微小RNA-106b靶向调控基质金属蛋白酶2对滋养细胞侵袭和增殖的影响

Effect of microRNA-106b on the invasion and proliferation of trophoblasts through targeting MMP-2

目的探讨子痫前期孕妇的胎盘组织中微小RNA-106b（miR-106b）、基质金属蛋白酶2（MMP-2）的表达及miR-106b靶向调控MMP-2的表达对滋养细胞侵袭和增殖的影响。方法共纳入30例轻度子痫前期（mPE）、30例重度子痫前期（sPE）和40例正常孕妇，分别为mPE组、sPE组及对照组，收集其胎盘组织；将绒毛膜癌细胞系JAR细胞和JEG3细胞各分为4组（miR-106b模拟物组、模拟物对照组、miR-106b抑制物组、抑制物对照组）。采用荧光素酶报告基因活性检测法验证miR-106b对其靶基因MMP-2基因表达的调控作用；采用实时荧光定量PCR技术、蛋白印迹法检测3组孕妇的胎盘组织和各组细胞中miR-106b和MMP-2 mRNA、蛋白的表达水平；并采用四甲基偶氮唑蓝（MTT）比色法检测各组细胞的相对增殖率，采用体外侵袭实验检测各组细胞的侵袭力。结果（1）荧光素酶报告基因活性检测法显示，含miR-106b的质粒和含MMP-2基因的重组质粒共同转染JAR细胞和JEG3细胞后，与模拟物对照比较，其荧光素酶水平下降（P〈0.01）。（2）实时荧光定量PCR技术显示：①mPE组、sPE组及对照组孕妇的胎盘组织中，miR-106b和MMP-2 mRNA的表达水平分别为2.89±0.04、1.96±0.03、1.01±0.03，1.87±0.05、0.69±0.03、2.78±0.03，3组分别比较，差异均有统计学意义（P〈0.05）。② 4组JAR细胞和4组JEG3细胞（miR-106b模拟物组、模拟物对照组、miR-106b抑制物、抑制物对照组）的miR-106b的表达水平分别为2.39±0.03、1.03±0.04、0.73±0.03、1.11±0.04，2.17±0.04、1.18±0.04、0.61±0.03、1.22±0.03，JAR、JEG3细胞4组间分别比较，差异有统计学意义（P〈0.05）。③4组JAR细胞和4组JEG3细胞的MMP-2 mRNA的表达水平分别为0.45±0.15、1.02±0.03、2.28±0.03、1.11±0.03，0.58±0.03、1.25±0.15、2.25±0.03、1.21±0.03，JAR、JEG3细胞的4组间分别比较，差异有统计学意义（P〈0.05）。（3）蛋白印迹法显示：①3组孕妇胎盘组织中MMP-2蛋白的表达水平分别为1.63±0.04、0.55±0.03、2.82±0.03，3组比较，差异有统计学意义（P〈0.05）。②4组JAR细胞的MMP-2蛋白表达水平分别为0.41±0.03、0.97±0.03、2.25±0.03、1.01±0.03，4组比较，差异有统计学意义（P〈0.05）。③4组JEG3细胞的MMP-2蛋白表达水平分别为0.53±0.03、1.20±0.03、2.31±0.04、1.19±0.03，差异有统计学意义（P〈0.05）。（4）MTT法检测显示：JAR细胞和JEG3细胞经miR-106b模拟物转染后（即miR-106b模拟物组）细胞增殖率均明显低于相应模拟物对照组，差异有统计学意义（P均〈0.05）；经miR-106b抑制物转染后（即miR-106b抑制物组）细胞增殖率均明显高于相应的抑制物对照组，差异有统计学意义（P均〈0.05）。（5）体外侵袭实验显示：①4组JAR细胞的穿膜细胞数分别为（61±15）、（79±13）、（134±13）、（80±12）个，差异有统计学意义（P〈0.05）；②4组JEG3细胞的穿膜细胞数分别为（57±12）、（71±15）、（128±15）、（70±14）个，差异有统计学意义（P〈0.05）。 结论miR-106b能够通过靶向调控MMP-2的表达抑制JAR细胞和JEG3细胞的侵袭、增殖，与子痫前期的发病相关。

ObjectiveTo investigate the expression of microRNA-106b （miR-106b） in the placentas of patients with pre-eclampsia and its relationship with matrix metallopeptidase （MMP） -2, and its effect on the invasion and proliferation of trophoblasts. Methods（1） Placental tissues were collected from patients with mild pre-eclampsia （mPE, n=30）, severe pre-eclampsia （sPE, n=30） and normal pregnant women （n=40）. Human choriocarcinoma cell lines JAR and JEG3 were assigned to the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group, respectively. （2） The target gene of miR-106b（such as MMP-2） was predicted by bioinformatics. Dual-luciferase reporting system was used to verify the regulation of miR-106b on the expression of MMP-2. （3） The expressions of miR-106b and MMP-2 were measured by quantitative real-time PCR （qRT-PCR） and western blot. （4） Cell proliferation was determined by MTT assay. （5） Invasive activities in each group were assessed by cell transwell invasion assays. Results（1） Predicting result of bioinformatics indicated that MMP-2 was one of the target genes of miR-106b. Dual-luciferase activity assay demonstrated that MMP-2 was the direct target of miR-106b （P〈0.01） .（2） The results of qRT-PCR.①The expression of miR-106b in the placentas of mPE, sPE, normal pregnant women were 2.89±0.04, 1.96±0.03, 1.01±0.03, respectively （P〈0.05）. And the expression of MMP-2 mRNA in the placentas of mPE, sPE, normal pregnant women were 1.87±0.05, 0.69±0.03, 2.78±0.03, respectively （P〈0.05）. ②The expression of miR-106b in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 2.39±0.03, 1.03±0.04, 0.73±0.03, 1.11±0.04, respectively （P〈0.05）. And its expression in the JEG3 cell line were 2.17±0.04, 1.18±0.04, 0.61±0.03 and 1.22±0.03, respectively （P〈0.05）. ③The expression of MMP-2 mRNA in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.45±0.15, 1.02±0.03, 2.28±0.03, 1.11±0.03, respectively （P〈0.05）. And its expression in the JEG3 cell line were 0.58±0.03, 1.25±0.15, 2.25±0.03, 1.21±0.03, respectively （P〈0.05）. （3） The results of western blot. ①The expression of MMP-2 protein in the placentas of mPE, sPE, normal pregnant women were 1.63±0.04, 0.55±0.03, 2.82±0.03, respectively （P〈0.05）. ②The expression of MMP-2 protein in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.41±0.03, 0.97±0.03, 2.25±0.03, 1.01±0.03, respectively （P〈0.05）. And its expression in the JEG3 cell line were 0.53±0.03, 1.20±0.03, 2.31±0.04, 1.19±0.03, respectively （P〈0.05）. （4） miR-106b could inhibit the proliferation of JAR and JEG3 cells, cell proliferation rates in the miR-106b mimics group were lower than that in the mimics negative control group （P〈0.05）. And cell proliferation rate in the miR-106b inhibitor group was higher than the inhibitor negative control group （P〈0.05）. （5） The numbers of JAR cell that passed the membrane in the miR-106b mimics group, the mimics negative control group. The miR-106b inhibitor group and the inhibitor negative control group were 61±15, 79±13, 134±13, 80±12, respectively（P〈0.05）. And the numbers of JEG3 cell that passed were 57±12, 71±15, 128±15, 70±14, respectively （P〈0.05）. ConclusionThe miR-106b could inhibit the invasion and proliferation of JAR and JEG3 cells through targeting MMP-2, and have a relationship with the pathogenesis of pre-eclampsia.