【作者】 陈晶；

【导师】 潘尚领；

【作者基本信息】 广西医科大学 ， 病理与病理生理学， 2023， 博士

【摘要】 背景:冠心病是常见和典型的衰老相关性疾病,其发生的首要风险因素是年龄,而长寿则是健康衰老。同时研究长寿的保护因素以及衰老相关性疾病的促发因素,有助于多维度解析衰老进程的分子机制。在本项目前期研究发现hsa-miR-26b-5p的血清表达水平在冠心病组和对照组中存在差异趋势。另据报道hsa-miR-26b-5p及其宿主基因CTDSP1(CTD small phosphatase 1)与脂肪细胞的分化相关,且hsa-miR-26b-5p参与了心血管疾病的疾病进程,因此有必要深入探讨hsa-miR-26b-5p在衰老相关性疾病/健康衰老过程中的作用,以期从新的视野诠释衰老的分子机制。目的:从多个维度,包括红水河流域群体/冠心病群体血清水平,宿主基因的SNPs分布、细胞生物学功能水平,高通量转录组测序结合双荧光素酶报告基因技术,研究hsa-miR-26b-5p与冠心病和健康衰老的关系。方法:(1)采集冠心病研究总体人群、红水河流域群体的血液样本和生物信息,提取其血清总RNA,利用RT-q PCR技术检测hsa-miR-26b-5p在血清中的表达水平。综合研究对象其他临床信息,用单因素方法分析对hsa-miR-26b-5p在不同分组中的表达水平进行统计分析,用Spearman相关分析对hsa-miR-26b-5p与各临床指标的相关关系进行分析;(2)提取两个群体外周血白细胞基因组DNA,首先使用改进型连接酶检测反应(improved ligase detection reaction,i MLDR)法进行基因分型,筛选hsa-miR-26b-5p宿主基因CTDSP1的部分SNPs,并针对筛选结果使用PCR-Sanger测序法检测其中有差异趋势的SNPs,随后使用卡方检验对各群体SNPs的分型结果进行比较;(3)合成以rs1809231为中心的一段长度为156 bp野生型片段,并以此片段为模板对rs1809231位点进行点突变,形成两个突变型片段,随后构建三个片段的质粒,进行双荧光素酶报告基因实验,验证不同rs1809231等位基因对下游基因表达水平的影响;最后经单因素方差分析前述两个群体的不同rs1809231基因型的hsa-miR-26b-5p表达水平差异,及不同rs1809231基因型的多个临床信息的差异;(4)在人脐静脉内皮细胞株EA.hy 926中转染慢病毒以过表达/沉默hsa-miR-26b-5p,野生型和转染空载病毒的EA.hy 926作为对照,检测细胞功能其包括增殖、迁移、细胞周期和凋亡在内的及衰老相关指标的改变,然后采用荧光原位杂交(Fluorescence in situ hybridization,FISH)技术确定EA.hy 926细胞中hsa-miR-26b-5p的定位,探究hsa-miR-26b-5p对EA.hy 926细胞功能的影响;(5)用RNA-Seq技术检测过表达hsa-miR-26b-5p/转染空载病毒的EA.hy 926的细胞转录水平的改变,针对转录组数据综合采用miRNAs靶基因预测、GO(Gene Ontology)富集分析和KEGG Pathway富集分析等生物信息学方法,分析hsa-miR-26b-5p的靶基因及其潜在生物功能,在此基础上使用RT-q PCR和双荧光素酶报告基因实验验证靶基因的表达水平、靶基因与hsa-miR-26b-5p结合的可能性和结合的部位。结果:(1)红水河流域群体中长寿组血清hsa-miR-26b-5p表达水平,显著高于老年组、年轻组(分别是0.31±0.37 vs 0.13±0.17,0.31±0.37vs 0.21±0.3,P<0.05);冠心病研究总体人群中冠心病组血清hsa-miR-26b-5p表达水平显著低于正常对照组(0.36±0.39 vs 0.49±0.40,P<0.05);相关分析显示红水河流域群体血清hsa-miR-26b-5p表达水平与FBG、Apo A、Apo B及CRP呈负相关关系(P<0.05),其与年龄呈正相关关系(P<0.05);而冠心病研究总体人群的血清hsa-miR-26b-5p表达水平与HDL-C呈正相关关系(P<0.05);(2)经i MLDR法检测发现hsa-miR-26b-5p宿主基因CTDSP1上游启动子至CTDSP1第一内含子区域内共4个SNPs的位点信息,分别是rs1809231、rs3795982、rs3795985、rs2739049;本研究涉及的两个群体的hsa-miR-26b-5p宿主基因CTDSP1的4个SNPs位点符合Hardy-Weinberg平衡;红水河流域群体中长寿组与老年组、长寿组与非长寿组,其rs1809231位点的等位基因频率均分别存在差异(P<0.0125);(3)红水河流域群体中rs1809231 GG基因型的hsa-miR-26b-5p表达量高于GC基因型(P<0.05)。冠心病研究总体人群中SNPs rs1809231GG基因型的hsa-miR-26b-5p表达量较GC基因型的高(P<0.05)。双荧光素酶报告基因检测结果则显示,相较于对照质粒p GL3-basic+PRL-TK组和CC型CTDSP1-MT1+PRL-TK组,GG型CTDSP1-WT+PRL-TK组的荧光量均显著升高(P<0.05),携带G等位基因的野生型序列上调了下游基因的表达,而携带与C等位基因的突变型序列则对下游基因的表达没有明显影响。(4)成功建立过表达hsa-miR-26b-5p的EA.hy 926细胞模型;过表达hsa-miR-26b-5p可抑制EA.hy 926细胞的增殖能力;划痕实验和Transwell迁移实验表明过表达hsa-miR-26b-5p可降低EA.hy 926细胞的迁移能力;过表达hsa-miR-26b-5p使得S期细胞比例增加,但并未增加G2/M期细胞;过表达hsa-miR-26b-5p能抑制EA.hy 926细胞早期凋亡,但未改变细胞的整体凋亡率;过表达hsa-miR-26b-5p可抑制细胞衰老,但并不体现在丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性的改变上;最后,FISH实验提示hsa-miR-26b-5p同时定位于细胞核和细胞质。(5)相较于感染空载病毒的EA.hy 926细胞,过表达hsa-miR-26b-5p的EA.hy 926细胞有231个基因表达量下调,247个基因的表达量上调;RNA-Seq差异表达的基因与预测得到的靶向3’-UTR区的靶基因的交集有23个基因;经过验证发现过表达hsa-miR-26b-5p可下调LYPD5的表达;GO富集分析结果表明LYPD5参与了cell-substrate adhesion和cell-matrix adhesion两个生物过程,二者是影响细胞迁移的重要生物过程,表明hsa-miR-26b-5p下调LYPD5的表达,并通过这两个生物过程影响细胞的迁移能力,该结果则与细胞功能实验的结果相互印证。结论:在群体水平,我们发现hsa-miR-26b-5p在红水河流域群体长寿组中高表达,在冠心病研究总体人群冠心病组中低表达;hsa-miR-26b-5p的宿主基因CTDSP1启动子的rs1809231的G等位基因在红水河流域长寿组富集、红水河流域群体和冠心病研究总体人群中rs1809231 GG基因型的hsa-miR-26b-5p表达水平均升高,且双荧光素酶报告基因结果显示rs1809231的G等位基因可显著上调其下游基因的表达。这些结果均有力的支撑了rs1809231不同基因型可影响血清hsa-miR-26b-5p的表达水平的科学假设。进一步的细胞功能学实验表明过表达hsa-miR-26b-5p可抑制EA.hy 926细胞的增殖能力、降低其迁移能力,且明显减少衰老细胞率,因此在细胞功能学层面上,hsa-miR-26b-5p有可能通过减轻内皮细胞的衰老进程、抑制内皮细胞不良的增殖和迁移行为,发挥保护内皮细胞的功能。基于高通量测序技术的RNA-Seq结果则在宏观层面展示了过表达hsa-miR-26b-5p对细胞转录组的影响。进一步的验证实验和联合分析结果则表明,hsa-miR-26b-5p可通过下调LYPD5的表达,影响细胞迁移相关的生物过程,并最终通过影响内皮细胞的迁移活动,进而参与冠心病的疾病进程。更多还原

【Abstract】 Background:longevity and aging are two side of the aging process.Insight into protective factors of longevity and causative factors of age-related disease will help to comprehend the potential mechanism of aging process in multiple dimensions.We had found that the expression differential trend of hsa-miR-26b-5p in different group in our early background research.In consideration of hsa-miR-26b-5p and its host gene CTDSP1 being related to adipocyte differentiation,and the fact of hsa-miR-26b-5p being involved in the cardiovascular disease process,it is necessary to further discuss the role and the potential pathogenesis of hsa-miR-26b-5p in the aging/longevity process.Objective:The aim of current study is to investigate the relationship between hsa-miR-26b-5p and age-related disease/longevity in different dimensions,which included population serum expression levels of hsa-miR-26b-5p,distribution of some SNPs of population,cell biological function and its underlying molecular pathogenesis involved in age-related disease/longevity.Methods:(1)The serum total RNAs were extracted from HongshuiRiver group and CHD group(including CHD patients and healthy control group),then RT-q PCR technology was employed to detect the serum expression level of hsa-miR-26b-5p.The hsa-miR-26b-5p expression level of different groups was compared by one-way ANOVA and the correlation between hsa-miR-26b-5p and different kind of clinical parameters was analyzed by Spearman correlation analysis.(2)Genomic DNA of peripheral blood leukocytes was extracted from the same samples.i MLDR was firstly utilized to screen the SNPs of host gene CTDSP1 of hsa-miR-26b-5p in a part of samples.According to the screening results,PCR-Sanger sequencing was furtherly used to detect the SNPs with differential trend.Finally,the distribution of these SNPs in different groups were calculated by chi-square test.(3)A 156 bp wild-type fragment centered on SNP rs1809231 was synthesized,and the point mutation was carried out on the SNP rs1809231 site using this fragment as a template to form two mutant fragments,then their plasmids were constructed.The double-luciferase reporter gene assay was carried out to verify the effect of different rs1809231 allele on the downstream gene expression level.Wilcoxon rank sum test were employed to reveal the relationships between the expression level of hsa-miR-26b-5p of these genotypes and multiple cardiovascular risk factors.(4)The Human umbilical vein endothelial cells(HUVECs)line EA.hy 926model of overexpression/silencing hsa-miR-26b-5p was constructed by lentivirus transfection technology,whereas HUVECs line infected with empty virus and HUVECs line uninfected with lentivirus were used as controls.Then cell proliferation,cell cycle,cell migration,cell apoptosis and cell aging-related parameters of these different cell models were detected respectively.Finally,the cell location of hsa-miR-26b-5p in EA.hy 926 was determined by Fluorescence in situ hybridization(FISH).All these data were analyzed to speculate on the potential molecular mechanism of hsa-miR-26b-5p.(5)Transcriptome data of hsa-miR-26b-5p overexpressed cell and its control cell was achieved through RNA-Seq technology,herein differential expression genes(DEGs)were obtained.Basing on the transcriptome data,different informatics analyses,including GO enrichment analysis,KEGG pathway enrichment analysis and the target genes of hsa-miR-26b-5p were calculated to demonstrate the potential biological function of hsa-miR-26b-5p in aging/longevity.Then RT-q PCR and double-luciferase reporter gene assay were employed to validate the expression level of the target gene and its binding site with hsa-miR-26b-5p.Results:(1)The serum hsa-miR-26b-5p expression level of longevity group was higher than elderly group and young-aged group respectively in Hongshuihe river group[0.31±0.37 vs 0.13±0.17,0.31±0.37 vs 0.21±0.3,P<0.05].The serum hsa-miR-26b-5p expression level of CHD group was dramatically lower than its control group[0.36±0.39 vs 0.49±0.40,P<0.05].In light of correlation analysis result,serum hsa-miR-26b-5p expression level of longevity group was respectively negatively linked to serum FBG,serum CRP,serum Apo A and serum Apo B,while it was positively correlated with age(P<0.05).But serum hsa-miR-26b-5p expression level of CHD group was positively linked to serum HDL-C.(2)Four SNPs distribution of hsa-miR-26b-5p host gene CTDSP1,which were rs1809231,rs3795982,rs3795985 and rs2739049,were obtained by i MLDR and sanger-sequencing.Four SNPs of CTDSP1 had reached Hardy-Weinberg equilibrium in both two cohorts(Hongshuihe river group and CHD group).Significant difference was found on the G allelic frequency of rs1809231 between longevity group and non-longevity group(P<0.0125).(3)The expression level of hsa-miR-26b-5p of rs1809231 GG genotype in Hongshuihe river group was higher than that of GC genotype(P<0.05),and the expression level of hsa-miR-26b-5p of rs1809231 GG genotype in longevity group was also higher than that of GC genotype(P<0.05).Comparing with genotype GC,the genotype GG of rs1809231 had higher serum expression level of hsa-miR-26b-5p in CHD group.The expression level of hsa-miR-26b-5p of SNPs rs1809231 GG genotype in the normal control group was higher than that of GC genotype(P<0.05).The results of double luciferase reporter gene assay showed that the fluorescence of CTDSP1-WT+PRL-TK was significantly higher than that of p GL3-basic+PRL-TK and CTDSP1-MT1+PRL-TK respectively(P<0.05).Therefore,the wild type sequence carrying G allele did up-regulate the expression of downstream genes.(4)The Human umbilical vein endothelial cells(HUVECs)line EA.hy 926model of over-expressed hsa-miR-26b-5p was constructed successfully.Overexpression of hsa-miR-26b-5p in EA.hy 926 inhibited the proliferation and the early apoptosis of EA.hy 926 cells.Meanwhile,Overexpression of hsa-miR-26b-5p decreased the cell migration of EA.hy 926.The rate of aging cells of Overexpression of hsa-miR-26b-5p was lower than that of controls,but the level of MDA and the activity of SOD of all groups were similar.Then,the result of FISH indicated hsa-miR-26b-5p located widely in cytoplasm and nucleus.(5)Total 478 DEGs,including 231 down-regulated DEGs and 247 up-regulated DEGs,were demonstrated after overexpression of hsa-miR-26b-5p in EA.hy 926.After overlapping differentially expressed genes and target genes of hsa-miR-26b-5p,23 genes were obtained.After verification,overexpression of hsa-miR-26b-5p in EA.hy 926 could down regulated the expression of LYPD5.The results of GO enrichment analysis showed that LYPD5 participated in the two biological processes of cell-substrate adhesion and cell-matrix adhesion,which are important biological processes of cell migration.Conclusions:At the population level,we prove the higher serum hsa-miR-26b-5p expression level in longevity group while lower serum hsa-miR-26b-5p expression level in CHD patients.When the SNPs of hsa-mir-26b-5p host gene reach hardy Weinberg equilibrium in these populations,the G allele of SNPs rs1809231 is enriched in the longevity Group.And comparing with genotype GC,the genotype GG of rs1809231 has higher serum expression level of hsa-miR-26b-5p in both two cohorts.In addition,the results of double luciferase reporter gene assay show that the fragment with G allele of rs1809231 can significantly up-regulate the expression of its downstream gene,which strongly supported the hypothesis that different genotypes of rs1809231 can affect the expression level of serum hsa-miR-26b-5p.The result from cell biology has demonstrated the cell proliferation and the cell migration is repressed in EA.hy926 of overexpressed hsa-mir-26b-5p.And the less aging cell rate is found in cell model of overexpressed hsa-mir-26b-5p,so hsa-mir-26b-5p is proved could protect endothelial cells by relieving the aging process and inhibiting harmful proliferation and migration of endothelial cells.The RNA-Seq results indicate the effect of overexpression of hsa-miR-26b-5p on cell transcriptome at the macro level.Further validation experiments and analysis show that hsa-miR-26b-5p down-regulates the expression of LYPD5,and ultimately affects the migration ability of EA.hy 926 by two biological processes of cell-substrate adhesion and cell-matrix adhesion.更多还原