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Title: [Mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1 induced invasion and metastasis of esophageal cancer cell EC-109].
Author: Zhang QQ, Cui YH, Wang Y, Kou WZ, Cao F, Cao XJ, Miao ZH, Kang XH.
Journal: Zhonghua Zhong Liu Za Zhi; 2017 Jun 23; 39(6):405-411. PubMed ID: 28635228.
Abstract:
Objective: To investigate the effect and mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1, (LncRNA-MALAT1) on invasion and metastasis of esophageal cancer cell EC-109. Methods: EC-109 cells were transfected with lentiviral vector carrying short hairpin RNA of MALAT1( shRNA-MALAT1) or a nonspecific shRNA control (shRNA-control). The expressions of MALAT1, microRNA-200a, ZEB1 and ZEB2 were detected by qRT-PCR. The effect of shRNA-MALAT1 on invasion of EC-109 cells was determined by transwell assay. The expressions of components of epithelial-msenchymal transition pathway in EC-109 cells were determined by immunofluorescence array and western blotting. The expression relationship between MALAT1 and miR-200a in EC-109 cells was detected by dual-luciferase reporter assay. Results: The result of qRT-PCR showed that the expressions levels of MALAT1, ZEB1 and ZEB2 in shRNA-MALAT1 group were 0.43±0.06, 0.64±0.04 and 0.51±0.04, respectively, significantly lower than 0.97±0.08, 1.06±0.07 and 0.98±0.05 in shRNA-control group and 1 in control group, respectively(all P<0.05). Transwell assay showed that the number of invaded cells in shRNA MALAT1 group was (96.81±10.43) per low-power field, markedly lower than that of (278.44±13.28) per low-power field in shRNA-control group (P<0.01). Immunofluorescence staining and Western blotting showed that MALAT1 downregulation significantly reduced the expressions of proteins related to EMT signal pathway in EC-109 cells.Dual luciferase reporter assay showed that compared to negative control, the activities of luciferase reporter in EC-109 cells co-transfected with pmirGLO-MALAT1-wt and miR-200a were significantly down-regulated. While co-transfected pmirGLO-MALAT1-mut with miR-200a mimics had no effect on the luciferase reporter activities of MALAT1. Conclusion: LncRNA MALAT1 functions as a competing endogenous RNA to regulate the expressions of ZEB1 and ZEB2 by sponging miR-200a and promotes invasion and migration of esophageal cancer cells through inducing epithelial-mesenchymal transition. 目的： 观察长链非编码RNA－肺腺癌转移相关转录子1(LncRNA－MALAT1)对食管癌细胞EC－109侵袭和转移的影响，并探讨其可能的作用机制。 方法： 应用慢病毒载体shRNA对照、shRNA－MALAT1转染EC－109细胞，采用实时荧光定量PCR法检测EC－109细胞中LncRNA－MALAT1、miR－200a、ZEB1和ZEB2 mRNA的表达；采用细胞侵袭实验检测EC－109细胞的侵袭能力；采用免疫荧光、Western blot法检测EC－109细胞中上皮间质转化(EMT)信号通路相关蛋白的表达；采用双荧光素酶报告基因实验检测EC－109细胞中MALAT1与miR－200a表达的关系。 结果： 实时荧光定量PCR检测结果显示，未转染组、转染shRNA对照组和转染shRNA－MALAT1组EC－109细胞中MALAT1 mRNA的相对表达量分别为1.00、0.97±0.08和0.43±0.06，转染shRNA－MALAT1组EC－109细胞中MALAT1 mRNA的表达明显降低(P<0.01)。未转染组、转染shRNA对照组和转染shRNA－MALAT1组EC－109细胞中ZEB1 mRNA的相对表达量分别为1.00、1.06±0.07和0.64±0.04，ZEB2 mRNA的相对表达量分别为1.00、0.98±0.05和0.51±0.04，转染shRNA－MALAT1组EC－109细胞中ZEB1和ZEB2 mRNA的相对表达量均明显降低(均P<0.05)。转染shRNA－MALAT1组的EC－109细胞的穿膜细胞数为(96.81±10.43)个/低倍视野，与转染shRNA对照组EC－109细胞的穿膜细胞数[(278.44±13.28)个/低倍视野]比较，差异有统计学意义(P<0.01)。免疫荧光和Western blot法检测结果显示，沉默EC－109细胞中MALAT1基因的表达可下调EMT信号通路相关蛋白的表达。双荧光素酶报告基因实验结果显示，与转染miRNA阴性对照组比较，共转染pmirGLO－MALAT1－wt和miR－200a模拟物可明显减少EC－109细胞的荧光素酶活性，而共转染pmirGLO－MALAT1－mut和miR－200a模拟物不能抑制EC－109细胞的荧光素酶活性。 结论： LncRNA－MALAT1可能作为竞争性内源RNA，竞争性结合miR－200a，从而上调EC－109细胞中ZEB1和ZEB2的表达，促进EMT进程，导致食管癌的侵袭和转移。.

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