miR-106b-5p通过靶向MMP-9调控HTR-8/SVneo细胞生物学行为的研究

miR-106b-5p regulates the sexual biological behavior of HTR-8/SVneo cells by targeting MMP-9

目的 探讨miR-106b-5p对HTR-8/SVneo细胞增殖、侵袭、迁移的影响及其与基质金属蛋白酶-9(MMP-9)的靶向关系。方法 收集80例子痫前期(PE)及30例正常孕妇胎盘组织。选取HTR-8/SVneo细胞,分为Vector组、MMP-9组、Scramble组、si-MMP-9组、miR-106b-5p组、si-miR-106b-5p组、miR-NC组、NC组、miR-106b-5p+si-NC组及miR-106b-5p+si-MMP-9组。CCK-8实验检测增殖能力,Transwell实验检测侵袭能力,划痕实验检测迁移能力,荧光定量聚合酶链反应(RT-PCR)检测细胞MMP-9基因mRNA及miR-106b-5p表达水平,Western blot检测细胞MMP-9蛋白表达水平,TargetScan在线网站预测miR-106b-5p与MMP-9结合位点,双萤光素酶报告基因实验验证miR-106b-5p与MMP-9的靶向关系。结果 轻度、重度PE孕妇胎盘组织MMP-9基因mRNA、蛋白及miR-106b-5p表达水平低于正常孕妇(P<0.01)。重度PE孕妇胎盘组织MMP-9基因mRNA、蛋白及miR-106b-5p表达水平低于轻度PE孕妇(P<0.01)。PE孕妇胎盘组织MMP-9及miR-106b-5p表达水平呈现正相关(r=0.735,P<0.001)。与对照组比较,miR-106b-5p过表达组、MMP-9过表达组miR-106b-5p水平、MMP-9水平升高(P<0.01),增殖、侵袭及迁移能力增强(P<0.01)。与对照组比较,miR-106b-5p下调组、MMP-9下调组miR-106b-5p水平、MMP-9水平降低(P<0.01),增殖、侵袭及迁移能力降低(P<0.01)。下调MMP-9逆转miR-106b-5p对HTR-8/SVneo细胞恶性表型、MMP-9表达的促进作用。miR-106b-5p可负性调控MMP-9表达。结论 PE胎盘组织低表达miR-106b-5p、MMP-9,下调miR-433-3p正性调控MMP-9抑制HTR-8/SVneo细胞的增殖、侵袭、迁移。

Objective To investigate the effects of miR-106b-5p on the proliferation,invasion and migration of HTR-8/SVneo cells and its targeting relationship with matrix metalloproteinase-9(MMP-9).Methods Placental tissues from 80 pregnant women with preeclampsia(PE)and 30 normal pregnant women were collected.HTR-8/SVneo cells were selected and divided into Vector group,MMP-9 group,Scramble group,si-MMP-9 group,miR-106b-5p group,si-miR-106b-5p group,miR-NC group,NC group,miR-106b-5p+si-NC group and miR-106b-5p+si-MMP-9 group.CCK-8 assay was used to detect proliferation ability.Transwell assay was used to detect invasion ability.Scratch assay was used to detect migration ability.Fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect MMP-9 gene mRNA and miR-106b-5p levels.Western blot was used to detect MMP-9 protein levels.TargetScan online website was used to predict miR-106b-5p and MMP-9 binding sites.Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-106b-5p and MMP-9.Results The MMP-9 mRNA,protein levels and miR-106b-5p level in placental tissues from pregnant women with mild and severe PE were lower than those from normal pregnant women(P<0.01).The MMP-9 mRNA,protein levels and miR-106b-5p level in placental tissue from pregnant women with severe PE were lower than those from pregnant women with mild PE(P<0.01).MMP-9 level bad a positive correlation with the level of miR-106b-5p(r=0.735,P<0.001).Compared with the control group,miR-106b-5p and MMP-9 levels increased,proliferation,invasion and migration ability enhanced in miR-106b-5p and MMP-9 overexpression groups(P<0.01).Compared with the control group,miR-106b-5p and MMP-9 levels,proliferation,invasion and migration abilities decreased(P<0.01).Down-regulation of MMP-9 reversed the promotive effect of miR-106b-5p on malignant phenotype,MMP-9 level in HTR-8/SVneo cells.miR-106b-5p negatively regulated MMP-9 expression.Conclusion The level of miR-106b-5p,MMP-9 in PE placental tissues were low.Down-regulation of miR-433-3p positively regulated MMP-9 to inhibit proliferation,invasion,and migration of HTR-8/SVneo cells.