南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (01): 61-66.doi: 10.12122/j.issn.1673-4254.2020.01.09

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miR-145-5p靶向双特异性磷酸酶6对人子宫内膜癌增殖、迁移、侵袭与凋亡的影响

门颖超，张 蕾，艾 浩   

  • 出版日期:2020-02-19 发布日期:2020-01-20
  • 基金资助:

MicroRNA-145-5p over-expression suppresses proliferation, migration and invasion and promotes apoptosis of human endometrial cancer cells by targeting dual specific phosphatase 6

  

  • Online:2020-02-19 Published:2020-01-20

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摘要： 目的 探讨微小RNA-145-5p（miR-145-5p）靶向双特异性磷酸酶6（DUSP6）对于人子宫内膜癌Ishikawa细胞增殖、迁移、侵袭与凋亡能力的影响。方法 人子宫内膜癌Ishikawa细胞分为A组（未转染组）、B组（mimic NC组）、C组（mimic组）、D组 （inhibitor NC组）、E组（inhibitor组），采用脂质体（Lipo 2000）细胞转染法转染。利用RT-PCR技术验证miR-145-5p的表达；过表达或抑制表达miR-145-5p对Ishikawa细胞增殖、迁移、侵袭及凋亡影响将运用MTT、伤口愈合、Transwell及凋亡实验检测；运用生物信息学方法预测miR-145-5p的潜在靶基因；运用RT-PCR及Western Blot技术检测细胞转染后下游靶基因DUSP6mRNA及蛋白的表达水平。结果 转染后48、72、96 h，C组比B组细胞增殖能力下降，E组比D组细胞的增殖能力增强，差异有统计学意义（P<0.05）。转染后24、48 h，C组比B组细胞迁移能力降低，E组比D组细胞迁移能力增强（P<0.05）。转染48 h后，C组比B组侵袭能力下降，E组比D组侵袭能力增强,差异有统计学意义（P<0.05）。转染48 h后，A组、B组、C组、D组、E组凋亡率分别为（0.66±0.05）%、（0.60±0.03）%、（17.74±1.02）%、（0.72±0.04）%、（0.31±0.02）%，差异有统计学意义（P<0.05）。经生物信息学在线软件预测DUSP6是miR-145-5p的潜在靶基因。转染48 h后，DUSP6 mRNA在A组、B组、C组、D组、E组中的相对表达水平分别为1.00±0.09、1.09±0.08、0.47±0.04、1.03±0.05、4.62±0.26，差异有统计学意义（P<0.05）。其蛋白的相对表达量为0.29± 0.03、0.28±0.04、0.16±0.03、0.32±0.05、0.74±0.05，差异有统计学意义（P<0.05）。结论 miR-145-5p过表达后能够抑制子宫内膜癌细胞增殖、迁移、侵袭并且促进癌细胞凋亡，其机制可能是通过负向调节DUSP6的表达而实现。

Abstract: Objective To investigate the role of microRNA-145-5p (miR-145-5p) in regulating the proliferation, migration, invasion and apoptosis of human endometrial carcinoma cells. Methods Human endometrial carcinoma Ishikawa cells were transfected with miR-145-5p mimic, miR-145-5p inhibitor, or their negative controls via liposome (Lipo2000), and the changes in the expression of miR-145-5p was verified by real-time PCR. The effects of overexpression or inhibition of miR-145-5p on the proliferation, migration, invasion and apoptosis of the cells were evaluated using MTT assay, wound healing assay, Transwell assay or flow cytometry. Bioinformatic analysis was performed to predict the target genes of miR-145-5p. The mRNA and protein expression levels of the downstream target of miR-145-5p, namely dual specific phosphatase 6 (DUSP6), were detected using real-time PCR and Western blotting. Results Transfection of the cells with miR-145-5p mimic significantly suppressed the proliferation of Ishikawa cells, while transfection with miR-145-5p inhibitor obvious enhanced the proliferation of the cells (P<0.05). Over-expression of miR-145-5p significantly suppressed the migration and invasion and promoted apoptosis of the cells, and inhibition of miR-145-5p caused the reverse changes (P<0.05). Bioinformatic analysis showed that DUSP6 was the potential target gene of miR-145-5p. Over-expression of miR-145-5p significantly lowered while inhibition of miR-145-5p significantly enhanced the expression of DUSP6 protein (P<0.05). Conclusion Overexpression of miR-145-5p inhibits the proliferation, migration and invasion and promotes apoptosis of endometrial cancer cells possibly by negative regulation of DUSP6 expression.