Li J, Wang J M, Liu Y H, Zhang Z, Han N, Wang J Y, Xue S H, Wang P
Department of Obstetrics, the Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Fu Chan Ke Za Zhi. 2017 May 25;52(5):327-332. doi: 10.3760/cma.j.issn.0529-567X.2017.05.007.
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. (1) Placental tissues were collected from patients with mild pre-eclampsia (mPE, 30), severe pre-eclampsia (sPE, 30) and normal pregnant women (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. (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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<0.05). And cell proliferation rate in the miR-106b inhibitor group was higher than the inhibitor negative control group (<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(<0.05). And the numbers of JEG3 cell that passed were 57±12, 71±15, 128±15, 70±14, respectively (<0.05). 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.
探讨微小RNA-106b（miR-106b）在子痫前期患者胎盘组织中的表达及其与基质金属蛋白酶（MMP）-2的关系，以及其对滋养细胞侵袭和增殖的影响。（1）收集轻度子痫前期（mPE，30例）、重度子痫前期（sPE，30例）患者及正常孕妇（40例）的胎盘组织。将人绒毛膜癌细胞系JAR和JEG3分别分为miR-106b模拟物组、模拟物阴性对照组、miR-106b抑制剂组和抑制剂阴性对照组。（2）通过生物信息学预测miR-106b的靶基因（如MMP-2）。采用双荧光素酶报告系统验证miR-106b对MMP-2表达的调控作用。（3）采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测miR-106b和MMP-2的表达。（4）采用MTT法检测细胞增殖。（5）采用细胞Transwell侵袭实验评估各组细胞的侵袭活性。（1）生物信息学预测结果显示，MMP-2是miR-106b的靶基因之一。双荧光素酶活性检测表明，MMP-2是miR-106b的直接靶基因（<0.01）。（2）qRT-PCR结果。①mPE、sPE、正常孕妇胎盘组织中miR-106b的表达分别为2.89±0.04、1.96±0.03、1.01±0.03（<0.05）。mPE、sPE、正常孕妇胎盘组织中MMP-2 mRNA的表达分别为1.87±0.05、0.69±0.03、2.78±0.03（<0.05）。②miR-106b模拟物组、模拟物阴性对照组、miR-106b抑制剂组和抑制剂阴性对照组JAR细胞系中miR-106b的表达分别为2.39±0.03、1.03±0.04、0.73±0.03、1.11±0.04（<0.05）。其在JEG3细胞系中的表达分别为2.17±0.04、1.18±0.04、0.61±0.03和1.22±0.03（<0.05）。③miR-106b模拟物组、模拟物阴性对照组、miR-106b抑制剂组和抑制剂阴性对照组JAR细胞系中MMP-2 mRNA的表达分别为0.45±0.15、1.02±0.03、2.28±0.03、1.11±0.03（<0.05）。其在JEG3细胞系中的表达分别为0.58±0.03、1.25±0.15、2.25±0.03、1.21±0.03（<0.05）。（3）蛋白质免疫印迹法结果。①mPE、sPE、正常孕妇胎盘组织中MMP-2蛋白的表达分别为1.63±0.04、0.55±0.03、2.82±0.03（<0.05）。②miR-106b模拟物组、模拟物阴性对照组、miR-106b抑制剂组和抑制剂阴性对照组JAR细胞系中MMP-2蛋白的表达分别为0.41±0.03、0.97±0.03、2.25±0.03、1.01±0.03（<0.05）。其在JEG3细胞系中的表达分别为0.53±0.03、1.20±0.03、2.31±0.04、1.19±0.03（<0.05）。（4）miR-106b可抑制JAR和JEG3细胞的增殖，miR-106b模拟物组细胞增殖率低于模拟物阴性对照组（<0.05）。miR-106b抑制剂组细胞增殖率高于抑制剂阴性对照组（<0.05）。（5）miR-106b模拟物组、模拟物阴性对照组、miR-106b抑制剂组和抑制剂阴性对照组JAR细胞穿膜数分别为61±15、79±13、134±13、80±12（<0.05）。JEG3细胞穿膜数分别为57±12、71±15、128±15、70±14（<0.05）。miR-106b可通过靶向MMP-2抑制JAR和JEG3细胞的侵袭和增殖，与子痫前期的发病机制有关。
