MicroRNA靶向PTEN/PI3K信号途径调控早期糖尿病肾病的研究
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摘要
第一部分早期糖尿病肾病miRNA差异表达谱分析
目的建立db/db小鼠早期糖尿病肾病(Diabetic nephropathy,DN)microRNA(miRNA)的差异表达谱,为进一步探讨microRNA在早期糖尿病肾病发生机制的作用提供依据。
方法db/db小鼠是Leptin受体基因缺陷导致的先天肥胖性2型糖尿病小鼠,4～5周后出现明显肥胖,血糖显著增高,8～10周后出现微量白蛋白尿,肾脏发生损害,其发病过程与人2型糖尿病肾病非常相似,是国际上广为采用的研究糖尿病肾病的动物模型。采用12只9周龄雄性有高血糖且24h尿白蛋白(24-h urine albumin excretion,UAE)显著增高的db/db小鼠作为早期糖尿病肾病动物模型,12只9周龄雄性背景具有同质性的db/m小鼠作为正常对照,一半用于miRNA芯片检测,另一半用于实时荧光定量RT-PCR检测。Trizol法抽提肾脏总RNA,YM-100微离心滤器富集小RNA,含568个探针的miRMouse_10.0_071620 miRNA芯片检测糖尿病肾病组和对照组miRNA的差异表达谱。利用TaqMan MicroRNA Assays定量RT-PCR试剂盒,以snoRNA202作为内标,对部分差异表达的miRNAs进行实时定量PCR检测。
结果9周龄db/db小鼠出现高血糖且UAE显著增高,提示进入早期糖尿病肾病阶段。miRNA芯片检测发现有480个miRNAs在糖尿病肾病组中表达,214个miRNAs在对照组表达。糖尿病肾病组和对照组肾脏组织表达的miRNA有66个miRNAs差异有显著性(p<0.01),其中35个miRNAs在糖尿病肾病组呈高表达,31个miRNAs在糖尿病肾病组呈低表达,差异倍数从1.1至10.2。在这66个miRNAs中有7个miRNAs在早期DN组和对照组的表达量信号值均大于1000,且差异显著(两组差异≥1.55倍),在早期DN组和对照组的表达量信号值均较高提示可靠性、可重复性较好;差异显著表明与DN关系密切。这7个miRNAs分别为:miR-196a,miR-98和miR-29c在糖尿病肾病组高表达,miR-21,miR-451,miR-709和miR-187在糖尿病肾病组低表达。利用TaqMan MicroRNA Assays定量RT-PCR试剂盒对部分miRNAs进行TaqMan探针荧光定量PCR,比较定量PCR结果与芯片结果,发现定量PCR结果与芯片结果趋势一致,表明芯片结果是可靠的,miRNAs参与了早期DN的发生。其中在细胞的增殖、分化方面尤为活跃的miR-21受到关注。
结论部分miRNAs在糖尿病肾病和正常对照肾脏组织中存在差异表达,可能参与了糖尿病肾病发生的分子机制。
第二部分MiR-21的构建及靶基因的预测研究
目的microRNA(miRNA)芯片结合实时荧光定量RT-PCR检测发现的糖尿病肾病相关miRNA-miR-21的基础上,构建miR-21真核表达载体,预测miR-21的靶基因,为进一步了解miR-21的生物学功能及在糖尿病肾病中的作用奠定基础。
方法人工合成小鼠糖尿病肾病相关的miRNA-miR-21基因序列,并添加酶切位点及poly T转录终止序列,经退火处理后形成双链结构,插入真核表达载体pGenesil-1中,经酶切鉴定和测序证实后,构建成为miR-21真核表达载体(pGenesil-miR-21)。运用生物信息学技术预测miR-21的靶基因。
结果合成的miR-21基因序列完全正确并成功克隆到真核表达载体pGenesil-1上。通过生物信息学技术,应用互联网上miRNA靶基因预测软件(Pictar、Targetscan、MiRbase、MiRanda等)在线服务站点搜索miR-21预测的作用靶基因,发现糖尿病肾病相关基因-张力蛋白在10号染色体上同源缺失的磷酸酶(Phosphatase and a tensin homolog deleted from chromosome 10, PTEN)基因3’-UTR区有16个碱基位点与mmu-miR-21匹配,结合NCBI文献检索对靶序列进行综合分析发现,有实验室通过构建双荧光素酶报告基因检测系统发现miR-21可通过结合与PTEN基因的3’UTR发挥其抑制靶基因表达的生物功能,因此PTEN基因可确立为miR-21的靶基因。
结论miR-21真核表达载体的构建,为进行miR-21在体内外表达的研究奠定基础,靶基因PTEN基因的初步确立,为进一步探讨miR-21在糖尿病肾病的信号途径分子机制提供依据。
第三部分MiR-21调控肾小球系膜细胞增殖的PTEN/PI3K信号途径研究
目的了解糖尿病肾病相关miRNA-miR-21在小鼠肾小球系膜细胞高低糖浓度培养下高表达对细胞生长、增殖的影响,及高表达miR-21的细胞PTEN/PI3K信号途径关键分子的变化,为探索miR-21调控肾小球系膜细胞增殖的分子机制提供线索。
方法采用高低糖浓度培养的小鼠肾小球系膜细胞模拟糖尿病及正常状态, Lipofectamine 2000转染miR-21真核表达载体(pGenesil-miR-21),G418筛选,稳定表达。细胞共分为4组,分别为:高糖肾小球系膜细胞转染pGenesil-miR-21组、高糖肾小球系膜细胞转染对照空质粒组、高糖肾小球系膜细胞未转染组和低糖肾小球系膜细胞对照组。运用MTT法检测细胞增殖,real-time RT-PCR检测转染质粒细胞miR-21的表达水平,western blot、免疫细胞组化检测PTEN/PI3K信号途径关键分子PTEN、p-Akt(Ser 473)和PI3K p85α的蛋白质表达水平,生物学软件分析图像结果。
结果实时荧光定量RT-PCR检测miR-21表达的结果显示,在转染pGenesil-miR-21重组质粒的小鼠肾小球系膜细胞中,miR-21的表达量比转染对照空质粒的系膜细胞高约12倍,具有显著性(p<0.01),提示筛选出的系膜细胞能稳定高表达miR-21。MTT法细胞增殖检测结果发现,高糖培养的转染对照空质粒和未转染系膜细胞增殖能力显著高于低糖下的系膜细胞,但在高糖培养的系膜细胞中miR-21重组质粒转染后其细胞增殖能力明显抑制(p均<0.01)。同时,western blot和免疫细胞化学结果显示,pGenesil-miR-21组PTEN蛋白质表达水平呈显著低于对照空质粒组和未转染组(p均<0.01),而p-Akt(Ser 473)和PI3K p85α蛋白质水平在pGenesil-miR-21组显著高于对照空质粒组和未转染组(p均<0.01)。
结论miR-21通过特异性靶向抑制PTEN蛋白表达,提高p-Akt(Ser 473)和PI3K p85α蛋白表达水平以减缓肾小球系膜细胞增殖,可能是一个预防和治疗糖尿病肾病新型的潜在靶点。
第四部分MiR-21调控db/db小鼠早期糖尿病肾病PTEN/PI3K信号途径研究
目的了解糖尿病肾病相关的miRNA-miR-21对db/db小鼠早期糖尿病肾病发病的影响,及尾静脉液压法注射pGenesil-miR-21的db/db小鼠24h尿白蛋白(24-h urine albumin excretion,UAE)、肾小球形态学、及PTEN/PI3K信号途径关键分子的变化,为探索miR-21调控糖尿病肾病肾小球系膜增生的分子机制提供线索。
方法24只5周龄雄性小鼠分为4组:6只db/m小鼠作为对照组,18只5周龄高血糖且无蛋白尿的db/db小鼠分别随机分到:pGenesil-miR-21质粒处理组、对照空质粒处理组及未处理组,每天尾静脉液压法注射质粒30mg/kg/d,直至未处理组db/db小鼠UAE显著高于db/m小鼠组。在光学显微镜下观察肾小球形态大小,计算机计算肾小球面积,电镜检测肾小球形态学改变,real-time RT-PCR检测肾脏组织miR-21的表达水平,western blot、免疫组化及激光共聚焦检测PTEN/PI3K信号途径关键分子PTEN、p-Akt(Ser 473)和PI3K p85α肾脏组织的蛋白质表达水平及定位。
结果5周龄db/db小鼠连续注射质粒4周后,未处理组db/db小鼠出现高血糖伴UAE显著增高,提示9周龄db/db小鼠进入早期糖尿病肾病阶段。Real-time RT-PCR结果显示,在注射pGenesil-miR-21重组质粒的db/db小鼠肾脏组织中,miR-21的表达量比注射对照空质粒的db/db小鼠肾脏组织高3倍多,差异具有显著性(p<0.01),提示液压法尾静脉注射的方法可以使miR-21在小鼠肾脏高表达。光镜观察和肾小球面积测量实验发现,早期DN的db/db小鼠肾小球较正常对照db/m小鼠增大,但注射pGenesil-miR-21重组质粒的db/db小鼠中,其肾小球较注射对照空质粒和未注射质粒的db/db小鼠肾小球均减小。进一步电镜检测发现,注射对照空质粒和未注射质粒的9周龄db/db小鼠肾小球上皮细胞足突融合,系膜增生,出现早期肾脏损害,但注射pGenesil-miR-21重组质粒的db/db小鼠中,系膜增生明显抑制,足突融合不明显,毛细血管内皮细胞无肿胀,系膜细胞无肿胀,提示连续4周每天液压法尾静脉注射miR-21质粒可抑制db/db小鼠肾小球系膜增生。western blot和免疫细胞化学结果显示,miR-21处理组的db/db小鼠肾脏组织中PTEN蛋白较注射对照空质粒和未注射质粒的db/db小鼠组显著降低,而PI3K p85α和phospho-Akt (Ser473)蛋白显著增高(p均<0.01),且激光共聚焦进行荧光双标实验检测发现PI3K p85α和phospho-Akt (Ser473)蛋白在miR-21处理组的db/db小鼠肾脏中呈共增长的状态。
结论miR-21通过特异性靶向调控PTEN/PI3K信号传导途径,以抑制肾小球系膜增生,可能是一个新的糖尿病肾病的保护因子。
Objective To explore the microRNA (miRNA) differential expression profile between the kidney tissues of db/db mice with diabetic nephropathy (DN) and the kidney tissues of db/m control mice, and to provide the evidence that miRNAs are involved in the molecular pathogensis of DN.
Methods Twelve 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion were used as animal model of the early stage of DN, and twelve 9-week-old male db/m mice were used as controls. Half of mice in each group were used in miRNA array, and the other half were used in real-time RT-PCR. mRNA was extracted from the kidney tissues of db/db mice and the db/m controls using Trizol reagent. MicroRNA differential expression profile in DN and normal controls were assayed by miRMouse_10.0_071620 miRNA array. Real-time RT-PCR was performed by TaqMan? MicroRNA Assays kits on the db/db DN mice and db/m controls. The relative expression was calculated using theΔΔCT method and normalized to the expression of snoRNA202.
Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. We first examined the expression of 568 miRNA species based on version 10.0 of the Sanger miRBase (http://microrna.sanger.ac.uk/sequences) by miRNA microarrays. 480 miRNAs were expressed in DN group, and 214 miRNAs were expressed in control group. Sixty-six miRNAs were differentially expressed with P<0.01. Of these, 35 were overexpressed and 31 were underexpressed in DN. The changes with P<0.01 ranged from 1.1-fold to 10.2-fold. Three upregulated (miR-196a, miR-98 and miR-29c) and four down-regulated miRNA species (miR-21, miR-451, miR-709 and miR-187) were examined. As anticipated, real-time RT-PCR confirmed the differences in expression of these miRNAs in the kidney tissues of mice in groups DN and controls.
Conclusion Some miRNAs expressed differently between DN and control groups, suggesting that they may be involved in the pathogensis of early DN.
PART II IDENTIFICATION AND TARGET PREDICTION OF MIR-21
Objective To identified the enkaryotic expression vector of miR-21 on the basis of the finding that miR-21 was a diabetic nephropathy related microRNA by microRNA microarray and real-time RT-PCR analysis. And to predicted the target gene of miR-21. To provide evidence for the potential role of miR-21 in DN.
Methods Mir-21 was chemically synthesized and contained a Sac I restriction enzyme cut site. After annealing, the DNA segments were cloned into pGenesil-1 expression vectors and confirmed by DNA sequencing. bioinformatics approach was used to identify potential candidate genes of miR-21.
Results The synthetic DNA was of the correct sequence and inserted into eukaryotic expession vector pGenesil-1 successfully. All known mRNA 3’-UTRs were scanned as potential targets for miR-21 by in silico analysis (Pictar, Targetscan, MiRbase, MiRanda, et al.). It was predicted phosphatase and a tensin homolog deleted from chromosome 10 (PTEN) gene, which contained the binding site of miR-21 in its 3’UTR, could be regulated by miR-21. With literature search, PTEN gene, a DN related gene was found a dirct target of miR-21 by luciferase report system. Therefore, PTEN gene was established the target gene of miR-21.
Conclusion The identification of pGenesil-miR-21 clone plasmid and the primary establishment that PTEN gene being the target prediction of miR-21 provide basis for the study of the molecular mechanism of miR-21 in DN.
PARTⅢEFFECT OF MIR-21 ON MESANGIAL CELL PROLIFERATION INDUCED BY HIGH GLUCOSE BY PTEN/PI3K SIGNAL PATHWAY
Objective To explore the effect of miR-21, the kidney specific microRNA on the cell growth and proliferation of the mesangial cell cultured with high glucose, and to study the regulation of high-expressed miR-21 on PTEN/PI3K signal pathway in mesangial cell cultured with high glucose. To provide the evidence for the molecular mechanism of miR-21 in mesangial cell proliferation induced by high glucose.
Methods Mesangial cells were maintained with high and low glucose to mimic diabetes mellitus and normal conditions. The expression of the enkaryotic expression vector of miR-21 (pGenesil-miR-21) was induced by transfection of the plasmid into the cells using Lipofectamine 2000, and the cells in the study was divided into 4 groups: cells in low glucose as the control group, a pGenesil-miR-21-transfected high glucose group, control empty plasmid transfected high glucose group, and untreated high glucose group. The cell proliferation was tested by MTT, the expression of miR-21 level was tested by real-time RT-PCR, the expression of PTEN, p-Akt (Ser 473) and PI3K p85αprotein levels were examined by western blot and immunohistochemistry.
Results Real-time RT-PCR showed that miR-21 was inceased in the miR-21-transfected group, suggesting miR-21 can be overexpressed by Lipofectamine 2000 transfection. MTT showed that miR-21 prevents the cell proliferation of mesangial cells cultured with high glucose. By western blot and immunohistochemistry, we found PTEN protein was reduced in the cells in the miR-21-transfected group compared with the cells in the control empty plasmid transfected group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the cells in miR-21-transfected group than the control empty plasmid group and the untreated group (all: p<0.01).
Conclusion MiR-21 may prevents mesangial cells proliferation reduced by high glucose through decreasing PTEN protein expression and increasing PI3K p85αand phospho-Akt (Ser 473) protein expressions, and it may be a novel potential DN preventative and therapic target.
PARTⅣMIR-21 REGULATES EARLY DIABETIC NEPHROPATHY BY PTEN/PI3K PATHWAY IN DB/DB MICE
Objective To explore the effect of miR-21, the kidney specific DN related microRNA on the mesangial hypertrophy of diabetic nephropathy db/db mice, and to study the regulation of high-expressed miR-21 on changes of 24-h urine albumin excretion (UAE), the morphology of glomeruli, PTEN/PI3K signal pathway in db/db mice with albuminuria. To provide the evidence for the molecular mechanism of miR-21 in DN.
Methods 24 5-week-old male mice were randomized into 4 groups: control group (6 normal untreated db/m mice), miR-21-treated db/db group (6 mice), control empty plasmid treated db/db group (6 mice), or untreated db/db group (6 mice). Mice were injected intraperitoneally using a hydrodynamics-based procedure with plasmids (30mg/kg/d of miR-21 or 30mg/kg/d of control plasmid) until albuminuria was detected in the untreated db/db mice. The glomeruli were observed under the light and electron microscope, and measured by the software in the computer. The expression of miR-21 was tested by real-time RT-PCR, the expression of PTEN, p-Akt(Ser 473) and PI3K p85αprotein levels were examined by western blot, immunohistochemistry and immunofluorescence staining.
Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. Real-time RT-PCR showed that miR-21 was inceased in the miR-21-treated group, suggesting miR-21 can be overexpressed by injected plasmids intraperitoneally using a hydrodynamics-based procedure. By measurement of glomeruli acreage, the glomeruli was smaller in miR-21-treated group than in the control empty plasmid treated and untreated groups (all: p<0.01). Additionally, miR-21 restrained mesangial hypertrophy under electron microscope. By western blot, immunohistochemistry, PTEN were reduced in the kidneys in the miR-21-treated group compared with the control empty plasmid treated group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the kidneys in miR-21-treated group than the control plasmid treated and the untreated groups (all: p<0.01). Moreover, by dual-fluorescence staining, PI3K p85αand phospho-Akt (Ser 473) were found to be co-increased in miR-21-treated group.
Conclusion MiR-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice, and it may be a novel DN protecting factor.
目的建立db/db小鼠早期糖尿病肾病(Diabetic nephropathy,DN)microRNA(miRNA)的差异表达谱,为进一步探讨microRNA在早期糖尿病肾病发生机制的作用提供依据。
方法db/db小鼠是Leptin受体基因缺陷导致的先天肥胖性2型糖尿病小鼠,4～5周后出现明显肥胖,血糖显著增高,8～10周后出现微量白蛋白尿,肾脏发生损害,其发病过程与人2型糖尿病肾病非常相似,是国际上广为采用的研究糖尿病肾病的动物模型。采用12只9周龄雄性有高血糖且24h尿白蛋白(24-h urine albumin excretion,UAE)显著增高的db/db小鼠作为早期糖尿病肾病动物模型,12只9周龄雄性背景具有同质性的db/m小鼠作为正常对照,一半用于miRNA芯片检测,另一半用于实时荧光定量RT-PCR检测。Trizol法抽提肾脏总RNA,YM-100微离心滤器富集小RNA,含568个探针的miRMouse_10.0_071620 miRNA芯片检测糖尿病肾病组和对照组miRNA的差异表达谱。利用TaqMan MicroRNA Assays定量RT-PCR试剂盒,以snoRNA202作为内标,对部分差异表达的miRNAs进行实时定量PCR检测。
结果9周龄db/db小鼠出现高血糖且UAE显著增高,提示进入早期糖尿病肾病阶段。miRNA芯片检测发现有480个miRNAs在糖尿病肾病组中表达,214个miRNAs在对照组表达。糖尿病肾病组和对照组肾脏组织表达的miRNA有66个miRNAs差异有显著性(p<0.01),其中35个miRNAs在糖尿病肾病组呈高表达,31个miRNAs在糖尿病肾病组呈低表达,差异倍数从1.1至10.2。在这66个miRNAs中有7个miRNAs在早期DN组和对照组的表达量信号值均大于1000,且差异显著(两组差异≥1.55倍),在早期DN组和对照组的表达量信号值均较高提示可靠性、可重复性较好;差异显著表明与DN关系密切。这7个miRNAs分别为:miR-196a,miR-98和miR-29c在糖尿病肾病组高表达,miR-21,miR-451,miR-709和miR-187在糖尿病肾病组低表达。利用TaqMan MicroRNA Assays定量RT-PCR试剂盒对部分miRNAs进行TaqMan探针荧光定量PCR,比较定量PCR结果与芯片结果,发现定量PCR结果与芯片结果趋势一致,表明芯片结果是可靠的,miRNAs参与了早期DN的发生。其中在细胞的增殖、分化方面尤为活跃的miR-21受到关注。
结论部分miRNAs在糖尿病肾病和正常对照肾脏组织中存在差异表达,可能参与了糖尿病肾病发生的分子机制。
第二部分MiR-21的构建及靶基因的预测研究
目的microRNA(miRNA)芯片结合实时荧光定量RT-PCR检测发现的糖尿病肾病相关miRNA-miR-21的基础上,构建miR-21真核表达载体,预测miR-21的靶基因,为进一步了解miR-21的生物学功能及在糖尿病肾病中的作用奠定基础。
方法人工合成小鼠糖尿病肾病相关的miRNA-miR-21基因序列,并添加酶切位点及poly T转录终止序列,经退火处理后形成双链结构,插入真核表达载体pGenesil-1中,经酶切鉴定和测序证实后,构建成为miR-21真核表达载体(pGenesil-miR-21)。运用生物信息学技术预测miR-21的靶基因。
结果合成的miR-21基因序列完全正确并成功克隆到真核表达载体pGenesil-1上。通过生物信息学技术,应用互联网上miRNA靶基因预测软件(Pictar、Targetscan、MiRbase、MiRanda等)在线服务站点搜索miR-21预测的作用靶基因,发现糖尿病肾病相关基因-张力蛋白在10号染色体上同源缺失的磷酸酶(Phosphatase and a tensin homolog deleted from chromosome 10, PTEN)基因3’-UTR区有16个碱基位点与mmu-miR-21匹配,结合NCBI文献检索对靶序列进行综合分析发现,有实验室通过构建双荧光素酶报告基因检测系统发现miR-21可通过结合与PTEN基因的3’UTR发挥其抑制靶基因表达的生物功能,因此PTEN基因可确立为miR-21的靶基因。
结论miR-21真核表达载体的构建,为进行miR-21在体内外表达的研究奠定基础,靶基因PTEN基因的初步确立,为进一步探讨miR-21在糖尿病肾病的信号途径分子机制提供依据。
第三部分MiR-21调控肾小球系膜细胞增殖的PTEN/PI3K信号途径研究
目的了解糖尿病肾病相关miRNA-miR-21在小鼠肾小球系膜细胞高低糖浓度培养下高表达对细胞生长、增殖的影响,及高表达miR-21的细胞PTEN/PI3K信号途径关键分子的变化,为探索miR-21调控肾小球系膜细胞增殖的分子机制提供线索。
方法采用高低糖浓度培养的小鼠肾小球系膜细胞模拟糖尿病及正常状态, Lipofectamine 2000转染miR-21真核表达载体(pGenesil-miR-21),G418筛选,稳定表达。细胞共分为4组,分别为:高糖肾小球系膜细胞转染pGenesil-miR-21组、高糖肾小球系膜细胞转染对照空质粒组、高糖肾小球系膜细胞未转染组和低糖肾小球系膜细胞对照组。运用MTT法检测细胞增殖,real-time RT-PCR检测转染质粒细胞miR-21的表达水平,western blot、免疫细胞组化检测PTEN/PI3K信号途径关键分子PTEN、p-Akt(Ser 473)和PI3K p85α的蛋白质表达水平,生物学软件分析图像结果。
结果实时荧光定量RT-PCR检测miR-21表达的结果显示,在转染pGenesil-miR-21重组质粒的小鼠肾小球系膜细胞中,miR-21的表达量比转染对照空质粒的系膜细胞高约12倍,具有显著性(p<0.01),提示筛选出的系膜细胞能稳定高表达miR-21。MTT法细胞增殖检测结果发现,高糖培养的转染对照空质粒和未转染系膜细胞增殖能力显著高于低糖下的系膜细胞,但在高糖培养的系膜细胞中miR-21重组质粒转染后其细胞增殖能力明显抑制(p均<0.01)。同时,western blot和免疫细胞化学结果显示,pGenesil-miR-21组PTEN蛋白质表达水平呈显著低于对照空质粒组和未转染组(p均<0.01),而p-Akt(Ser 473)和PI3K p85α蛋白质水平在pGenesil-miR-21组显著高于对照空质粒组和未转染组(p均<0.01)。
结论miR-21通过特异性靶向抑制PTEN蛋白表达,提高p-Akt(Ser 473)和PI3K p85α蛋白表达水平以减缓肾小球系膜细胞增殖,可能是一个预防和治疗糖尿病肾病新型的潜在靶点。
第四部分MiR-21调控db/db小鼠早期糖尿病肾病PTEN/PI3K信号途径研究
目的了解糖尿病肾病相关的miRNA-miR-21对db/db小鼠早期糖尿病肾病发病的影响,及尾静脉液压法注射pGenesil-miR-21的db/db小鼠24h尿白蛋白(24-h urine albumin excretion,UAE)、肾小球形态学、及PTEN/PI3K信号途径关键分子的变化,为探索miR-21调控糖尿病肾病肾小球系膜增生的分子机制提供线索。
方法24只5周龄雄性小鼠分为4组:6只db/m小鼠作为对照组,18只5周龄高血糖且无蛋白尿的db/db小鼠分别随机分到:pGenesil-miR-21质粒处理组、对照空质粒处理组及未处理组,每天尾静脉液压法注射质粒30mg/kg/d,直至未处理组db/db小鼠UAE显著高于db/m小鼠组。在光学显微镜下观察肾小球形态大小,计算机计算肾小球面积,电镜检测肾小球形态学改变,real-time RT-PCR检测肾脏组织miR-21的表达水平,western blot、免疫组化及激光共聚焦检测PTEN/PI3K信号途径关键分子PTEN、p-Akt(Ser 473)和PI3K p85α肾脏组织的蛋白质表达水平及定位。
结果5周龄db/db小鼠连续注射质粒4周后,未处理组db/db小鼠出现高血糖伴UAE显著增高,提示9周龄db/db小鼠进入早期糖尿病肾病阶段。Real-time RT-PCR结果显示,在注射pGenesil-miR-21重组质粒的db/db小鼠肾脏组织中,miR-21的表达量比注射对照空质粒的db/db小鼠肾脏组织高3倍多,差异具有显著性(p<0.01),提示液压法尾静脉注射的方法可以使miR-21在小鼠肾脏高表达。光镜观察和肾小球面积测量实验发现,早期DN的db/db小鼠肾小球较正常对照db/m小鼠增大,但注射pGenesil-miR-21重组质粒的db/db小鼠中,其肾小球较注射对照空质粒和未注射质粒的db/db小鼠肾小球均减小。进一步电镜检测发现,注射对照空质粒和未注射质粒的9周龄db/db小鼠肾小球上皮细胞足突融合,系膜增生,出现早期肾脏损害,但注射pGenesil-miR-21重组质粒的db/db小鼠中,系膜增生明显抑制,足突融合不明显,毛细血管内皮细胞无肿胀,系膜细胞无肿胀,提示连续4周每天液压法尾静脉注射miR-21质粒可抑制db/db小鼠肾小球系膜增生。western blot和免疫细胞化学结果显示,miR-21处理组的db/db小鼠肾脏组织中PTEN蛋白较注射对照空质粒和未注射质粒的db/db小鼠组显著降低,而PI3K p85α和phospho-Akt (Ser473)蛋白显著增高(p均<0.01),且激光共聚焦进行荧光双标实验检测发现PI3K p85α和phospho-Akt (Ser473)蛋白在miR-21处理组的db/db小鼠肾脏中呈共增长的状态。
结论miR-21通过特异性靶向调控PTEN/PI3K信号传导途径,以抑制肾小球系膜增生,可能是一个新的糖尿病肾病的保护因子。
Objective To explore the microRNA (miRNA) differential expression profile between the kidney tissues of db/db mice with diabetic nephropathy (DN) and the kidney tissues of db/m control mice, and to provide the evidence that miRNAs are involved in the molecular pathogensis of DN.
Methods Twelve 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion were used as animal model of the early stage of DN, and twelve 9-week-old male db/m mice were used as controls. Half of mice in each group were used in miRNA array, and the other half were used in real-time RT-PCR. mRNA was extracted from the kidney tissues of db/db mice and the db/m controls using Trizol reagent. MicroRNA differential expression profile in DN and normal controls were assayed by miRMouse_10.0_071620 miRNA array. Real-time RT-PCR was performed by TaqMan? MicroRNA Assays kits on the db/db DN mice and db/m controls. The relative expression was calculated using theΔΔCT method and normalized to the expression of snoRNA202.
Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. We first examined the expression of 568 miRNA species based on version 10.0 of the Sanger miRBase (http://microrna.sanger.ac.uk/sequences) by miRNA microarrays. 480 miRNAs were expressed in DN group, and 214 miRNAs were expressed in control group. Sixty-six miRNAs were differentially expressed with P<0.01. Of these, 35 were overexpressed and 31 were underexpressed in DN. The changes with P<0.01 ranged from 1.1-fold to 10.2-fold. Three upregulated (miR-196a, miR-98 and miR-29c) and four down-regulated miRNA species (miR-21, miR-451, miR-709 and miR-187) were examined. As anticipated, real-time RT-PCR confirmed the differences in expression of these miRNAs in the kidney tissues of mice in groups DN and controls.
Conclusion Some miRNAs expressed differently between DN and control groups, suggesting that they may be involved in the pathogensis of early DN.
PART II IDENTIFICATION AND TARGET PREDICTION OF MIR-21
Objective To identified the enkaryotic expression vector of miR-21 on the basis of the finding that miR-21 was a diabetic nephropathy related microRNA by microRNA microarray and real-time RT-PCR analysis. And to predicted the target gene of miR-21. To provide evidence for the potential role of miR-21 in DN.
Methods Mir-21 was chemically synthesized and contained a Sac I restriction enzyme cut site. After annealing, the DNA segments were cloned into pGenesil-1 expression vectors and confirmed by DNA sequencing. bioinformatics approach was used to identify potential candidate genes of miR-21.
Results The synthetic DNA was of the correct sequence and inserted into eukaryotic expession vector pGenesil-1 successfully. All known mRNA 3’-UTRs were scanned as potential targets for miR-21 by in silico analysis (Pictar, Targetscan, MiRbase, MiRanda, et al.). It was predicted phosphatase and a tensin homolog deleted from chromosome 10 (PTEN) gene, which contained the binding site of miR-21 in its 3’UTR, could be regulated by miR-21. With literature search, PTEN gene, a DN related gene was found a dirct target of miR-21 by luciferase report system. Therefore, PTEN gene was established the target gene of miR-21.
Conclusion The identification of pGenesil-miR-21 clone plasmid and the primary establishment that PTEN gene being the target prediction of miR-21 provide basis for the study of the molecular mechanism of miR-21 in DN.
PARTⅢEFFECT OF MIR-21 ON MESANGIAL CELL PROLIFERATION INDUCED BY HIGH GLUCOSE BY PTEN/PI3K SIGNAL PATHWAY
Objective To explore the effect of miR-21, the kidney specific microRNA on the cell growth and proliferation of the mesangial cell cultured with high glucose, and to study the regulation of high-expressed miR-21 on PTEN/PI3K signal pathway in mesangial cell cultured with high glucose. To provide the evidence for the molecular mechanism of miR-21 in mesangial cell proliferation induced by high glucose.
Methods Mesangial cells were maintained with high and low glucose to mimic diabetes mellitus and normal conditions. The expression of the enkaryotic expression vector of miR-21 (pGenesil-miR-21) was induced by transfection of the plasmid into the cells using Lipofectamine 2000, and the cells in the study was divided into 4 groups: cells in low glucose as the control group, a pGenesil-miR-21-transfected high glucose group, control empty plasmid transfected high glucose group, and untreated high glucose group. The cell proliferation was tested by MTT, the expression of miR-21 level was tested by real-time RT-PCR, the expression of PTEN, p-Akt (Ser 473) and PI3K p85αprotein levels were examined by western blot and immunohistochemistry.
Results Real-time RT-PCR showed that miR-21 was inceased in the miR-21-transfected group, suggesting miR-21 can be overexpressed by Lipofectamine 2000 transfection. MTT showed that miR-21 prevents the cell proliferation of mesangial cells cultured with high glucose. By western blot and immunohistochemistry, we found PTEN protein was reduced in the cells in the miR-21-transfected group compared with the cells in the control empty plasmid transfected group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the cells in miR-21-transfected group than the control empty plasmid group and the untreated group (all: p<0.01).
Conclusion MiR-21 may prevents mesangial cells proliferation reduced by high glucose through decreasing PTEN protein expression and increasing PI3K p85αand phospho-Akt (Ser 473) protein expressions, and it may be a novel potential DN preventative and therapic target.
PARTⅣMIR-21 REGULATES EARLY DIABETIC NEPHROPATHY BY PTEN/PI3K PATHWAY IN DB/DB MICE
Objective To explore the effect of miR-21, the kidney specific DN related microRNA on the mesangial hypertrophy of diabetic nephropathy db/db mice, and to study the regulation of high-expressed miR-21 on changes of 24-h urine albumin excretion (UAE), the morphology of glomeruli, PTEN/PI3K signal pathway in db/db mice with albuminuria. To provide the evidence for the molecular mechanism of miR-21 in DN.
Methods 24 5-week-old male mice were randomized into 4 groups: control group (6 normal untreated db/m mice), miR-21-treated db/db group (6 mice), control empty plasmid treated db/db group (6 mice), or untreated db/db group (6 mice). Mice were injected intraperitoneally using a hydrodynamics-based procedure with plasmids (30mg/kg/d of miR-21 or 30mg/kg/d of control plasmid) until albuminuria was detected in the untreated db/db mice. The glomeruli were observed under the light and electron microscope, and measured by the software in the computer. The expression of miR-21 was tested by real-time RT-PCR, the expression of PTEN, p-Akt(Ser 473) and PI3K p85αprotein levels were examined by western blot, immunohistochemistry and immunofluorescence staining.
Results 9-week-old male db/db mice with hyperglycemia and significant elevation of urinary albumin excretion showed features similar to the early stage of diabetic nephropathy. Real-time RT-PCR showed that miR-21 was inceased in the miR-21-treated group, suggesting miR-21 can be overexpressed by injected plasmids intraperitoneally using a hydrodynamics-based procedure. By measurement of glomeruli acreage, the glomeruli was smaller in miR-21-treated group than in the control empty plasmid treated and untreated groups (all: p<0.01). Additionally, miR-21 restrained mesangial hypertrophy under electron microscope. By western blot, immunohistochemistry, PTEN were reduced in the kidneys in the miR-21-treated group compared with the control empty plasmid treated group and untreated group (all: p<0.01). Whereas PI3K p85αand phospho-Akt (Ser 473) were increased in the kidneys in miR-21-treated group than the control plasmid treated and the untreated groups (all: p<0.01). Moreover, by dual-fluorescence staining, PI3K p85αand phospho-Akt (Ser 473) were found to be co-increased in miR-21-treated group.
Conclusion MiR-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice, and it may be a novel DN protecting factor.
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[3] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell. 2004, 116(2):281-297.
[4] Poy MN, Eliasson L, Krutzfeldt J, et al. A pancreatic islet-specific microRNA regulates insulin secretion[J]. Nature. 2004, 432 (7014): 226-230.
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