miR-26a-1-3p调控XIAP促进滋养层细胞凋亡而引起复发性流产的研究

miR-26a-1-3p regulates XIAP to promote trophoblast cell apoptosis and induce recurrent abortion

目的 探讨miR-26a-1-3p通过调节滋养层细胞凋亡引发复发性流产的分子调控机制。方法 体外培养人正常绒毛膜滋养层细胞HTR-8/Svneo,并将细胞随机分为miR-NC mimic组(转染miR-NC mimic)、miR-26a-1-3p mimic组(转染miR-26a-1-3p mimic)、miR-26a-1-3p mimic+pcDNA-NC组(转染miR-26a-1-3p mimic、pcDNA-NC)、miR-26a-1-3p mimic+pcDNA-XIAP组(转染miR-26a-1-3p mimic、pcDNA-XIAP)。用流式细胞术检测各组细胞凋亡情况;用蛋白质印迹(Western blot)法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的胱天蛋白酶-3(Cl-caspaes-3)、裂解的胱天蛋白酶-9(Cl-caspaes-9)、X连锁凋亡抑制蛋白(XIAP);用实时荧光定量聚合酶链反应(RT-qPCR)检测miR-26a-1-3p表达水平;用Transwell小室实验检测各组细胞侵袭能力;用双荧光素酶报告基因实验检测miR-26a-1-3p与XIAP靶向结合。结果 miR-NC mimic组、miR-26a-1-3p mimic组、miR-26a-1-3p mimic+pcDNA-NC组、miR-26a-1-3p mimic+pcDNA-XIAP组的细胞凋亡率分别为(4.97±0.40)%,(16.52±1.08)%,(16.67±1.88)%,(5.99±0.40)%;Bax蛋白表达水平分别为0.34±0.03,0.82±0.11,0.87±0.08,0.37±0.06;Cl-caspaes-3蛋白表达水平分别为0.31±0.02,0.91±0.11,1.01±0.07,0.42±0.04;Cl-caspaes-9蛋白表达水平分别为0.31±0.02,0.91±0.11,0.88±0.08,0.45±0.05;Bcl-2蛋白表达水平分别为0.84±0.06,0.42±0.05,0.43±0.06,0.80±0.08;细胞侵袭数目分别为(100.83±8.35),(46.17±3.39),(47.50±5.28),(84.67±8.22)个。以上指标,miR-NC mimic组与miR-26a-1-3p mimic组相比、miR-26a-1-3p mimic+pcDNA-NC组与miR-26a-1-3p mimic+pcDNA-XIAP组相比,差异均有统计学意义(均P<0.05)。双荧光素酶报告基因结果证实miR-26a-1-3p与XIAP靶向结合。结论 miR-26a-1-3p可通过调控XIAP的表达加速滋养层细胞凋亡、抑制侵袭而引发复发性流产。

Objective To investigate the molecular regulatory mechanism of microRNA-26 a-1-3 p(miR-26 a-1-3 p) that induces recurrent abortion by regulating trophoblast cell apoptosis. Methods Human normal chorionic trophoblast cells HTR-8/Svneo were cultured in vitro. The cells were randomly divided into miR-NC mimic group(transfected with miR-NC mimic), miR-26 a-1-3 p mimic group(transfected with miR-26 a-1-3 p mimic), miR-26 a-1-3p mimic + pc DNA-NC group( transfected with mi R-26a-1-3p mimic and pc DNA-NC) and mi R-26a-1-3p mimic + pc DNA-XIAP group( transfected with mi R-26a-1-3p mimic and pc DNA-XIAP). Cell apoptosis was detected by flow cytometry. Western blot was used to detect B cell lymphoma-2( Bcl-2),Bcl-2 associated X protein( Bax),cleaved cysteine protease-3( Cl-caspaes-3),cleaved cysteine protease-9( Cl-caspaes-9),X-linked inhibitor of apoptosis protein( XIAP). The expression level of mi R-26a-1-3p was detected by real-time fluorescence quantitative polymerase chain reaction( RT-q PCR). Transwell chamber assay was used to detect the invasiveness of cells in each group. The double luciferase reporter gene assay was used to detect the targeted binding of mi R-26a-1-3p to XIAP. Results Apoptosis rates of mi R-NC mimic group,mi R-26a-1-3p mimic group,mi R-26a-1-3p mimic + pc DNA-NC group and mi R-26a-1-3p mimic + pc DNA-XIAP group were( 4. 97 ± 0. 40) %,( 16. 52 ± 1. 08) %,( 16. 67 ± 1. 88) %,( 5. 99 ± 0. 40) %;Bax protein expression levels were0. 34 ± 0. 03,0. 82 ± 0. 11,0. 87 ± 0. 08,0. 37 ± 0. 06;Cl-caspaes-3 protein expression levels were 0. 31 ± 0. 02,0. 91 ± 0. 11,1. 01 ± 0. 07,0. 42 ± 0. 04;Cl-caspaes-9 protein expression levels were 0. 31 ± 0. 02,0. 91 ± 0. 11,0. 88 ± 0. 08,0. 45 ± 0. 05;Bcl-2 protein expression levels were 0. 84 ± 0. 06,0. 42 ± 0. 05,0. 43 ± 0. 06,0. 80 ± 0. 08;the number of cell invasion were 100. 83 ± 8. 35,46. 17 ± 3. 39,47. 50 ± 5. 28,84. 67 ± 8. 22,respectively. There were statistically significant differences between mi R-NC mimic and mi R-26a-1-3p mimic +pc DNA-NC group,between mi R-26a-1-3p mimic + pc DNA-NC group and mi R-26a-1-3p mimic + pc DNA-XIAP group( all P < 0. 05). Meanwhile,double luciferase reporter gene results confirmed the targeted binding of mi R-26A-1-3p to XIAP. Conclusion mi R-26a-1-3p can accelerate trophoblast cell apoptosis and inhibit invasion and induce recurrent abortion by regulating XIAP expression.