Objective: To 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). Conclusion: The 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.
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。.
Keywords: Cell proliferation; Matrix metalloproteinase 2; MicroRNAs; Pre-eclampsia; Trophoblasts.