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微小RNA-433-3p靶向滑蛋白调控胰腺癌PANC-1细胞恶性生物学行为的研究

MicroRNA-433-3p regulates malignant biological behavior of pancreatic cancer PANC-1 cells by targeting Smoothened

    摘要
  • 摘要:
    目的 探讨微小RNA-433-3p(miR-433-3p)对胰腺癌细胞增殖、侵袭、迁移的影响及其与滑蛋白(SMO)的靶向关系。
    方法 培养胰腺癌PANC-1细胞、人正常胰腺上皮细胞HPNE。将miR-NC、miR-433-3p mimics、si-NC、si-miR-433-3p、敲低空载Scramble、si-SMO、Vector、SMO过表达(OE-SMO)质粒分别转染至PANC-1细胞，设为miR-NC组、miR-433-3p组、si-NC组、si-miR-433-3p组、Scramble组、si-SMO组、Vector组及SMO组。将Vector、OE-SMO质粒分别转染至miR-433-3p组细胞，设为si-miR-433-3p+Scramble组、si-miR-433-3p+si-SMO组。采用四甲基偶氮唑蓝(MTT)检测细胞增殖能力, Transwell实验检测细胞侵袭、迁移能力，荧光定量聚合酶链反应(RT-PCR)检测细胞SMO基因mRNA及miR-433-3p表达水平, Western blot检测细胞SMO蛋白表达水平, TargetScan在线网站预测miR-433-3p与SMO结合位点，双荧光素酶报告基因实验验证miR-433-3p与SMO的靶向关系。
    结果 PANC-1细胞SMO基因mRNA及其蛋白表达水平高于HPNE细胞，差异有统计学意义(P < 0.05); PANC-1细胞miR-433-3p表达水平低于HPNE细胞，差异有统计学意义(P < 0.05)。下调miR-433-3p、上调SMO可促进细胞的增殖、侵袭及迁移，上调miR-433-3p、沉默SMO可抑制细胞的增殖、侵袭及迁移。下调SMO逆转了miR-433-3p低表达对细胞增殖、侵袭及迁移的促进作用。miR-433-3p可负性调控SMO表达。
    结论 miR-433-3p负性调控SMO抑制胰腺癌细胞的增殖、侵袭及迁移。

     

    Abstract:
    Objective To explore the effect of microRNA-433-3p (miR-433-3p) on proliferation, invasion and migration of pancreatic cancer cells and its targeting relationship with Smoothened (SMO).
    Methods Pancreatic cancer PANC-1 cells and human normal pancreatic epithelial cells HPNE were cultured. The miR-NC, miR-433-3p mimics, si-NC, si-miR-433-3p, Scramble (knocked down and empty loaded), si-SMO, Vector and SMO overexpression (OE-SMO) plasmids were transfected into PANC-1 cells respectively, which were recorded as miR-NC group, miR-433-3p group, si-NC group, si-miR-433-3p group, Scramble group, si-SMO group, Vector group and SMO group. Vector and OE-SMO plasmids were transfected into cells in the miR-433-3p group respectively, and were recorded as si-miR-433-3p+Scramble group and si-miR-433-3p+si-SMO group. Tetramethylazo blue (MTT) was used to detect cell proliferation ability, the Transwell test was used to detect cell invasion and migration abilities, the fluorescent quantitative polymerase chain reaction (RT-PCR) was used to detect the expression levels of cell SMO gene mRNA and miR-433-3p, the Western blot was used to detect the expression level of cell SMO protein, the TargetScan online website was used to predict the binding site of miR-433-3p and SMO, and double luciferase reporter gene test was used to verify the targeting relationship between miR-433-3p and SMO.
    Results The expression levels of mRNA and protein of SMO gene in PANC-1 cells were significantly higher than those in HPNE cells (P < 0.05); the expression level of miR-433-3p in PANC-1 cells was significantly lower than that in HPNE cells (P < 0.05). Down-regulation of miR-433-3p and up-regulation of SMO were able to promote cell proliferation, invasion and migration, while up-regulation of miR-433-3p and silencing SMO were able to inhibit cell proliferation, invasion and migration. Down-regulation of SMO reversed the promotion of low expression of miR-433-3p on cell proliferation, invasion and migration. The miR-433-3p was able to negatively regulate SMO expression.
    Conclusion The miR-433-3p can negatively regulate SMO to inhibit the proliferation, invasion and migration of pancreatic cancer cells.

     

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