长链非编码RNA SCAMP1-AS1通过靶向调控微小RNA-483-5p对食管癌细胞增殖和迁移的影响

The effect of long-chain noncoding RNA SCAMP1-AS1 on the proliferation and migration of esophageal cancer cells by targeting and regulating miR-483-5p

目的观察长链非编码RNA(lncRNA)SCAMP1-AS1在食管癌组织中的表达,探讨SCAMP1-AS1对食管癌细胞增殖和迁移的影响及可能的分子机制。方法采用实时荧光定量聚合酶链反应(RT-qPCR)检测鄂东医疗集团黄石市中心医院2017年3月至2020年8月手术切除的37例食管癌组织及癌旁组织、4种食管癌细胞(EC9706、TE-13、KYSE30、Eca109)及正常食管上皮细胞HET-1A中SCAMP1-AS1的表达水平。选择表达最低的细胞,以阴性对照慢病毒(LV-NC)感染为对照组,以携带SCAMP1-AS1序列的重组慢病毒(LV-SCAMP1-AS1)感染作为实验组。RT-qPCR检测感染后食管癌细胞中SCAMP1-AS1的表达。细胞计数试剂盒8(CCK-8)和Transwell小室法分别检测食管癌细胞的增殖能力和迁移能力。生物信息学方法预测SCAMP1-AS1的靶基因,双荧光素酶报告实验验证SCAMP1-AS1与靶基因的相互作用。RT-qPCR检测靶基因的表达,Western blotting检测细胞增殖和迁移表型蛋白的表达。结果食管癌组织中SCAMP1-AS1的相对表达水平明显低于癌旁组织(1.26±0.48比8.03±1.17),差异有统计学意义(P<0.01)。食管癌细胞EC9706、TE-13、KYSE30、Eca109中SCAMP1-AS1的相对表达水平均低于正常食管上皮细胞(0.54±0.05、0.14±0.02、0.46±0.07、0.77±0.05比1.00±0.06),差异有统计学意义(P<0.05),TE-13细胞中SCAMP1-AS1的表达最低(P<0.01)。与对照组比较,实验组TE-13细胞中SCAMP1-AS1的表达上调(P<0.01),细胞的增殖能力降低(P<0.01),细胞的迁移能力降低(P<0.01)。miR-483-5p是SCAMP1-AS1的直接作用靶点(P<0.01)。与对照组比较,实验组TE-13细胞中miR-483-5p的表达下调(P<0.01),细胞增殖和迁移表型蛋白表达降低。结论lncRNA SCAMP1-AS1在食管癌中表达下调,SCAMP1-AS1可通过靶向调控miR-483-5p的表达抑制食管癌TE-13细胞的增殖能力和迁移能力,SCAMP1-AS1有望成为食管癌潜在的分子治疗靶点。

Objective To observe the expression of long-chain noncoding RNA(lncRNA)SCAMP1-AS1 in esophageal cancer tissues,and explore the effect of SCAMP1-AS1 on the proliferation and migration of esophageal cancer cells and the possible molecular mechanism.Methods Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression level of SCAMP1-AS1 in 37 cases of esophageal cancer tissues and adjacent tissues surgically resected in Huangshi Central Hospital of Edong Medical Group from March 2017 to August 2020.RT-qPCR was also used to detect the expression level of SCAMP1-AS1 in 4 types of esophageal cancer cells(EC9706,TE-13,KYSE30,Eca109)and normal esophageal epithelial cells HET-1A.The cells with the lowest expression were selected,the negative control lentivirus(LV-NC)infection was used as the control group,and the recombinant lentivirus carrying SCAMP1-AS1 sequence(LV-SCAMP1-AS1)infection was used as the experimental group.RT-qPCR was used to detect the expression of SCAMP1-AS1 in esophageal cancer cells after infection.Cell counting kit 8(CCK-8)and Transwell chamber method were used to detect the proliferation and migration ability of esophageal cancer cells.Bioinformatics methods predicted the target genes of SCAMP1-AS1,and dual luciferase reporter experiments verified the interaction of SCAMP1-AS1 with target gene.RT-qPCR detected the expression of target genes.Western blotting detected the expression of cell proliferation and migration phenotype proteins.Results The relative expression level of SCAMP1-AS1 in esophageal cancer tissue was significantly lower than that in adjacent tissues(1.26±0.48 vs.8.03±1.17,P<0.01).The relative expression levels of SCAMP1-AS1 in esophageal cancer cells EC9706,TE-13,KYSE30,Eca109 were all lower than that in normal esophageal epithelial cells(0.54±0.05,0.14±0.02,0.46±0.07,0.77±0.05 vs.1.00±0.06,P<0.05),and the expression of SCAMP1-AS1 in TE-13 cells was the lowest(P<0.01).Compared with the control group,the expression of SCAMP1-AS1 in TE-13 cells in the experimental group was up-regulated(P<0.01),the proliferation ability of the cells was reduced(P<0.01),and the migration ability of the cells was reduced(P<0.01).miR-483-5p was the direct target of SCAMP1-AS1.Compared with the control group,the expression of miR-483-5p was down-regulated in TE-13 cells in the experimental group(P<0.01),and the expression of cell proliferation and migration phenotype proteins was down-regulated.Conclusions The expression of lncRNA SCAMP1-AS1 is down-regulated in esophageal cancer.SCAMP1-AS1 can inhibit the proliferation and migration of esophageal cancer TE-13 cells by targeting the expression of miR-483-5p.SCAMP1-AS1 is expected to become a potential molecular therapeutic target for esophageal cancer.