miRNA-144在复发性自然流产绒毛组织中的表达及其功能研究

发布时间：2018-01-22 00:06

本文关键词： 复发性自然流产绒毛组织 miR-144 STAT1　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：1.探讨miR-144在复发性自然流产与正常早期妊娠绒毛组织中的表达差异。 2.在人滋养细胞HTR-8中探讨miR-144对其可能的靶基因STAT1的调控作用。 3.了解miR-144对人滋养细胞HTR-8凋亡的影响,以探讨miR-144在复发性自然流产发病过程中的作用。方法：1.采用实时荧光定量PCR(real-time PCR)技术检测miR-144在15例正常早期妊娠和15例复发性自然流产患者绒毛组织中的表达。 2.采用瞬时转染技术在人正常滋养细胞系HTR-8细胞中分别转染miR-144mimics与miR-144inhibitor,即过表达与抑制miR-144的表达后,RT-PCR技术检测HTR-8细胞中miR-144及STAT1mRNA的表达水平,并采用Western-blot技术检测Statl蛋白的表达水平。 3.在HTR-8细胞中过表达与抑制miR-144的表达后,采用流式细胞术检测细胞凋亡情况。结果：1.实时荧光定量PCR结果显示,复发性自然流产患者绒毛组织中miR-144的表达低于对照组(P0.001)。 2.在人滋养细胞系HTR-8细胞中过表达与抑制miR-144的表达后,RT-PCR技术检测miR-144及STAT1mRNA的表达量,结果显示与对照组相比,过表达组miR-144的表达量升高,而STAT1mRNA的表达量降低,差异均有统计学意义(P0.001)；抑制组miR-144的表达量降低,而STAT1mRNA的表达量升高,差异均有统计学意义(P0.001); Western-blot技术检测STAT1蛋白的表达水平,结果显示与对照组相比,过表达组Statl蛋白的表达量降低,抑制组STAT1蛋白的表达量升高,差异均有统计学意义(P0.05)。提示STAT1在mRNA和蛋白水平的表达受到miR-144的负调控。 3.在HTR-8细胞中过表达与抑制miR-144的表达后,流式细胞术检测细胞凋亡指数,结果显示,上调miR-144的表达后细胞凋亡率较对照组降低,差异有统计学意义(P0.05),而下调miR-144的表达后细胞凋亡率较对照组增加,差异有统计学意义(P0.001)。结论：1.复发性自然流产患者绒毛组织中miR-144的表达降低,提示miR-144可能参与了复发性自然流产的发生。 2.STAT1是miR-144的一个作用靶点,且在mRNA和蛋白水平,miR-144均对STAT1有着明显的负性调控作用。 3.miR-144可以抑制滋养细胞的凋亡,提示miR-144的低表达可能通过增加滋养细胞的凋亡而参与了复发性自然流产的发病过程。
[Abstract]:Objective 1. To investigate the expression of miR-144 in villi of recurrent spontaneous abortion and normal early pregnancy. 2. To investigate the regulation of miR-144 on its possible target gene STAT1 in human trophoblastic HTR-8. 3. To investigate the effect of miR-144 on the apoptosis of human trophoblastic HTR-8 and to explore the role of miR-144 in the pathogenesis of recurrent spontaneous abortion. Method 1. Real-time fluorescence quantitative PCR(real-time PCR was used. The expression of miR-144 in chorionic villi of 15 cases of normal early pregnancy and 15 cases of recurrent spontaneous abortion was detected. 2. Transient transfection technique was used to transfect miR-144mimics and miR-144inhibitor into human normal trophoblast cell line HTR-8 cells. The expression of miR-144 and STAT1mRNA in HTR-8 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) after over-expression and inhibition of miR-144 expression. Western-blot technique was used to detect the expression of Statl protein. 3. After overexpression and inhibition of miR-144 expression in HTR-8 cells, apoptosis was detected by flow cytometry. Results the results of real-time fluorescence quantitative PCR showed that the expression of miR-144 in villi of recurrent spontaneous abortion patients was lower than that of control group (P 0.001). 2. After overexpression and inhibition of miR-144 expression in human trophoblast cell line HTR-8, RT-PCR was used to detect the expression of miR-144 and STAT1mRNA. The results showed that the expression of miR-144 in overexpression group was higher than that in control group, but the expression of STAT1mRNA was decreased, the difference was statistically significant (P 0.001). In the inhibitory group, the expression of miR-144 decreased, but the expression of STAT1mRNA increased, and the difference was statistically significant (P 0.001). The expression level of STAT1 protein was detected by Western-blot. The results showed that the expression of Statl protein in overexpression group was lower than that in control group. The expression of STAT1 protein in the inhibitory group was significantly higher than that in the control group (P 0.05), suggesting that the expression of STAT1 in mRNA and protein level was negatively regulated by miR-144. 3. After overexpression and inhibition of miR-144 expression in HTR-8 cells, apoptosis index was detected by flow cytometry. After upregulating the expression of miR-144, the rate of apoptosis was lower than that of the control group, the difference was statistically significant (P 0.05), while the rate of apoptosis after down-regulating the expression of miR-144 was higher than that of the control group. The difference was statistically significant (P 0.001). Conclusion 1. The expression of miR-144 in villi of recurrent spontaneous abortion patients was decreased, suggesting that miR-144 may be involved in the occurrence of recurrent spontaneous abortion. 2. STAT1 is a target of miR-144 and has a negative regulatory effect on STAT1 at both mRNA and protein levels. 3. MiR-144 can inhibit the apoptosis of trophoblast, suggesting that the low expression of miR-144 may be involved in the pathogenesis of recurrent spontaneous abortion by increasing the apoptosis of trophoblast.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.21

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