子痫前期相关miR-30a-3p靶基因的预测和验证
发布时间：2018-09-04 14:39
【摘要】：子痫前期（pre-eclampsia，PE）是一组妊娠与高血压共存的疾病，这种疾病严重影响着母婴健康，是孕产妇及围产儿死亡率升高的主要原因之一。然而，子痫前期的发病机制迄今为止尚未完全明确，该疾病的预防和治疗的进展受到严重阻碍，因此，深入研究子痫前期的发生发展机制具有重要的临床指导意义。微小RNA(microRNA，miRNA)是一种能够调节蛋白表达但不能编码蛋白的小RNA，能够降解靶基因mRNA或者阻碍其翻译，在基因表达调控中具有重要的作用。有研究表明miRNAs在调控细胞周期和生命体的生长发育中起了重要的作用，子痫前期以及其它妊娠相关疾病的发生与胎盘中miRNAs的表达失衡密切相关[1]。我们课题组在前期研究中利用基因芯片技术及荧光实时定量PCR技术证实了miR-30a-3p在子痫前期患者胎盘中的表达特异性升高，并开展体外实验，研究miR-30a-3p表达水平对人正常滋养细胞系HTR-8/SVneo细胞功能的影响，结果发现，miR-30a-3p表达水平与HTR-8/SVneo细胞的凋亡率呈正相关，我们推测在胎盘中miR-30a-3p表达的升高与子痫前期发病相关。然而，miR-30a-3p具体是通过调控哪个或者哪些靶基因而起作用？相关靶基因又在子痫前期的发病中扮演了怎样的角色？为解决这些问题,我们进一步开展了对miR-30a-3p子痫前期相关靶基因的预测和验证的研究。 目的 1.预测miR-30a-3p的靶基因，并从中筛查与子痫前期有关的基因为目的基因。 2.明确miR-30a-3p对靶基因的调控关系和作用位点。 方法 1.利用生物信息软件预测miR-30a-3p的靶基因，根据候选靶基因在生物信息软件中的分值及其与子痫前期相关性进行筛选。 2．利用脂质体转染技术在人正常滋养细胞系HTR-8/Svneo细胞中建立miR-30a-3p过表达及抑制表达模型，利用荧光实时定量PCR技术和Western-blot技术检测细胞模型中的miR-30a-3p与靶基因的表达水平的相关性。 3．构建目的基因的荧光素酶报告载体，利用双荧光素酶载体报告系统验证miR-30a-3p与靶基因的靶向关系和结合位点。 结果 1．生物信息预测软件预测结果表明：IGF-1为miR-30a-3p的理论靶基因之一，其mRNA序列的3′UTR区含有五个理论上可以与miR-30a-3p相结合的位点。 2．荧光实时定量PCR及Western-blot结果显示，过表达组中miR-30a-3p的表达量显著上升（P＜0.05），而IGF-1在mRNA和蛋白水平上的表达显著下降（P＜0.05）；抑制表达组中miR-30a-3p的表达量显著下降（P＜0.05），IGF-1在mRNA水平及蛋白水平的表达上显著升高（P＜0.05)。 3．双荧光素酶载体报告实验结果表明: miR-30a-3p和IGF-1有确切的靶向关系；其中IGF-1mRNA序列的3′UTR区有四个可能与miR-30a-3p相结合的位点。 结论 IGF-1为miR-30a-3p的靶基因之一，miR-30a-3p对IGF-1在其mRNA水平和蛋白水平上均有明显的负性调控作用。胎盘中表达升高的miR-30a-3p通过负性调控IGF-1，，使其表达水平下降，滋养细胞功能发生障碍，从而导致了子痫前期的发生。
[Abstract]:Preeclampsia (PE) is a group of pregnant and hypertension coexisting diseases, which seriously affects maternal and infant health and is one of the main causes of maternal and perinatal mortality. MicroRNA (microRNA) is a small RNA that can regulate protein expression but can not encode protein. It can degrade the target gene mRNA or hinder its translation. It plays an important role in the regulation of gene expression. Preeclampsia and other pregnancy-related diseases are closely related to the unbalanced expression of microRNAs in the placenta [1].In our previous study, microarray and real-time fluorescent quantitative PCR were used to confirm the presence of microRNAs-30a-3p in the placenta of preeclampsia patients. In vitro experiments were carried out to investigate the effect of the expression of microRNAs-30a-3p on the function of human normal trophoblast HTR-8/SVneo cells. The results showed that the expression of microRNAs-30a-3p was positively correlated with the apoptosis rate of HTR-8/SVneo cells. We speculated that the increased expression of microRNAs-30a-3p in placenta was associated with the pathogenesis of preeclampsia. Mi-30a-3p plays a role in the pathogenesis of pre-eclampsia by regulating which or which target genes? To solve these problems, we have further carried out the prediction and validation of the target genes related to pre-eclampsia of Mi-30a-3p.
objective
1. predict the target genes of miR-30a-3p and screen the genes associated with preeclampsia as the target genes.
2. clarify the regulatory relationship between miR-30a-3p and target genes.
Method
1. Bioinformatics software was used to predict the target genes of microRNAs-30a-3p. The candidate genes were screened according to their scores in bioinformatics software and their correlation with preeclampsia.
2. Liposome transfection technique was used to establish an overexpression and inhibition model of microRNAs-30a-3p in human normal trophoblastic cell line HTR-8/Svneo. The correlation between microRNAs-30a-3p and target gene expression was detected by real-time fluorescence quantitative PCR and Western-blot.
3. To construct luciferase reporter vectors for target genes and validate the targeting relationship and binding sites between microRNAs-30a-3p and target genes by double luciferase reporter system.
Result
1. Prediction results of bioinformatics software showed that IGF-1 was one of the theoretical target genes of microarray-30a-3p. The 3'-UTR region of its mRNA sequence contained five sites that could theoretically bind to microarray-30a-3p.
2. The results of real-time PCR and Western-blot showed that the expression of miR-30a-3p increased significantly in the overexpression group (P < 0.05), whereas the expression of IGF-1 decreased significantly in the mRNA and protein levels (P < 0.05); the expression of miR-30a-3p decreased significantly in the inhibition group (P < 0.05), and the expression of IGF-1 increased significantly in the mRNA and protein levels. High (P < 0.05).
3. The results of double luciferase vector report showed that there was a definite targeting relationship between microRNAs-30a-3p and IGF-1, and there were four sites in the 3'UTR region of the IGF-1 mRNA sequence that might bind to microRNAs-30a-3p.
conclusion
IGF-1 is one of the target genes of microRNAs-30a-3p. MicroRNAs-30a-3p can negatively regulate the expression of IGF-1 at both mRNA and protein levels.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244
本文编号：2222455
[Abstract]:Preeclampsia (PE) is a group of pregnant and hypertension coexisting diseases, which seriously affects maternal and infant health and is one of the main causes of maternal and perinatal mortality. MicroRNA (microRNA) is a small RNA that can regulate protein expression but can not encode protein. It can degrade the target gene mRNA or hinder its translation. It plays an important role in the regulation of gene expression. Preeclampsia and other pregnancy-related diseases are closely related to the unbalanced expression of microRNAs in the placenta [1].In our previous study, microarray and real-time fluorescent quantitative PCR were used to confirm the presence of microRNAs-30a-3p in the placenta of preeclampsia patients. In vitro experiments were carried out to investigate the effect of the expression of microRNAs-30a-3p on the function of human normal trophoblast HTR-8/SVneo cells. The results showed that the expression of microRNAs-30a-3p was positively correlated with the apoptosis rate of HTR-8/SVneo cells. We speculated that the increased expression of microRNAs-30a-3p in placenta was associated with the pathogenesis of preeclampsia. Mi-30a-3p plays a role in the pathogenesis of pre-eclampsia by regulating which or which target genes? To solve these problems, we have further carried out the prediction and validation of the target genes related to pre-eclampsia of Mi-30a-3p.
objective
1. predict the target genes of miR-30a-3p and screen the genes associated with preeclampsia as the target genes.
2. clarify the regulatory relationship between miR-30a-3p and target genes.
Method
1. Bioinformatics software was used to predict the target genes of microRNAs-30a-3p. The candidate genes were screened according to their scores in bioinformatics software and their correlation with preeclampsia.
2. Liposome transfection technique was used to establish an overexpression and inhibition model of microRNAs-30a-3p in human normal trophoblastic cell line HTR-8/Svneo. The correlation between microRNAs-30a-3p and target gene expression was detected by real-time fluorescence quantitative PCR and Western-blot.
3. To construct luciferase reporter vectors for target genes and validate the targeting relationship and binding sites between microRNAs-30a-3p and target genes by double luciferase reporter system.
Result
1. Prediction results of bioinformatics software showed that IGF-1 was one of the theoretical target genes of microarray-30a-3p. The 3'-UTR region of its mRNA sequence contained five sites that could theoretically bind to microarray-30a-3p.
2. The results of real-time PCR and Western-blot showed that the expression of miR-30a-3p increased significantly in the overexpression group (P < 0.05), whereas the expression of IGF-1 decreased significantly in the mRNA and protein levels (P < 0.05); the expression of miR-30a-3p decreased significantly in the inhibition group (P < 0.05), and the expression of IGF-1 increased significantly in the mRNA and protein levels. High (P < 0.05).
3. The results of double luciferase vector report showed that there was a definite targeting relationship between microRNAs-30a-3p and IGF-1, and there were four sites in the 3'UTR region of the IGF-1 mRNA sequence that might bind to microRNAs-30a-3p.
conclusion
IGF-1 is one of the target genes of microRNAs-30a-3p. MicroRNAs-30a-3p can negatively regulate the expression of IGF-1 at both mRNA and protein levels.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244
【参考文献】
相关硕士学位论文 前1条
1 栾丽霞;子痫前期相关miR-30a-3p的功能研究[D];第四军医大学;2012年
本文编号：2222455
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