微小RNA-425-5p靶向调控PTEN表达促进宫颈癌海拉细胞增殖、侵袭和迁移的实验研究

miR-425-5p promotes proliferation,invasion and migration of HeLa cells by targeting regulation of PTEN expression

目的探讨微小RNA(miR)-425-5p对宫颈癌海拉细胞增殖、侵袭和迁移的影响及其机制。方法2018年10月至2019年10月,利用Lipofectamine^(TM) 2000将miR-425-5p模拟物(mimics)、抑制剂(inhibitor)及其阴性对照转染至海拉细胞中,采用实时荧光定量PCR检测细胞中miR-425-5p的表达情况,MTT法检测细胞增殖活力,平板克隆形成实验检测细胞克隆形成能力,Transwell小室实验检测细胞侵袭和迁移,蛋白质印迹法(Western blotting)检测细胞中磷酸酶及张力蛋白同源基因(PTEN)蛋白的表达。采用生物信息学软件预测、双荧光素酶报告基因实验验证miR-425-5p和PTEN的靶向关系。将PTEN过表达质粒pcDNA3.1-PTEN和PTEN干扰序列siRNA-PTEN转染至海拉细胞后,观察PTEN对海拉细胞增殖活力、克隆形成能力、侵袭能力和迁移能力的影响。结果与各自阴性对照比较,miR-425-5p过表达可促进海拉细胞增殖[(142.25±11.32)%比(100.00±6.67)%]、克隆形成、侵袭[(133.28±9.86)个比(77.46±5.32)个]和迁移[(187.56±15.12)个比(115.35±10.26)个]并下调PTEN蛋白表达,而miR-425-5p低表达则抑制海拉细胞增殖[(58.38±3.45)比(100.00±5.74)]、克隆形成、侵袭[(43.32±3.62)个比(75.65±6.15)个]和迁移[(62.28±4.05)个比(109.72±9.84)个]并上调PTEN蛋白表达(P<0.05)。双荧光素酶报告基因实验证实,PTEN是miR-425-5p的靶基因;PTEN过表达可促进海拉细胞增殖[(66.52±4.36)%比(100.00±6.18)%]、克隆形成、侵袭[(46.23±3.13)个比(71.65±5.24)个]和迁移[(62.65±4.26)个比(108.42±8.57)个],而PTEN低表达则抑制海拉细胞增殖[(136.52±9.85)个比(100.00±7.05)个]、克隆形成、侵袭[(110.27±9.85)个比(70.52±6.18)个]和迁移[(168.78±16.96)个比(113.26±10.13)个]。结论miR-425-5p可通过靶向调控PTEN表达促进宫颈癌海拉细胞增殖、侵袭和迁移。

Objective To investigate the effect of miR-425-5p on the proliferation,invasion and migration of cervical cancer HeLa cells and its mechanism.Methods From October 2018 to October 2019,Lipofectamine ^(TM) 2000 was used to transfect miR-425-5p mimics,inhibitor and its negative control into HeLa cells.The expression of miR-425-5p in the cells was detected by real-time fluorescent quantitative PCR,and the cell proliferation activity was detected by methyl thiazolyl tetrazolium(MTT)assay.Plate colony formation assay was used to detect cell clone formation ability,Transwell chamber experiment was used to detect cell invasion and migration,and the expression levels of phosphatase and tensin homolog deleted on chromosome ten(PTEN)protein in cells was detected by Western blotting.Bioinformatics software prediction and dual luciferase reporter gene experiments were used to verify the targeting relationship between miR-425-5p and PTEN.The PTEN overexpression plasmids(pcDNA3.1-PTEN)and PTEN interference sequence(siRNA-PTEN)were transfected into HeLa cells,and the effects of PTEN on HeLa cell proliferation viability,clone formation ability,invasion ability and migration ability were observed.Results Compared with their negative controls,overexpression of miR-425-5p promoted HeLa cell proliferation[(142.25±11.32)%vs.(100.00±6.67)%],colony formation,invasion[(133.28±9.86)vs.(77.46±5.32)]and migration[(187.56±15.12)vs.(115.35±10.26)],and down-regulated PTEN protein expression,while low expression of miR-425-5p inhibited HeLa cell proliferation[(58.38±3.45)vs.(100.00±5.74)],clone formation,invasion[(43.32±3.62)vs.(75.65±6.15)]and migration[(62.28±4.05)vs.(109.72±9.84)],and up-regulated PTEN protein expression(P<0.05).Double luciferase reporter gene experiments confirmed that PTEN was a target gene of miR-425-5p;PTEN overexpression promoted HeLa cell proliferation[(66.52±4.36)%vs.(100.00±6.18)%],clone formation,invasion[(46.23±3.13)vs.(71.65±5.24)]and migration[(62.65±4.26)vs.(108.42±8.57)indivual],while low PTEN expression inhibited HeLa cell proliferation[(136.52±9.85)vs.(100.00±7.05)],clone formation,invasion[(110.27±9.85)vs.(70.52±6.18)]and migration[(168.78±16.96)vs.(113.26±10.13).Conclusion miR-425-5p can promote the proliferation,invasion and migration of cervical cancer HeLa cells through targeted regulation of PTEN expression.