H19介导miR-let-7的分子机制及其在妊娠期糖尿病中的作用研究
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摘要
背景妊娠期糖尿病作为临床中常见的妊娠期合并症,其严重威胁产妇及胎儿的生命健康。H19作为一种印记基因,进一步分析其为妊娠期糖尿病靶向治疗工作的开展提供依据。目的研究H19介导miR-let-7的分子机制及其在妊娠期糖尿病中的作用。方法 2017年1月—2018年1月于北京大学医学部购进50只SPF级小鼠,根据小鼠的血糖以及血脂水平等将其分为正常组(n=20)和高脂组(n=30)。采用实时荧光定量反转录聚合酶链式反应(RT-qPCR)技术分析正常组和高脂组小鼠H19、let-7、靶基因脂蛋白脂肪酶(LPL)以及肝脏胰岛素受体β(INSR-β)和胰岛素通路相关因子2(IRS-2)、胰岛素受体(INSR)的相对m RNA表达水平。同时于小鼠成肌细胞株中,分别通过细胞转染技术转染干扰RNA(siRNA),特异性敲除小鼠H19,以及转染反义链抑制剂ilet-7,特异性阻断let-7结合靶基因,共转染后分别在第3、6周时收获细胞,观察不同时间点H19、let-7以及靶基因LPL、INSR-β、IRS-2和INSR的相对m RNA表达水平。在人卵巢畸胎瘤细胞系PA-1中,分别通过细胞转染技术转染siRNA,特异性敲除人H19,以及转染反义链抑制剂ilet-7,特异性阻断let-7结合靶基因,共转染后分别在第3、6周时收获细胞,观察各组中H19、let-7以及靶基因LPL、INSR-β、IRS-2及INSR的相对mRNA表达水平。结果高脂组小鼠H19、let-7及LPL mRNA表达水平高于正常组,INSR-β、IRS-2及INSR mRNA表达水平低于正常组(P<0.05)。不同时间点小鼠成肌细胞株H19、let-7、LPL、INSR-β、IRS-2及INSR mRNA表达水平比较,差异均有统计学意义(P<0.05)。不同时间点人卵巢畸胎瘤细胞系PA-1 H19、let-7、LPL、INSR-β、IRS-2及INSR mRNA表达水平比较,差异均有统计学意义(P<0.05)。结论 H19在miR-let-7中发挥分子介导机制,且在妊娠期糖尿病的参与中发挥至关重要的作用,临床中应当加强对妊娠期孕妇的H19以及miR-let-7等相关指标的检测,实现对妊娠期糖尿病的早期预测。
Background Gestational diabetes mellitus(GDM) is a gestational complication commonly seen in clinical practice,which seriously threatens the life and health of the mother and the fetus.The analysis of the molecular mechanism of miR-let-7 mediated by H19(an imprinted gene) and its role in the development of GDM can provide a basis for targeted therapy for this disease.Objective To study the molecular mechanism of H19-mediated miR-let-7 and its role in GDM.Methods This experiment was conducted from January 2017 to January 2018.The 50 SPF grade experimental mice were purchased from Peking University Health Science Center.After 7 days of free diet,the mice were divided into normal-fat group(20 cases) and high-fat group(30 cases) according to their blood glucose and lipid levels.RT-qPCR was used to analyze the relative mRNA expression levels of H19,let-7,target genes LPL,INSR-β in the liver and IRS-2 in mice of the two groups.At the same time in mice myoblast cell lines,through cell transfection technique,small interfering RNA(siRNA) was transfected for the knockdown of the H19,and antisense strand iLet-7 was transfected for specifically blocking the let-7 to combine with target genes.3 and 6 weeks after transfection,cells were harvested,The relative mRNA expression levels of H19,let-7 and target genes LPL,INSR-β,IRS-2 and INSR were observed at different time points.And the same experimental procedure was carried out in human ovarian teratoma-derived cell line PA-1.Results The normal-fat group showed significantly lower mRNA expression levels of H19,let-7 and LPL,and much higher mRNA expression levels of INSR-β,IRS-2 and INSR compared with the high-fat group(P<0.05).The mRNA expression levels of the above-mentioned 6 indicators at baseline,and at the end of 3 weeks and 6 weeks of intervention were all significantly different in mice myoblast cell lines(P<0.05),as did they in human ovarian teratoma-derived cell line PA-1(P<0.05).Conclusion H19 mediates the molecular mechanism in miR-let-7 and plays a vital role in the development of GDM.Clinical detection of relevant indicators such as H19 and miR-let-7 in pregnant women should be strengthened in order to identify GDM as early as possible.
Background Gestational diabetes mellitus(GDM) is a gestational complication commonly seen in clinical practice,which seriously threatens the life and health of the mother and the fetus.The analysis of the molecular mechanism of miR-let-7 mediated by H19(an imprinted gene) and its role in the development of GDM can provide a basis for targeted therapy for this disease.Objective To study the molecular mechanism of H19-mediated miR-let-7 and its role in GDM.Methods This experiment was conducted from January 2017 to January 2018.The 50 SPF grade experimental mice were purchased from Peking University Health Science Center.After 7 days of free diet,the mice were divided into normal-fat group(20 cases) and high-fat group(30 cases) according to their blood glucose and lipid levels.RT-qPCR was used to analyze the relative mRNA expression levels of H19,let-7,target genes LPL,INSR-β in the liver and IRS-2 in mice of the two groups.At the same time in mice myoblast cell lines,through cell transfection technique,small interfering RNA(siRNA) was transfected for the knockdown of the H19,and antisense strand iLet-7 was transfected for specifically blocking the let-7 to combine with target genes.3 and 6 weeks after transfection,cells were harvested,The relative mRNA expression levels of H19,let-7 and target genes LPL,INSR-β,IRS-2 and INSR were observed at different time points.And the same experimental procedure was carried out in human ovarian teratoma-derived cell line PA-1.Results The normal-fat group showed significantly lower mRNA expression levels of H19,let-7 and LPL,and much higher mRNA expression levels of INSR-β,IRS-2 and INSR compared with the high-fat group(P<0.05).The mRNA expression levels of the above-mentioned 6 indicators at baseline,and at the end of 3 weeks and 6 weeks of intervention were all significantly different in mice myoblast cell lines(P<0.05),as did they in human ovarian teratoma-derived cell line PA-1(P<0.05).Conclusion H19 mediates the molecular mechanism in miR-let-7 and plays a vital role in the development of GDM.Clinical detection of relevant indicators such as H19 and miR-let-7 in pregnant women should be strengthened in order to identify GDM as early as possible.
引文
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[11]张洁,李慧阁,傅力,等.高脂饮食干预父代C57BL/6小鼠对子代肝脏脂肪沉积的影响[J].中华肝脏病杂志,2017,25(2):139-144.DOI:10.3760/cma.j.issn.1007-3418.2017.02.012.ZHANG J,LI H G,FU L,et al.Influence of high-fat diet in paternal C57BL/6 mice on liver fat deposition in offspring[J].Chinese Journal of Hepatology,2017,25(2):139-144.DOI:10.3760/cma.j.issn.1007-3418.2017.02.012.
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[2]王杏,王超,宋光耀,等.氧化苦参碱对高脂诱导胰岛素抵抗ApoE-/-小鼠肝脏脂代谢相关基因的影响[J].中国药理学通报,2015,31(12):1688-1692.DOI:10.3969/j.issn.1001-1978.2015.12.012.WANG X,WANG C,SONG G Y,et al.Effect of oxymatrine on lipid metabolism regulated genes in liver of fat-induced insulin resistance in ApoE-/-mice[J].Chinese Pharmacological Bulletin,2015,31(12):1688-1692.DOI:10.3969/j.issn.1001-1978.2015.12.012.
[3]MORGADO A L,RODRIGUES C M,SOLA S.MicroRNA-145regulates neural stem cell differentiation through the Sox2-Lin28/let-7signaling pathway[J].Stem Cells,2016,34(5):1386-1395.
[4]王喜欢,张金华,胡亚南,等.大蒜素对高脂饮食ApoE-/-小鼠动脉粥样硬化形成的影响[J].中国动脉硬化杂志,2017,28(2):140-144.WANG X H,ZHANG J H,HU Y N,et al.Effect and mechanisms of allicin on atherosclerosis in high-fat fed mice[J].Chinese Journal of Atherosclerosis,2017,28(2):140-144.
[5]孙婧,刘素娟,牛燕媚,等.Wnt信号通路在有氧运动改善高脂饮食诱导C57BL/6小鼠胰岛素抵抗中的作用[J].中国运动医学杂志,2015,34(3):219-227.SUN J,LIU S J,NIU Y M,et al.The role of Wnt signaling pathway in attenuating high-fat diet induced-insulin resistance in C57BL/6 mice after aerobic exercise[J].Chinese Journal of Sports Medicine,2015,34(3):219-227.
[6]汤月霞,孟建忠,丁亚娟,等.电针对高脂饮食诱导的肥胖小鼠白色脂肪组织中腺苷受体表达的影响[J].针刺研究,2017,42(1):39-44.DOI:10.13702/j.1000-0607.2017.01.007.TANGYX,MENGJZ,DINGYJ,etal.Effectof electroacupuncture on adenosine receptor expression in white adipose tissue of dietinduced obese mice[J].Acupuncture Study,2017,42(1):39-44.DOI:10.13702/j.1000-0607.2017.01.007.
[7]ECSEDI M,RAUSCH M,GROBHANS H.The let-7,microRNA directsvulvaldevelopmentthroughasingletarget[J].Developmental Cell,2015,32(3):335-344.
[8]张浩强,王茹,张龙,等.TLR2基因敲除下调高脂饮食诱导的肥胖小鼠脂肪组织炎症因子表达和细胞凋亡[J].免疫学杂志,2017,33(2):164-168.ZHANG H Q,WANG R,ZHANG L,et al.TLR2 knockouts down the expression of inflammatory factors and apoptosis in adipose tissue of obese mice induced by high-fat diet[J].Journal of Immunology,2017,33(2):164-168.
[9]朱振梁,尹洪萍,陈如程,等.Toll样受体3对高脂饮食小鼠棕色脂肪组织中解偶联蛋白1表达的影响[J].现代预防医学,2017,44(13):2401-2405.ZHU Z L,YIN H P,CHEN R C,et al.Effects of Toll like receptor3 on the uncoupling protein 1 expression in brown adipose tissue in mice with high-fat diet[J].Modern Preventive Medicine,2017,44(13):2401-2405.
[10]崔菊,陈爱群,庞婧,等.高脂饮食影响小鼠体内能量代谢的性别差异[J].山西医科大学学报,2017,48(7):658-664.DOI:10.13753/j.issn.1007-6611.2017.07.005.CUI J,CHEN A Q,PANG J,et al.Difference of energy metabolism between male and female mice exposed to high fat diet[J].Journal of Shanxi Medical University,2017,48(7):658-664.DOI:10.13753/j.issn.1007-6611.2017.07.005.
[11]张洁,李慧阁,傅力,等.高脂饮食干预父代C57BL/6小鼠对子代肝脏脂肪沉积的影响[J].中华肝脏病杂志,2017,25(2):139-144.DOI:10.3760/cma.j.issn.1007-3418.2017.02.012.ZHANG J,LI H G,FU L,et al.Influence of high-fat diet in paternal C57BL/6 mice on liver fat deposition in offspring[J].Chinese Journal of Hepatology,2017,25(2):139-144.DOI:10.3760/cma.j.issn.1007-3418.2017.02.012.
[12]POBEZINSKY L A,ETZENSPERGER R,JEURLING S,et al.Let-7 miRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function[J].Nat Immunol,2015,16(5):517-524.DOI:10.1038/ni.3146.
[13]黄珊珊,鲁一兵.长链非编码RNA与糖尿病[J].国际内分泌代谢杂志,2015,35(4):271-274.DOI:10.3760/cma.j.issn.1673-4157.2015.04.016.HUANG S S,LU Y B.Long non-coding RNA and diabetes mellitus[J].International Journal of Endocrinology and Metabolism,2015,35(4):271-274.DOI:10.3760/cma.j.issn.1673-4157.2015.04.016.
[14]童佩,刘云,孙丽洲,等.Lin28/let-7调控区SNP对妊娠期糖尿病易感性的影响与机制研究[J].临床和实验医学杂志,2017,16(1):20-23.DOI:10.3969/j.issn.1671-4695.2017.01.006.TONG P,LIU Y,SUN L Z,et al.Effect and mechanism of Lin28/let-7 SNP on the susceptibility of gestational diabetes mellitus in SNP control area[J].Journal of Clinical and Experimental Medicine,2017,16(1):20-23.DOI:10.3969/j.issn.1671-4695.2017.01.006.
[15]吕洋,弓春玲,孙佳佳,等.Let-7的靶基因研究以及与妊娠的相关性[J].中国生育健康杂志,2016,27(2):193-195.DOI:10.3969/j.issn.1671-878X.2016.02.028.LYU Y,GONG C L,SUN J J,et al.Target gene study of let-7 and its correlation with pregnancy[J].Chinese Journal of Reproductive Health,2016,27(2):193-195.DOI:10.3969/j.issn.1671-878X.2016.02.028.
[16]刘奇云,马敬,陶艳萍,等.葛根素对妊娠期糖尿病大鼠胰腺组织let-7f及免疫因子IL-17、IL-23表达的影响[J].中国免疫学杂志,2017,33(12):1843-1848.DOI:10.3969/j.issn.1000-484X.2017.12.018.LIU Q Y,MA J,TAO Y P,et al.Influence of puerarin on pancreatic tissue let-7f and immune factors IL-17 and IL-23expression of rats with gestational diabetes mellitus[J].Chinese Journal of Immunology,2017,33(12):1843-1848.DOI:10.3969/j.issn.1000-484X.2017.12.018.
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