史秋香,吴艳峰,谢丽华
SHI Qiuxiang,WU Yanfeng,XIE Lihua

• 1:吉林省公安消防总队长春支队卫生队
• 2:吉林大学白求恩第二医院呼吸与危重症医学科
• 3:中南大学湘雅三医院

摘要(Abstract)：

目的:探究mi RNA-21过表达对TGF-β/Smad信号通路Smad7蛋白的影响。方法:采用mi RNA-21过表达慢病毒载体和慢病毒空载体分别感染高糖和低糖培养的人肾近端小管上皮细胞HK-2细胞,根据培养基类型和是否感染病毒将细胞分组为高糖mi RNA-21过表达慢病毒组、低糖mi RNA-21过表达慢病毒组、高糖空载组、低糖空载组、高糖细胞组和低糖细胞组,用荧光倒置显微镜观察感染后表达绿色荧光蛋白(GFP)细胞数,通过流式细胞术检测转染率;采用实时荧光定量PCR检测感染后细胞mi RNA-21的表达量,采用Western blot检测Smad7蛋白的表达。结果:感染mi RNA-21过表达慢病毒后,荧光显微镜下HK-2细胞有明显绿色荧光(约为60%),感染率结果显示转染率>60%,实时荧光定量PCR结果显示感染mi RNA-21过表达慢病毒后,高糖转染组细胞mi RNA-21表达量高于高糖细胞组和高糖空载组(P<0.05);Weston blot结果显示高糖转染组Smad 7蛋白的表达低于高糖转染空载病毒组、低糖细胞组、高糖细胞组(P<0.05)。结论:mi RNA-21可以通过抑制Smad7蛋白的表达来调控TGF-β/Smad信号通路。
Objective: To explore the effect of mi RNA-21 overexpression on TGF-β/Smad signaling pathway Smad7 protein. Methods: The mi RNA-21 overexpression lentiviral vector was used to infect HK-2 cells cultured with high glucose and low sugar respectively. According to the type of culture medium and whether or not the virus was infected,the cells were divided into high glucose mi RNA-21 overexpression lentivirus group,low glucose mi RNA-21 overexpression lentiviral group,high glucose and empty virus group,low sugar empty virus group,high glucose cell group and low glucose cell group. The expression of green fluorescent protein( GFP) cells( green fluorescence) after infection was observed by fluorescence inverted microscope,and the transfection rate was detected by flow cytometry. The expression of mi RNA-21 in infected cells was detected by real-time quantitative PCR,and the expression of Smad7 protein was detected by Western blot. Results: When infected with mi RNA-21 overexpressing lentivirus,HK-2 cells showed significant green fluorescence( about 60%) under fluorescence microscope. The infection rate showed that transfection rate was 60% or more. The results of real-time quantitative PCR showed that the expression of mi RNA-21 in the high glucose transfection group was higher than that in the high glucose cell group as well as the high glucose and empty virus group after infection with mi RNA-21 overexpression lentivirus( P < 0. 05). The results of Weston blot showed that the expression of Smad 7 protein in high glucose transfection group was lower than that in high glucose empty virus group,low sugar cell group and high glucose cell group( P < 0. 05). Conclusion: mi RNA-21 can regulate the TGF-β/Smad signaling pathway by inhibiting the expression of Smad7 protein.

关键词(KeyWords)： TGF-β/Smad信号通路;Smad7蛋白;miRNA-21;慢病毒表达载体;HK-2细胞;实时荧光定量PCR
TGF-β/Smad signaling pathway;Smad7 protein;miRNA-21;lentivirus expression vector;HK-2 cell;real-time fluorescence quantification PCR

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81660810)

作者(Author): 史秋香,吴艳峰,谢丽华
SHI Qiuxiang,WU Yanfeng,XIE Lihua

DOI: 10.19367/j.cnki.1000-2707.2017.10.005

参考文献(References)：

• [1]魏锦锦,李佳鑫,刘巨源.肺间质纤维化中肌成纤维细胞的来源及调控因素[J].新乡医学院学报,2009(6):633-636.
• [2]B9ttinger EP.TGF-beta in renal injury and disease.[J].Seminars in Nephrology,2007(3):309.
• [3]杨力,李荟元.Smad蛋白家族与TGF-β信号转导[J].中国美容医学杂志,2001(6):547-549.
• [4]Blobe GC,Schiemann WP,Lodish HF.Mechanisms of disease:Role of Transforming Growth Factorßin human disease[J].New England Journal of Medicine,2000(18):1350-1358.
• [5]Drummond A,Findlay J.Focus on TGF-βsignalling[J].Reproduction,2006(2):177-178.
• [6]杨秀华,孟涛.TGF-β2和TGFβRⅡ在子痫前期胎盘中的表达[J].贵阳医学院学报,2016(2):185-189.
• [7]Eulertaimor G,Heger J.The complex pattern of SMAD signaling in the cardiovascular system[J].Cardiovascular Research,2006(1):15.
• [8]邹崎葩.甲基化抑制剂5-氮杂-2-脱氧胞苷对瘢痕疙瘩成纤维细胞TGF-β/smad信号通路的影响[D].重庆:重庆医科大学,2014.
• [9]黄静,蔡颖,朱丽华,等.Micro RNA在肿瘤EMT调控中的作用[J].分子诊断与治疗杂志,2014(6):420-423.
• [10]高冬梅,朱明玥,唐努尔,等.mi RNA-196a与宫颈癌的关系研究[J].贵阳医学院学报,2014(4):522-525.
• [11]甄滢滢,姚广涛,金若敏,等.mi RNA-21和mi RNA-34a在环孢素A致大鼠肾损伤肾脏中的表达[Z].中国海南海口,2015:2.
• [12]成学琴.核心蛋白聚糖在肾小管间质损害中的作用[D].南京:南京医科大学,2007.
• [13]陈静,覃远汉,赵艳君,等.PAX2在单侧输尿管梗阻大鼠肾小管间质纤维化中的作用[J].实用儿科临床杂志,2009(5):355-357.
• [14]Wang B,Jha JC,Hagiwara S,et al.Transforming growth factor-β1-mediated renal fibrosis is dependent on the regulation of transforming growth factor receptor 1 expression by let-7b[J].Kidney international,2014(2):352-361.
• [15]Ding Y,Choi ME.Regulation of autophagy by TGF-β:emerging role in kidney fibrosis[J]Seminars in nephrology.WB Saunders,2014(1):62-71.
• [16]李莹莹.转化生长因子β_1对子宫内膜腺癌细胞增殖、细胞周期、凋亡的影响[D].沈阳:中国医科大学,2010.
• [17]Zhang J,Zhang X,Xie F,et al.The regulation of TGF-β/SMAD signaling by protein deubiquitination[J].Protein&cell,2014(7):503-517.
• [18]Wu TT,Lu J,Zheng PQ,et al.Yiqi Huayu Jiedu decoction inhibits the invasion and metastasis of gastric Cancer cells through TGF-β/Smad Pathway[J].Evidence-Based Complementary and Alternative Medicine,2017(19):1-8.
• [19]颜小明,张立婷,李敏,等.TGF-β1/Smad信号通路在肝纤维化中的研究进展[J].现代生物医学进展,2016(9):1778-1781.
• [20]Poznansky M,Lever A,Bergeron L,et al.Gene transfer into human lymphocytes by a defective human immunodeficiency virus type 1 vector[J].Journal of Virology,1991(1):532-536.
• [21]赖靖,訾聃,杨英捷.短发夹RNA干扰CXCR4表达对卵巢癌SKOV3细胞凋亡及侵袭能力的影响[J].贵州医科大学学报,2016(6):653-659.
• [22]Wang JY,Gao YB,Zhang N,et al.mi R-21 overexpression enhances TGF-β1-induced epithelial-to-mesenchymal transition by target smad7 and aggravates renal damage in diabetic nephropathy[J].Molecular&Cellular Endocrinology,2014(2):163-172.
• [23]Han M,Liu M,Wang Y,et al.Re-expression of mi R-21contributes to migration and invasion by inducing epithelial-mesenchymal transition consistent with cancer stem cell characteristics in MCF-7 cells[J].Molecular and Cellular Biochemistry,2012(1):427.
• [24]Han M,Wang Y,Liu M,et al.Mi R-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible factor-1αexpression in third-sphere forming breast cancer stem cell-like cells[J].Cancer Science,2012(6):1058-1064.
• [25]Costa PM,Cardoso AL,Custódia C,et al.Mi RNA-21silencing mediated by tumor-targeted nanoparticles combined with sunitinib:a new multimodal gene therapy approach for glioblastoma[J].Journal of Controlled Release,2015(207):31-39.

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