.The PCR revealed that the distribution of GG,AG and AA genotypes were 50(66.67%),16(21.33%)and 9(12%).The immunohistochemical results showed that the positive-expression rate of Bax in esophageal squamous cell carcinoma was 42.67%.There was a correlation between the expression of Bax and Bax SNP in ESCC.Bax gene polymorphism was associated with external profile infiltration,differentiation,lymph node metastasis and clinical stage.The survival analysis revealed that the prognosis of patients with AG+AA genotypes was favorable,while that of patients with GG genotype was poor.These results suggest that,lncRNA GAS8-AS1 can regulate the expression of Bax,while Bax gene polymorphism was associated with Bax gene expression,tumor staging and lymphatic metastasis in patients with ESCC.Thus,lncRNA GAS8-AS1 may affect the proliferation and apoptosis of ESCC by regulating the expression of Bax.We further explored the mechanism of GAS8-AS1 regulating Bax.GAS8-AS1 is predicted to interact with miR-573,which can target Bax.We found that GAS8-AS1 may form base pairing with miR-573.Correlation analysis showed that AS8-AS1 was positively correlated with Bax,which is a target of miR-573.QPCR and western blot was performed to analyze the effects of GAS8-AS1 and miR-573 overexpression on the expression of Bax.GAS8-AS1 overexpression resulted in the upregulated Bax at both m RNA and protein levels.Mi R-573 overexpression played an opposite role and reduce the effects of GAS8-AS1 overexpression.Cell apoptosis assays showed increase apoptotic rate of ESCC cells after GAS8-AS1 and Bax overexpression.MiR-573 overexpression led to attenuated effects of GAS8-AS1 overexpression.Therefore,GAS8-AS1 may promoted ESCC apoptosis by sponging miR-573 to upregulate Bax.ConclusionsIn this study,lncRNA GAS8-AS1 was found to have low expression in ESCC,and inhibited the proliferation,survival and formation of transplanted tumor by promoting the expression of apoptotic protein BAX.lncRNA GAS8-AS1 promoted ESCC apoptosis by sponging miR-573 to upregulate Bax.Therefore,lncRNA GAS8-AS1 could be used as a potential molecular marker for clinical treatment and prognosis evaluation of ESCC. |
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