微小RNA-1291对胃癌细胞增殖、侵袭和迁移的影响及机制研究

Effect of microRNA-1291 on cell proliferation, invasion and migration of gastric cancer cells

  • 摘要:
    目的 探讨微小RNA-1291(miR-1291)对胃癌细胞增殖、迁移和侵袭能力的影响及机制。
    方法 通过公共数据库分析miR-1291在胃癌组织、正常胃组织中的表达。培养人胃癌细胞SGC-7901、人胃黏膜上皮细胞GES-1, 比较miR-1291表达水平。将miR-1291 mimic、miR-NC、miR-1291 inhibitor、miR inhibitor质粒分别转染至SGC-7901细胞,设为miR-1291组、miR-NC组、miR-1291 inhibitor组、miR inhibitor组。将BMP激活素膜结合抑制因子(BAMBI) mimic质粒、Vector质粒、sh-BAMBI质粒、sh-NC质粒分别转染至SGC-7901细胞中,设为BAMBI组、Vector组、sh-BAMBI组、sh-NC组。采用CCK-8法检测细胞增殖能力,采用Transwell实验检测细胞侵袭和迁移能力,采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-1291和BAMBI mRNA表达水平,采用蛋白质印迹法(Western blot)检测BAMBI、转化生长因子-β(TGF-β)、SMAD同源物4(SMAD4)蛋白水平,通过双荧光素酶报告基因实验检测miR-1291和BAMBI的靶向关系。
    结果 胃癌组织miR-1291表达水平为(0.85±0.11), 低于正常胃组织的(2.02±0.23), 差异有统计学意义(t=18.33, P < 0.01)。SGC-7901细胞miR-1291表达水平低于GES-1细胞, BAMBI蛋白、BAMBI mRNA表达水平高于GES-1细胞,差异有统计学意义(P < 0.01)。miR-1291组细胞72 h时光密度(OD)值、侵袭细胞数、迁移细胞数低于或少于miR-NC组, miR-1291 inhibitor组细胞72 h时OD值、侵袭细胞数、迁移细胞数高于或多于miR inhibitor组,差异有统计学意义(P < 0.01)。BAMBI组细胞72 h时OD值、侵袭细胞数、迁移细胞数高于或多于Vector组, sh-BAMBI组细胞72 h时OD值、侵袭细胞数、迁移细胞数低于或少于sh-NC组,差异有统计学意义(P < 0.01)。miR-1291组细胞BAMBI蛋白、BAMBI mRNA表达水平低于miR-NC组, miR-1291 inhibitor组细胞BAMBI蛋白、BAMBI mRNA表达水平高于miR inhibitor组,差异有统计学意义(P < 0.01)。双荧光素酶报告基因实验结果显示, miR-1291与BAMBI存在靶向关系。BAMBI组TGF-β、SMAD4蛋白水平低于Vector组, sh-BAMBI组TGF-β、SMAD4蛋白水平高于sh-NC组,差异有统计学意义(P < 0.01)。
    结论 miR-1291可抑制胃癌细胞增殖、迁移和侵袭能力,其机制可能与负性调控BAMBI表达进而抑制TGF-β通路有关。

     

    Abstract:
    Objective To investigate the effects and mechanism of microRNA-1291(miR-1291) on the cell proliferation, migration and invasion of gastric cancer cells.
    Methods The public database was used to analyze the expression of miR-1291 in gastric cancer and normal gastric tissues. Human gastric cancer SGC-7901 cells and human gastric mucosal epithelial cells GES-1 cells were selected, and the levels of miR-1291 expression were compared. The miR-1291 mimic, miR-NC, miR-1291 inhibitor and miR inhibitor plasmids were transfected into SGC-7901 cells, and these cells were divided into miR-1291 group, miR-NC group, miR-1291inhibitor group, miR inhibitor group. BMP activator membrane-bound inhibitor (BAMBI) mimic plasmid, Vector plasmid, sh-BAMBI plasmid, and sh-NC plasmid were transfected into cells, and were set as BAMBI group, Vector group, sh-BAMBI group, and sh-NC group. CCK-8 assay was applied to detect cell proliferation ability; transwell assays were applied to detect cell invasion and migration ability; quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) was applied to detect miR-1291 and BAMBI mRNA levels; Western blot was applied to detect BAMBI, transforming growth factor β (TGF-β) and SMAD homolog 4 (SMAD4) protein levels; dual luciferase reporter gene assay was applied to detect the targeting relationship between miR-1291 and BAMBI.
    Results The expression level of miR-1291 in gastric cancer tissues was (0.85±0.11), which was lower than (2.02±0.23) in normal gastric tissues (t=18.33, P < 0.001). The expression level of miR-1291 in SGC-7901 cells was lower than that in GES-1 cells, and the levels of BAMBI protein and BAMBI mRNA were higher than that in GES-1 cells (P < 0.01). The optical density (OD) value, invasion and migration cell numbers in the miR-1291 group at 72 h were lower or less than those in the miR-NC group, and miR-1291 inhibitor group had higher or more OD value, and the number of invasion and migration cells at 72 h than miR inhibitor group (P < 0.01). The OD value and the number of invasion and migration cells at 72 h were higher or more in the BAMBI group than Vector group, and were lower or less in the sh-BAMBI group than those in the sh-NC group (P < 0.01). The expression levels of BAMBI protein and BAMBI mRNA in cells of miR-1291 group were lower than those of miR-NC group, while the expression levels of BAMBI protein and BAMBI mRNA in cells of miR-1291 inhibitor group were higher than those of miR inhibitor group (P < 0.01). The results of double luciferase reporter gene experiment showed that miR-1291 had a targeting relationship with BAMBI. The protein levels of TGF-β and SMAD4 in BAMBI group were lower than those in the Vector group, and the protein levels of TGF-β and SMAD4 in sh-BAMBI group were higher than those in the sh-NC group, the difference was statistically significant (P < 0.01).
    Conclusion MiR-1291 can inhibit the proliferation, migration and invasion ability of gastric cancer cells, and the mechanism may be related to the upregulation of BAMBI expression, thereby inhibiting TGF-β pathway.

     

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