MiR-29b与妊娠糖尿病的相关性及其分子机制的研究

发布时间：2018-05-29 20:52

本文选题：妊娠糖尿病 + 胎盘　；参考：《北京协和医学院》2017年博士论文

【摘要】：妊娠糖尿病(gestational diabetes mellitus,GDM)是指妊娠期首次发生或发现的任何程度的糖耐量受损,包含了部分妊娠前已患有糖尿病但孕期首次被诊断的患者。GDM临床经过复杂,容易造成多种不良结局,对母婴有严重的危害甚至有长远的影响。目前关于GDM的发病机制尚不明确,已有的研究表明,GDM的病因是多源性的,可能是遗传因素和环境因素共同作用的结果。微小RNA(microRNA,miRNA)是一类长度为18-23个核苷酸的,进化过程中高度保守的,在表观遗传学水平发挥负调控作用的单链小分子非编码RNA。近年来的研究发现越来越多的miRNAs在GDM发病过程中起了重要作用。胎盘是唯一连接母胎的界面,是母胎物质交换的场所,因此成为研究GDM这类糖代谢异常疾病发病机制的重要器官。为了探索在GDM中,miRNAs的分子生物学调控机制,前期我们利用miRNAs芯片筛选了在GDM和正常胎盘中差异表达的miRNAs。在本研究中我们利用qRT-PCR和原位杂交在GDM和正常人群样本中进行验证。对于差异表达的miRNAs,采用生物信息学预测其靶基因,并通过构建靶基因的3'非翻译区表达质粒在双荧光素酶报告系统中验证候选miRNAs对其靶基因表达的调控。利用候选miRNAs及其靶基因的过表达、敲低及恢复实验,研究候选miRNA通过靶基因对胎盘来源的人绒毛膜滋养层细胞细胞增殖、活性、凋亡、迁移和侵袭等功能的影响。根据miRNAs芯片结果,我们选择了 let-7b、miR-1202和miR-29b 3个差异表达的miRNAs利用qRT-PCR在大规模的人群中进行验证,结果显示miR-29b在GDM中的表达水平显著低于正常对照,年轻正常产妇的表达水平显著高于年龄较大的产妇。原位杂交结果显示miR-29b定位于胎盘滋养层细胞。MiR-29b过表达可以抑制细胞增殖、迁移和侵袭,促进细胞凋亡;MiR-29b敲低可以促进细胞迁移和侵袭。为了研究miR-29b发挥作用的分子机制,我们通过miRBase和TargetScan预测了 miR-29b的靶基因,筛选出了 3个与糖尿病和GDM相关的候选miR-29b靶基因:ING2、ING3和HIF3A。双荧光素酶报告系统分析显示miR-29b模拟物与HIF3A 3'-UTR共转染细胞,可以降低荧光素酶活性;MiR-29b抑制剂与HIF 3'-UTR共转染细胞可以升高荧光素酶活性;而miR-29b模拟物或抑制剂与ING2或INNG3 3'-UTR共转染细胞,对荧光素酶活性没有显著影响。这些结果提示了HIF3 可能是miR-29b的靶基因。为了进一步验证二者的关系,我们将HIF3'-UTR上miR-29b的识别、结合位点突变,发现其突变后与miR-29b的结合能力显著降低。同时,我们利用Western blot分析了 miR-29b对内源性靶基因表达的影响,结果显示miR-29b可以反向调节HIF3A的表达,这些结果证实了HIF3 是miR-29b的靶基因。HIF3A过表达可以促进细胞增殖、迁移和侵袭抑制细胞凋亡;HIF3A敲低可以抑制细胞迁移和侵袭。为了研究HIF3A是否为miR-29b功能性的靶基因,我们利用基因补偿实验分析了 miR-29b和其靶基因对细胞生物学行为的影响。当滋养层细胞HTR8-/SVneo过表达miR-29b,补充外源性的HIF3A可以拮抗miR-29b过表达引起的细胞增殖、迁移和侵袭抑制;当HTR8-/SVneo低表达miR-29b,抑制内源性HIF3A表达可以使细胞迁移和侵袭基本恢复正常水平。本研究的结果显示在GDM胎盘中miR-29b表达水平降低,其通过上调HIF3A的表达,促进胎盘滋养层细胞的增殖、迁移和侵袭。本研究从分子流行病学的角度研究了 miR-29b与GDM的相关性。同时,也确定了 miR-29b对胎盘来源的滋养层细胞功能的影响。
[Abstract]:Gestational diabetes mellitus (GDM) refers to any degree of impaired glucose tolerance that occurs for the first time or found during pregnancy. It includes the complication of.GDM in the patients who have been diagnosed with diabetes before pregnancy but the first diagnosis of pregnancy. It is easy to cause a variety of bad knot, serious harm to mother and baby and even a long-term shadow. The pathogenesis of GDM is not yet clear. Previous studies have shown that the etiology of GDM is multisource and may be the result of the combination of genetic and environmental factors. Small RNA (microRNA, miRNA) is a class of 18-23 nucleotides, highly conserved during evolution, and plays a negative regulatory role at epigenetic level. The study of single strand small molecule non coding RNA. in recent years has found that more and more miRNAs plays an important role in the pathogenesis of GDM. The placenta is the only interface connecting the mother fetal and the place for the exchange of maternal fetal substances. Therefore, it has become an important organ for the study of the pathogenesis of GDM, such as glucose metabolism disorder. In order to explore the molecular birth of miRNAs in GDM. We used miRNAs chip to screen the differentially expressed miRNAs. in GDM and normal placenta at the early stage. In this study, we used qRT-PCR and in situ hybridization to verify the GDM and normal population samples. For differentially expressed miRNAs, the target gene was predicted by bioinformatics and the 3'non reversal of the target gene was constructed. The expression plasmid in the double luciferase reporter system validates the regulation of the candidate miRNAs for the target gene expression in the double luciferase reporter system. Using the overexpression of the candidate miRNAs and its target gene, the knockdown and recovery experiments are used to study the function of the candidate miRNA on the proliferation, activity, apoptosis, migration and invasion of human chorionic trophoblast cells from the placental source by the target gene. Effect. According to the results of miRNAs chip, we selected 3 differentially expressed miRNAs of let-7b, miR-1202 and miR-29b to use qRT-PCR to verify in a large population. The results showed that the expression level of miR-29b in GDM was significantly lower than that of normal control. The expression of young normal women was significantly higher than that of older women. In situ hybridization node was significantly higher than that of older women. The results show that the overexpression of miR-29b in placental trophoblast cells.MiR-29b can inhibit cell proliferation, migration and invasion, and promote cell apoptosis; MiR-29b knockdown can promote cell migration and invasion. In order to study the molecular mechanism of miR-29b, we have predicted the target gene of miR-29b through miRBase and TargetScan, and screened 3 of them. The candidate miR-29b target genes related to diabetes and GDM: ING2, ING3 and HIF3A. double luciferase reporter system analysis showed that the miR-29b mimics and HIF3A 3'-UTR co transfected cells could reduce the luciferase activity, and MiR-29b inhibitors and HIF 3'-UTR co transfected cells could increase the luciferase activity; miR-29b analogue or inhibitor was associated with the HIF3A 3'-UTR. NNG3 3'-UTR co transfected cells have no significant effect on luciferase activity. These results suggest that HIF3 may be the target gene for miR-29b. In order to further verify the relationship between the two, we identified the miR-29b identification on HIF3'-UTR and the mutation of the binding site, and found that the binding capacity of the miR-29b was significantly reduced after the mutation. At the same time, we used Western. Blot analyzed the effect of miR-29b on the expression of endogenous target genes. The results showed that miR-29b could reverse the expression of HIF3A. These results confirm that HIF3 is the target gene.HIF3A overexpression of miR-29b, which can promote cell proliferation, migration and invasion to inhibit cell apoptosis; HIF3A knockdown can inhibit cell migration and invasion. In order to study HIF3A, As a target gene for miR-29b function, we used gene compensation experiments to analyze the effect of miR-29b and its target gene on cell biological behavior. When the trophoblast HTR8-/SVneo overexpressed miR-29b, supplementation of exogenous HIF3A could antagonize cell proliferation, migration and invasion inhibition caused by miR-29b overexpression; when HTR8-/SVneo was low in expression of miR- 29b, inhibiting the expression of endogenous HIF3A can make cell migration and invasion basically restored to normal levels. The results of this study showed that the level of miR-29b expression in GDM placenta decreased, and it promoted the proliferation, migration and invasion of placental trophoblast cells by up regulation of HIF3A expression. This study studied miR-29b and GDM from the molecular epidemiology point of view. At the same time, we also confirmed the effect of miR-29b on the function of trophoblast cells from placenta.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.256

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