| Background and purpose:Esophageal cancer was one of the most common malignant tumors in China.The main pathological type was esophageal squamous cell carcinoma(ESCC).Although the diagnosis and treatment techniques of ESCC had been gradually improved in recent years,the overall survival of patients was still short and the treatment effect was not good.The molecular mechanism of ESCC development had not yet been fully elucidated.Abnormal expression of long-chain non-coding RNA(LncRNA)was closely related to many malignant tumors such as ESCC.Long-chain non-coding RNA X chromosome inactivation-specific transcript gene(LncRNA XIST)may play an oncogene role in the development and progression of esophageal cancer.But its specific mechanism of action in the development and development of ESCC was still not fully understood.Therefore,this study was to investigate the mechanism of action of LncRNA XIST in the regulation of ESCC development,and further analyze its interaction with micro RNA-129-5p(miR-129-5p)/cyclin D1(CCND1)during ESCC development and progression.The role of the relationship,in order to reveal the pathogenesis of ESCC,help guide the clinical development of ESCC treatment options.This subject included the following three parts: Part Ⅰ: si-XIST inhibits the growth of ESCC cells;Part Ⅱ: XIST/miR-129-5p regulates the mechanism of ESCC cell growth;Part Ⅲ: XIST regulates miR-129-5p regulates the expression of CCND1.Part Ⅰ: si-XIST inhibits the growth of ESCC cellsMETHODS:A total of 42 patients with esophageal cancer who were admitted to our hospital from May 2013 to December 2013 were enrolled in the esophageal cancer group.All subjects underwent resection of esophageal cancer tissues and adjacent tissues.In esophageal cancer tissues and adjacent tissues,real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression level of LncRNA XIST.According to the detection results,LncRNA XIST was divided into high expression group(23 cases)and low expression group(19 cases).At the same time,the pathological and clinical data of patients with esophageal cancer were collected,and the relationship between the expression of LncRNA XIST and the clinicopathological features of patients was compared.All patients with esophageal cancer were followed up for 5 years.Kaplan-Meier method was used to analyze the relationship between LncRNA XIST expression and prognosis.The human esophageal epithelial cells Het-1A and esophageal squamous cell carcinoma cell lines EC9706,KYSE510,KYSE450,Eca-109 and qRT-PCR were cultured in vitro to detect the expression of XIST in different cells.Si-NC and si-XIST were transfected into esophageal squamous cell carcinoma EC9706 and Eca-109 cells by lipofection.The cell proliferation activity of each group after transfection was detected by MTT assay.The cell cycle changes of each group after transfection were detected by flow cytometry.Transwell cells were used to detect the migration and invasion ability of each group after transfection.The Annexin V-FITC/PI double staining technique was used to detect the apoptosis of each group after transfection.RESULTS:Compared with normal tissues adjacent to the cancer,the expression level of LncRNA XIST in esophageal cancer tissues was significantly increased(P<0.001).XIST expression was positively correlated with clinical stage of patients with esophageal cancer.The survival rate of patients with XIST low expression group was significantly higher than that of patients with high expression group(P<0.05).Compared with Het-1A cells,the expression levels of XIST in esophageal cancer cells EC9706,KYSE510,KYSE450 and Eca-109 were significantly increased(P<0.01),and the expression levels of XIST in esophageal cancer EC9706 and Eca-109 were increased significantly,so EC9706 and Eca-109 cells were selected as research materials in subsequent experiments.The expression level of XIST in esophageal cancer EC9706 and Eca-109 cells was significantly lower than that of transfected si-NC(P<0.05).Compared with the si-NC group,the cell proliferation ability of the si-XIST group was significantly decreased(P<0.05),the proportion of cells in the G0/G1 phase was increased significantly,while the proportion of cells in the S phase was decreased significantly,and the number of cell migration and invasion were significantly reduced(P<0.05),the apoptotic rate was significantly increased(P<0.05).Conclusion:XIST is highly expressed in esophageal carcinoma tissues and cells.The high expression level is related to the clinicopathological features and prognosis of patients.The silent of XIST expression can inhibit the proliferation,invasion and migration of esophageal cancer cells and induce apoptosis and inhibit esophageal cancer development process.Part Ⅱ: XIST/miR-129-5p regulates the mechanism of ESCC cell growthMETHODS:Esophageal squamous cell carcinoma EC9706 and Eca-109 cells were transfected with miR-NC and miR-129-5p mimics,respectively,into miR-NC group and miR-129-5p group;transfected empty plasmids(Vector group)and XIST Expression plasmid(XIST group);si-XIST and anti-miR-NC were co-transfected into esophageal squamous cell carcinoma EC9706,Eca-109 cells(si-XIST+anti-miR-NC group),si-XIST and anti-miR-129-5p was co-transfected into esophageal squamous cell carcinoma EC9706,Eca-109 cells(si-XIST+anti-miR-129-5p group).Dual luciferase reporter assay validated the targeting relationship between XIST and miR-129-5p,and RIP assay verified the targeted binding relationship between XIST and miR-129-5p.qRT-PCR was used to detect the expression of miR-129-5p in esophageal carcinoma tissues and different cell lines.The relationship between miR-129-5p expression and prognosis was analyzed by Kaplan-Meier method.The Pearson method was used to analyze the correlation between miR-129-5p and XIST expression levels in ESCC tissues.MTT assay and Transwell assay were used to detect the proliferation,migration and invasion of each group.Flow cytometry was used to detect apoptosis and cell cycle changes.RESULTS:The dual luciferase reporter assay demonstrated that XIST binds to miR-129-5p and regulates its activity.RIP experiments demonstrated that endogenous XIST can directly target miR-129-5p.Compared with normal tissues adjacent to the cancer,the expression level of miR-129-5p was significantly decreased in esophageal cancer tissues(P<0.01).Pearson analysis showed that miR-129-5p was significantly negatively correlated with XIST(P<0.05).The overall survival rate of esophageal cancer patients with miR-129-5p high expression group was significantly higher than that of miR-129-5p low expression group(P<0.05).Compared with Het-1A cells,the expression level of miR-129-5p was significantly decreased in different esophageal squamous carcinoma cells(P<0.05).Compared with the Vector group,the expression level of miR-129-5p in the XIST group was significantly lower(P<0.05).Compared with the si-NC group,the expression level of miR-129-5p in the si-XIST group was significantly increased(P<0.05).Compared with miR-NC group,the cell proliferation activity of miR-129-5p group was significantly decreased(P<0.05),the cell cycle G0/G1 phase cell percentage was significantly increased,while the S phase cell ratio was significantly decreased.The number of migrations and the number of invasions were significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05).Compared with the si-XIST+anti-miR-NC group,the cell proliferation activity of the si-XIST+anti-miR-129-5p group was significantly increased with the prolongation of culture time(P<0.05).The proportions of G0/G1 phase cells were significantly decreased in cell cycle,while the proportion of cells in S phase was increased significantly,the number of cell migration and invasion were increased significantly(P<0.05),and the apoptosis rate was significantly decreased(P<0.05).Conclusion:Mi R-129-5p is down-regulated in esophageal carcinoma tissues and cells.Up-regulation of miR-129-5p expression can inhibit the development of esophageal carcinoma.Silencing XIST can up-regulate the expression of miR-129-5p and inhibit the development of esophageal cancer process.Part Ⅲ: XIST regulates miR-129-5p regulates the expression of CCND1.METHODS:Si-XIST was co-transfected with anti-miR-NC and anti-miR-129-5p into esophageal squamous cell carcinoma EC9706 and Eca-109 cells,respectively,si-XIST+anti-miR-NC group,si-XIST+anti-miR-129-5p group;XIST overexpression plasmid and miR-NC,miR-129-5p mimics were co-transfected into esophageal squamous cell carcinoma EC9706,Eca-109 cells respectively XIST+miR-NC group,XIST+miR-129-5p group.Dual luciferase reporter assay and RIP assay verified the targeted binding relationship of miR-129-5p to CCND1.qRT-PCR assay was used to detect the expression of CCND1 mRNA in esophageal squamous cancer tissues and cells.The correlation between CCND1 mRNA,XIST and miR-129-5p expression levels was analyzed by Pearson method.The relationship between CCND1,XIST and miR-129-5p was detected by Western blot.The sh-XIST overexpression vector was constructed and transfected into esophageal squamous cell carcinoma EC9706 cells,and injected into the right axillary fossa of nude mice for nude mice xenograft experiments.The volume and weight of the transplanted tumor were measured.Western blot and qRT-PCR were used to detect the expression of CCND1,XIST and miR-129-5p in transplanted tumors.RESULTS:Dual luciferase reporter assay and RIP assay demonstrated that CCND1 was the target gene of miR-129-5p,and miR-129-5p binds to CCND1.The expression level of CCND1 in esophageal cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05).Compared with normal esophageal epithelial cells Het-1A,the expression level of CCND1 in ESCC cells was significantly increased(P<0.05).The expression of CCND1 mRNA was negatively correlated with the expression of miR-129-5p(P<0.05),but positively correlated with the expression level of XIST(P<0.05).XIST could positively regulate the expression of CCND1 by negatively regulating miR-129-5p expression.Compared with sh-NC group,the volume of xenografts in sh-XIST group was decreased significantly(P<0.05),the weight of transplanted tumors was significantly decreased(P<0.05),and the expression level of XIST was significantly decreased in nude mice xenografts(P<0.05),the expression level of miR-129-5p was significantly increased(P<0.05),while the expression level of CCND1 protein was significantly decreased(P<0.05).Conclusion:Silencing XIST can inhibit the growth of xenografts in esophageal squamous cell carcinoma by regulating miR-129-5p/CCND1 axis. |
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