miR-324-3P对宫颈癌细胞增殖、侵袭和转移的作用及其机制研究

Effect and mechanism of miR-324-3P on proliferation, invasion and metastasis of cervical cancer cells

  • 摘要:
      背景   miR-324-3P在多种恶性肿瘤中发挥着重要作用,调控Wnt/β-catenin信号通路可能是其参与肿瘤进展的重要途径,miR-324-3P在宫颈癌中的具体作用尚不清楚。
      目的   研究miR-324-3P对宫颈癌细胞增殖、侵袭和转移的作用及其机制。
      方法   收集入住我院宫颈癌患者的宫颈癌组织和正常宫颈组织各40例,TargetScan7.1预测软件及双荧光素酶报告基因试验检测miR-324-3P对WNT3a基因的靶向调控作用。实时荧光定量PCR(qRT-PCR)检测宫颈癌组织和正常宫颈组织中miR-324-3P、WNT3a mRNA的表达水平。Pearson法分析宫颈癌组织中miR-324-3P与WNT3a表达的相关性。常规培养宫颈癌细胞(HeLa细胞),将其分为对照组(NC组)、miR-324-3P mimic组和miR-324-3P mimic+oe-WNT3a组,采用Lipofectamine 2000转染试剂进行各组质粒的转染。四唑盐比色法(MTT)检测各组细胞的增殖能力,Boyden实验检测各组细胞的侵袭能力,Transwell实验检测各组细胞的转移能力。Western-blot法检测各组细胞中WNT3a和β-catenin的表达。
      结果   宫颈癌组织中miR-324-3P、WNT3a的相对表达量分别为0.79±0.39、1.46±0.53,与正常宫颈组织相比,miR-324-3P表达降低、WNT3a表达升高。Targetscan7.1预测软件、双荧光素酶报告基因试验及qRT-PCR表明miR-324-3P靶向调控WNT3a,且Pearson分析显示二者呈负相关。与NC组相比,miR-324-3P mimic组和miR-324-3P mimic+oe-WNT3a组宫颈癌细胞增殖、侵袭和转移能力降低。与miR-324-3P mimic 组相比,miR-324-3P mimic+oe-WNT3a组宫颈癌细胞增殖、侵袭和转移能力增加,WNT3a削弱miR-324-3P对宫颈癌细胞增殖、侵袭和转移能力的抑制作用。与NC组相比,miR-324-3P mimic组和miR-324-3P mimic+oe-WNT3a组细胞中WNT3a和β-catenin表达降低。而与miR-324-3P mimic 组相比,miR-324-3P mimic+oe-WNT3a组WNT3a和β-catenin蛋白表达增加。WNT3a削弱miR-324-3P对β-catenin的抑制作用。
      结论   miR-324-3P靶向调控WNT3a表达,抑制宫颈癌细胞增殖、侵袭和转移能力。

     

    Abstract:
      Background   MicroRNA (miR)-324-3P plays an important role in a variety of malignant tumors, regulation of Wnt/β- catenin signaling pathway may be an important pathway involved in tumor progression, while the specific role of miR-324-3P in cervical cancer remains unclear.
      Objective   To study the effect of microRNA (miR)-324-3P on the proliferation, invasion and metastasis of cervical cancer cells and its mechanism.
      Methods   Forty cases of cervical cancer tissues and normal cervical tissues were collected from patients with cervical cancer admitted to our hospital. TargetScan 7.1 prediction software and double luciferase reporter gene test were used to detect the targeting regulation of miR-324-3P on WNT3a gene. Real time fluorescent quantitative PCR (QRT PCR) was used to detect expression of miR-324-3P and WNT3a mRNA in cervical cancer tissues and normal cervical tissues. Pearson was used to analyze the correlation between miR-324-3P and WNT3a expression in cervical cancer tissues. HeLa cells were routinely cultured and divided into control group (NC group), miR-324-3P mimic group and miR-324-3P mimic+oe-WNT3a group. Lip2000 was used to transfect the plasmids of each group, Tetrazolium salt colorimetry (MTT) test was used to detect the cell proliferation ability, Boyden test was used to detect the invasion ability, Transwell test was used to detect the metastasis ability, and Western blot (Western-blot) was used to detect the expression of WNT3a and β-catenin in each group.
      Results   The relative expression levels of miR-324-3P and WNT3a were (0.79±0.39) and (1.46±0.53), respectively, compared with normal cervical tissues, the expression of miR-324-3P decreased, and the expression of WNT3a increased. Targetscan7.1 prediction software, dual luciferase reporter assay and qRT-PCR showed that miR-324-3P targeted WNT3a, and Pearson analysis showed that there was a negative correlation between them. Compared with the NC group, the proliferation, invasion and metastasis ability of cervical cancer cells in the miR-324-3P mimic group and the miR-324-3P mimic+oe-WNT3a group were reduced. Compared with the miR-324-3P mimic group, the proliferation, invasion and metastasis ability of cervical cancer cells in the miR-324-3P mimic+oe-WNT3a group were increased, and WNT3a attenuated the effect of miR-324-3P on the inhibition proliferation, invasion and metastasis ability of cervical cancer cells. Compared with NC group, the expressions of WNT3a and β-catenin decreased in miR-324-3P mimic group and miR-324-3P mimic+oe-WNT3a group. Compared with miR-324-3P mimic group, the expression of WNT3a and β-catenin protein increased in miR-324-3P mimic+oe-WNT3a group. WNT3a attenuated the inhibitory effect of miR-324-3P on β-catenin.
      Conclusion   miR-324-3P targets WNT3a expression and inhibits the proliferation, invasion and metastasis of cervical cancer cells.

     

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