miR-152靶向下调DNMT1表达对宫颈癌细胞株Siha侵袭转移的影响

Effect of miR-152 on invasion and metastasis of the Siha cervical cancer cell line by targeted down-regulation of DNMT1 expression

目的研究miR-152靶向下调DNA甲基化转移酶1(DNMT1)表达对宫颈癌细胞株Siha侵袭转移的影响。方法采用实时荧光定量PCR(qRT-PCR)检测miR-152在宫颈癌细胞株Siha及人正常宫颈上皮细胞HUCEC中的表达水平;体外培养Siha细胞,随机分为对照组、miR-152 mimics阴性对照组、miR-152 mimics组,分组转染后采用CCK-8试验检测细胞增殖情况,采用Transwell侵袭试验和细胞划痕试验检测细胞侵袭转移情况,采用qRT-PCR检测miR-152及DNMT1 mRNA水平,采用Western blot检测DNMT1和上皮-间质转化(EMT)相关蛋白表达水平。结果与HUCEC细胞相比,miR-152在Siha细胞中[(0.43±0.08)比(1.02±0.25)]表达水平明显下降(P<0.05);与对照组相比,miR-152 mimics组细胞miR-152[(2.61±0.47)比(1.00±0.21)]和E-cadherin表达水平[(0.85±0.16)比(0.42±0.07)]显著升高(均P<0.05),相对生存率[(45.53±10.15)%比(100)%]、迁移细胞数[(28.86±4.91)个比(387.75±56.43)个]、侵袭细胞数[(108.54±19.53)个比(273.68±47.36)个]、DNMT1 mRNA[(0.46±0.09)比(1.01±0.23)]及DNMT1[(0.28±0.05)比(1.01±0.22)]和Vimentin蛋白表达[(0.12±0.03)比(0.94±0.15)]水平均显著降低(均P<0.05)。miR-152 mimics阴性对照组细胞各指标均无显著变化(均P>0.05)。结论 miR-152在宫颈癌细胞株Siha中低表达,上调其表达可下调DNMT1表达,抑制癌细胞的增殖,降低癌细胞的侵袭转移能力。

Objective To investigate the effect of miR-152 on the invasion and metastasis of the Siha cervical cancer cell line as a result of targeted down-regulation of the expression of DNA methyltransferase 1(DNMT1). Methods Real-time quantitative PCR(qRT-PCR) was used to determine the level of expression of miR-152 in the Siha cervical cancer cell line and HUCEC normal human cervical epithelial cells. Siha cells were cultured in vitro and randomly divided into a control group, a miR-152 mimic-negative control group, and a miR-152 mimic group. After the groups of cells were transfected, the CCK-8 assay was used to detect cell proliferation. The Transwell invasion assay and the cell scratch assay were used to detect invasion and metastasis in each group. qRT-PCR was used to detect the levels of miR-152 and DNMT1 mRNA. Western blotting was used to detect the level of expression of proteins related to DNMT1 and EMT. Results The level of expression of miR-152 in Siha cells was significantly lower than that in HUCEC cells(0.43±0.08 vs. 1.02±0.25, P<0.05). The level of expression of miR-152(2.61±0.47 vs. 1.00±0.21) and the level of expression of E-cadherin(0.85±0.16 vs. 0.42±0.07) were significantly higher in the miR-152 mimic group than in the control group(P<0.05). The relative survival rate(45.53%±10.15% vs. 100%), the number of migrating cells(28.86±4.91 vs. 387.75±56.43), the number of invading cells(108.54±19.53 vs. 273.68±47.36), the level of DNMT1 mRNA(0.46±0.09 vs. 1.01±0.23), and the level of expression of the proteins DNMT1(0.28±0.05 vs. 1.01±0.22) and vimentin(0.12±0.03 vs. 0.94±0.15) were markedly lower(P<0.05). There were no marked changes in cell indices in the miR-152 mimic-negative control group(P>0.05). Conclusion MiR-152 is down-regulated in the Siha cervical cancer cell line. Up-regulation of miR-152 can down-regulate the expression of DNMT1, inhibit its proliferation, and reduce the invasion and metastasis of cancer cells.