摘要
子痫前期(Preeclampsia, PE)是人类妊娠期特有的常见病和多发病。在我国,其发生率为9.7%,在孕产妇死亡原因中占第二位,严重威胁母婴健康[1]。目前,PE的病因和发病机理仍未阐明,研究表明胎盘滋养细胞功能障碍是PE发病的病理基础。微小RNA(microRNA,miRNA)是一类长约22个核苷酸的单链非编码RNA,通过与靶mRNA完全或不完全互补配对,导致靶基因降解或抑制其翻译,从而在基因的表达调控中发挥着重要作用。最近有研究表明在人类子痫前期胎盘与正常胎盘组织中,miR-210与miR-182的表达水平存在差异[2],提示miRNA的表达失衡可能参与了PE的发病过程。但以往研究检测的miRNA数量有限,并且国内外尚无miRNA在PE胎盘组织中功能研究的报告。因此有必要深入开展miRNA在PE胎盘组织中的表达和功能研究,为深入研究PE的发病机理提供新的线索。
目的
1.了解miRNA在正常与PE胎盘组织中的表达差异,进一步筛选与PE发病相关的miRNA。
2.了解miRNA的作用靶点以及对胎盘滋养细胞功能的调控作用,探讨miRNA在PE发生发展中的作用。
方法
1.应用Exiqon 8.1版的miRNA表达谱芯片筛选8对重度PE胎盘与正常胎盘组织中差异表达的miRNA,并采用TargetScan与PITA预测系统筛选差异miRNA的靶基因。
2.选取10个miRNA,应用实时荧光定量RT-PCR(Real-time RT-PCR)方法加以验证,分别定量检测miRNA在15例重度子痫前期患者(sPE组)、8例轻度子痫前期患者(mPE组)及11例正常妊娠胎盘组织(对照组)中的表达水平。
3.选择PE与正常胎盘组织中表达差异较为显著的miR-152进行靶基因验证。采用miRNA过表达技术在人滋养细胞肿瘤细胞系(JEG-3)中过表达miR-152,RT-PCR与免疫印迹(Western blot)技术验证其靶基因的表达水平,并通过荧光素酶报告系统进一步验证其作用位点。
4.在JEG-3细胞中过表达miR-152,检测JEG-3细胞浸润能力的变化,并检测NK-92MI细胞杀伤JEG-3细胞的能力是否改变。
结果
1.芯片结果显示:重度子痫前期胎盘与正常胎盘组织中存在34个差异表达的miRNA,其中,11个miRNA在sPE胎盘组织中表达升高,23个miRNA表达下降。通过靶基因预测发现:在miR-30a-3p、miR-152与miR-18a的可能的作用靶点中,分别包括IGF1、HLA-G与ESR1等与PE发病相关的基因。
2.选取10个miRNA,应用Real-time RT-PCR进行验证,其中8个miRNA与芯片结果相符:miR-210、miR-152与miR-518b在sPE胎盘中表达升高(P<0.05),miR-377、miR-411、miR-542-3p、miR-18a与miR-363在sPE胎盘中表达下降(P<0.05);miR-152在mPE胎盘组织中表达升高(P<0.05),miR-210、miR-377、miR-411在mPE中表达下降(P<0.05)。
3.将pre-miR-152转染入JEG-3细胞,使JEG-3细胞中的miR-152表达升高,RT-PCR结果显示转染前后JEG-3细胞中HLA-G mRNA的水平无显著差异(P>0.05),但Western blot结果发现在JEG-3中过表达miR-152之后,HLA-G的蛋白表达水平明显下降(P<0.05)。采用pMIR-REPORT荧光素酶报告基因系统进行靶点验证,结果显示转染pre-miR-152与重组载体的实验组其荧光素酶活性下降约47%,表明HLA-G为miR-152的作用靶点。
4.细胞浸润实验发现过表达miR-152前后,JEG-3细胞的浸润能力无显著改变(P>0.05)。然而,在JEG-3细胞中过表达miR-152之后,NK-92MI细胞对JEG-3细胞的杀伤能力显著提高(P<0.05)。
结论
1.子痫前期胎盘组织中存在一系列表达失衡的miRNA,这一异常可能参与了子痫前期的发生和病理生理过程。
2.实验结果显示HLA-G为miR-152的作用靶点,表明miR-152可能通过调控免疫耐受因子HLA-G的表达参与了子痫前期的发生。
3. miR-152可以通过降低靶基因HLA-G的表达,从而提高NK细胞对滋养细胞的杀伤作用,为基因治疗在子痫前期与绒毛膜癌等疾病中的应用奠定了理论与实验基础。
Preeclampsia (PE) is a pregnancy specific disorder which has a high incidence, approximately 9.7% in China[1]. It is the second cause of maternal mortality and is a major threat to maternal and fetus health. The aetiology and pathogenesis of this disease has remained unknown. Evidence showed that the disorder of trophoblast function is associated with the pathogenesis of PE. MicroRNA (miRNA) are noncoding RNAs of 21 to 24 nucleotides, which function as negative regulators of gene expression by antisense complimentarily to specific messenger RNAs and subsequent induction of translation inhibition, which can also be associated with transcript destabilization. It has been reported previously that miR-210 and miR-182 are expressed differentially in the human placentas of patients with PE compared with controls using real time RT-PCR analysis[2], suggesting that PE is associated with alterations in placental microRNA expression. However, neither a comprehensive list of the human miRNA nor the function study of placental microRNA has been screened in PE placentas. Thus, it is necessary to unravel the expression and function of the miRNA in PE placentas. Our study may provide a useful tool in studing the aetiology of PE.
Objective
1. To investigate the expression of miRNA in placentas from women with severe PE and normal pregnancy, and discuss the roles of miRNA in the pathogenesis of PE.
2. To investigate the roles of miRNA in the regulation of target gene expression and functions of trophoblast function.
Methods
1. MicroRNA microarray (Exiqon, release 8.1) analysis was performed on 8 of the matched tissue pairs from the control and sPE groups. Two softwares for bioinformatics anaylsis, targetscan and PITA, were used for miRNA target prediction.
2. Expression of ten miRNA in fifteen placentas from severe PE (sPE group), eight placentas from mild PE (mPE group) and eleven from gestational normal pregnancies (control group) were confirmed using real time RT-PCR.
3. The differentially expressed miRNA in PE placentas, miR-152, was selected for target verification. We transfected pre-miR-152 into JEG-3 cells, and then determined the levels of HLA-G mRNA and protein in JEG-3 cells using RT-PCR and Western blot analysis, respectively. The verification of miRNA-152 target site was performed using luciferase reporter vector.
4. After transfection of pre-miR-152 into JEG-3 cells, invasion assay and NK cell cytotoxicity assay were performed to investigate the effect of miR-152 on JEG-3 cell invasion and whether overexpression of miR-152 in JEG-3 cells could be associated with increased susceptibility to NK lysis.
Results
1. Thirty-four microRNA were differentially expressed in preeclamptic placentas compared with that of normal placentas. Of these, 11 were overexpressed and 23 were underexpressed in preeclamptic pregnancies. The potential targets of miR-30a-3p, miR-152 and miR-182, including IGF1, HLA-G and ESR1, respectively, were predicted using bioinformatics anaylsis.
2. Among the ten miRNA which selected for real-time RT-PCR verification, 8 of 10 real-time RT-PCR data were concordant with the microarray data: sPE-overexpressed miR-210, miR-152, miR-518b and sPE-underexpressed miR-377, miR-411, miR-542-3p, miR-18a, miR-363 (P<0.05); mPE-overexpressed miR-152 and mPE-underexpressed miR-210, miR-377, miR-411 (P<0.05).
3. We transfected pre-miR-152 into JEG-3 cells. RT-PCR analysis showed that the transfection was effective, while no significant changes were observed in the mRNA level of HLA-G (P>0.05). However, the decreased HLA-G protein level was observed using western blot analysis (P<0.05). We performed a pMIR-REPORT luciferase reporter assay and observed a significant decrease (≈47%) of luciferase activity in the presence of pre-miR-152 in JEG-3 cells. These results experimentally confirm that HLA-G is a direct target for miR-152 in JEG-3 cells.
4. The results of invasion assay showed that there was no difference in invasion among the test groups (P>0.05). However, after transfection of pre-miR-152 into JEG-3 cells, the lytic activity of NK-92 cells toward JEG-3 cells was dramatically enhanced (P<0.05) versus that in the control groups.
Conclusion
1. The results show that different microRNA are deregulated in preeclamptic pregnancies, suggesting the involvement of these microRNA in the pathogenesis of preeclampsia.
2. These results experimentally confirm that HLA-G is a direct target for miR-152, suggesting the potential role of miR-152 in the etiology of PE through regulating HLA-G.
3. Our results showed that overexpression of miR-152 led to increased NK cell mediated cytolysis in JEG-3 cells through regulating HLA-G. This study may provide a useful tool in the gene treatment of PE and choriocarcinoma.
引文
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