MicroRNA-204-5p及其靶基因SIRT1在糖尿病角膜病变中的作用及机制研究

发布时间：2018-04-21 15:16

本文选题：MicroRNA-204-5p + SIRT1　；参考：《华中科技大学》2015年博士论文

【摘要】：糖尿病(Diabetes Mellitus, DM)是一组以慢性血糖水平增高为特征的代谢性疾病,是由于胰岛素分泌和(或)作用缺陷所引起。近年来糖尿病发病率持续增高,人们日益对糖尿病及其并发症防治予以重视。在眼科领域,糖尿病角膜病变在糖尿病患者比较常见,糖尿病患者眼部手术术后角膜上皮的迁延不愈是临床棘手的难题。糖尿病角膜病变主要临床表现为浅层点状角膜炎或持续性上皮缺损以及角膜知觉的减退。很多研究表明角膜上皮基底膜的异常增厚和角膜上皮细胞功能改变共同导致了糖尿病角膜病变。目前糖尿病角膜病变的发病机制尚不明确,其发生的机制以及治疗对策仍是临床中亟待解决的科学问题。沉默信号调控因子1(silence signal regulating factor1, SlRT1)是一种组蛋白脱乙酰化酶,参与糖代谢和胰岛素分泌等多条代谢通路,被认为是治疗糖尿病的新靶点。已有研究证实SIRT1在糖尿病角膜中表达显著下调,过量表达SIRT1后可以显著促进糖尿病小鼠角膜上皮的损伤修复。但引起糖尿病角膜SIRT1表达下调的原因尚不清楚。 MicroRNAs(miRNAs, miRs)是一类长度约为22个核苷酸(nucleotide, nt)的内源性非编码RNA,通过与其靶蛋白的3端非编码区(Untranslated Regions, UTRs)区结合,完成转录后水平的基因调控。它参与了多个生物学进程如发育、细胞死亡、细胞增殖、中枢神经系统功能及肿瘤形成等。它能通过调控众多靶基因从而发挥生物学效应。目前对miRNAs的研究虽广泛,但有关糖尿病角膜病变的miRNAs研究少且不深入。因此本研究选择围绕SIRT1这一糖尿病的重要靶点,拟从表观遗传学的角度探讨是否在角膜组织中存在某种或某些miRNAs,它能否通过影响SIRT1的表达进而改变糖尿病角膜上皮的损伤修复能力。本研究目的是筛选角膜上皮中可能调控SIRTl的miRNAs,探讨所筛选出的miRNAs通过SIRT1在糖尿病角膜病变中的作用,以期探明其是否参与糖尿病角膜病变以及和提供新的治疗靶点。第一部分筛选microRNAs及对靶基因SIRT1的作用研究目的：筛选糖尿病角膜病变中调控Sirt1表达的microRNA (miRNA) 方法：采用生物信息学的方法预测调控Sirtl的可能miRNAs,通过实时定量PcR(Quantitative ReaI time PCR,qRT-PCR)的方法在糖尿病小鼠角膜上皮组织进行验证；采用miRNA的模拟物或抑制剂转染正常小鼠角膜缘上皮干／祖细胞系(TKE2)细胞,从而实现miRNA在TKE2细胞的过量或者下降的表达结果：从生物信息学结果中挑选出9个miRNAs用于其后的qRT-PCR的验证,其中miR-204-5p用于后续的研究,与非糖尿病的对照小鼠角膜上皮组织相比,其表达是糖尿病角膜上皮组织的5.16倍。正常TKE2转染miR-204-5p的模拟物或抑制物后,SIRT1表达显著下调或上调。双荧光素酶报告基因分析结果显示miR-204-5p的靶基因是Sirt1。结论：在糖尿病角膜中miR-204-5p表达显著上调。MiR-204-5p能负性调控SIRT1,其高表达是导致糖尿病角膜上皮中Sirtl低表达的原因之一。第二部分调控microRNA-204-5p通过SIRT1促进高糖环境下小鼠角膜缘上皮干／祖细胞系(TKE2)细胞周期循环目的：探究miR-204-5p通过SIRTl是否能影响因高糖而被阻滞TKE2细胞周期循环。方法：MiR-204-5p的抑制剂转染高糖环境下的TKE2细胞：q RT-PCR/Western blot检测SIRT1,细胞周期相关蛋白cyclin D1.p21.p16的表达；MTT法检测细胞增殖变化；PI染色法流失细胞仪检测细胞周期变化。结果：TKE2细胞转染miR-204-5p的抑制物后显著促进了高糖环境下细胞生长与细胞周期的循环,Sirtl与Cyclin D1的表达上调,而p16的表达下调。结论：抑制miR-204-5p的表达,可通过对Sirtl的调控,促进高糖环境下被阻滞的角膜上皮细胞周期循环第三部分调控microRNA-204-5p通过SIRT1促进糖尿病小鼠C57BL/6J-INS2AKita (INS2Akita/+)角膜上皮损伤修复究目的：探究在糖尿病角膜中miR-204-5p通过SIRTl是否能影响角膜上皮损伤修复。方法：建立机械性角膜上皮损伤小鼠动物模型,结膜下注射miR-204-5p,观察建模成功后0-72小时内角膜上皮缺损面积,用于评价该miRNA对糖尿病角膜上皮损伤修复的作用。qRT-PCR/Western blot检测角膜中SIRT1,细胞周期相关蛋白cyclin D1、 p21、 p16的表达。结果：对Ins2AKita/+小鼠结膜下注射miR-204-5p的模拟物后,显著促进了角膜上皮的损伤修复,该过程中SIRTl的表达增加,激活Cyclin D1/CDK1途径,Cyclin D1的表达增加而p16的表达下降。结论：抑制miR-204-5p的表达,能通过Sirt1促进糖尿病角膜上皮的损伤修复。
[Abstract]:Diabetes Mellitus ( DM ) is a group of metabolic diseases characterized by elevated levels of chronic blood glucose , which are caused by insulin secretion and / or deficiency of action . In the field of ophthalmology , diabetic keratopathy is more common in diabetic patients . In the field of ophthalmology , diabetic keratopathy is more common in diabetic patients .

The silencing signal regulating factor 1 ( SlRT1 ) is a new target for the treatment of diabetes , which is a new target for the treatment of diabetes mellitus .

MicroRNAs ( miRs ) are endogenous non - coding RNAs with a length of about 22 nucleotides ( nucleotides , nt ) , which are combined with the 3 - terminal non - coding region of their target protein to complete the gene regulation at post - transcriptional level . It has been involved in many biological processes such as development , cell death , cell proliferation , central nervous system function and tumor formation .

The aim of this study was to screen the possible regulation of SIRTl in corneal epithelium and investigate the role of SIRT1 in diabetic keratopathy , with a view to exploring whether it was involved in diabetic keratopathy and providing a new treatment target .

The first part of screening microRNAs and the effect on target gene SIRT1

Objective : To screen the microRNA ( miRNA ) regulating Sirt1 expression in diabetic keratopathy .

Methods : Using bioinformatics methods to predict the possible expression of Sirtl , we used real - time quantitative PCR ( qRT - PCR ) to verify the corneal epithelial tissue of diabetic mice .
miRNA - based mimics or inhibitors were used to transfected normal mouse limbal epithelial stem / progenitor cell line ( TKE2 ) cells , thereby enabling overexpression or decreased expression of miRNA in TKE2 cells

Results : The expression of miR - 204 - 5p was 5 . 16 times that of non - diabetic control mice . The expression of miR - 204 - 5p was significantly downregulated or up - regulated after normal TKE2 transfection of miR - 204 - 5p . Conclusion : The expression of miR - 204 - 5p in diabetic cornea is significantly up - regulated . Conclusion : The high expression of miR - 204 - 5p is one of the causes of low expression of Sirtl in diabetic corneal epithelium .

The second part regulates microRNA - 204 - 5p to promote cell cycle circulation of mouse corneal epithelial stem / progenitor cell line ( TKE2 ) in high - sugar environment through SIRT1 .

Objective : To investigate the effect of miR - 204 - 5p on cell cycle of TKE2 blocked by high glucose .

Methods : The inhibitors of MiR - 204 - 5p were transfected into TKE2 cells in high - sugar environment : q - RT - PCR / Western blot was used to detect the expression of cyclin D1.p21 . p16 .
MTT assay was used to detect cell proliferation .
Cell cycle changes were detected by PI staining .

Results : After transfection of the inhibitor of miR - 204 - 5p by TKE2 cells , the cycle of cell growth and cell cycle in high glucose environment was significantly promoted , and the expression of Sirtl and Cyclin D1 was up - regulated , while the expression of p16 was down regulated .

Conclusion : The expression of miR - 204 - 5p can be inhibited and the cycle of corneal epithelial cells blocked in high glucose environment can be promoted by regulating Sirtl .

The third part regulates microRNA - 204 - 5p to promote the repair of corneal epithelial damage in diabetic mice C57BL / 6 J - INS2AKita ( INS2Akita / + ) by SIRT1 .

Objective : To investigate whether miR - 204 - 5p can influence corneal epithelial damage repair by SIRTl in diabetic cornea .

Methods : To establish an animal model of mechanical corneal epithelium injury in mice . miR - 204 - 5p was injected into the conjunctiva , and the area of corneal epithelium defect was observed within 0 - 72 hours after successful modeling . The expression of SIRT1 , cyclin D1 , p21 and p16 in the cornea was detected by qRT - PCR / Western blot .

Results : After injection of miR - 204 - 5p in the conjunctiva of Ins2AKita / + mice , the repair of corneal epithelium was significantly promoted , the expression of SIRTl increased , the expression of Cyclin D1 / CDK1 and Cyclin D1 increased , and the expression of p16 was decreased .

Conclusion : Inhibition of miR - 204 - 5p expression can promote the repair of diabetic corneal epithelium by Sirt1 .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R772.2

【相似文献】