miR-21靶向内源性脂蛋白受体相关蛋白6突变在非酒精性脂肪性肝病发病中的作用
发布时间：2019-02-16 18:47
【摘要】：目的:非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)是以肝脏脂肪蓄积和异常脂质代谢为特征的慢性肝脏疾病。随着生活方式的全球转型,NAFLD患者人口急速上升,现已成为21世纪全球重要的公共健康问题之一,给社会带来了巨大的经济负担。近年来,大量研究已经显示NAFLD中微小RNA(microRNA,miRNA)谱发生改变,探索通过miRNA靶向治疗NAFLD及相关病变的新兴分子机制引起了研究者的注意。有研究报道,miR-21及低密度脂蛋白(Low-density lipoprotein,LDL)受体相关蛋白6(LDL receptor-related protein 6,LRP6)均参与NAFLD的发生、发展,但miR-21是否可以作为其治疗靶标以及miR-21与LRP6之间的关系均是未知的。在本研究中,使用NAFLD的体外细胞模型进行实验,以研究mi R-21对脂质合成和分泌的影响,并确定miR-21对NAFLD发挥其作用的新靶标。方法:(1)使用软脂酸(Palmitic acid,PA)和油酸(Oleic acid,OA)(1:2)混合物(PA/OA)干预Hep G2细胞建立NAFLD体外细胞模型。用siRNA技术沉默PA/OA干预后HepG2细胞中的miR-21表达,用miR-21 mimic或对照(control)mimic转染PA/OA干预后Hep G2细胞使miR-21表达上调,然后监测miR-21对胆固醇(Cholesterol,C),胆固醇酯(Cholesteryl ester,CE),甘油三酯(Triglyceride,TG)和磷脂(Phospholipid,PL)合成和分泌的影响;(2)miR-21 mimic或controlmimic,mir-21antagomir或controlantagomir转染pa/oa干预后hepg2细胞分别上调和阻断mir-21的表达,检测lrp6表达;(3)实时荧光定量pcr和蛋白免疫印迹(westernblot)分析检测参与脂质代谢的基因,包括肝脏x受体α(liverxreceptorα,lxrα),硬脂酰辅酶a去饱和酶1(stearoyl-coadesaturase1,scd1),乙酰辅酶a羧化酶1(acetyl-coacarboxylase1,acc1),和固醇调节元件结合蛋白1(sterolregulatoryelementbindingprotein1,srebp1)等,以及lrp6在mrna和蛋白质水平的表达;(4)运行targetscan程序,之后将lrp63’非翻译区(untranslatedarea,utr)或lrp6突变的3'-utr克隆到荧光素酶报告基因载体中,lxra3’-utr克隆到荧光素酶报告基因载体作为阴性对照,之后分别与mir-21mimic或controlmimic共转染pa/oa干预后hepg2细胞进行双荧光素酶报告基因检测,探讨mir-21表达水平对lrp6的影响。结果:(1)mir-21mimic转染pa/oa干预后hepg2细胞可以使除了ce之外的脂类,包括c,tg,pl合成和分泌增加,实时荧光定量pcr显示mir-21表达上调可以诱导编码脂肪生成酶基因,包括acc1,scd1,srebp1和lxrα等的表达增加;(2)mir-21mimic转染pa/oa干预后的hepg2细胞,实时荧光定量pcr和westernblot显示lrp6在mrna和蛋白质水平表达降低。反之,mir-21antagomir转染却使lrp6的表达水平增加;(3)运行targetscan程序,显示lrp6为mir-21的潜在靶标。双荧光素酶报告基因检测显示,lrp63'-utr和mir-21mimic共转染的细胞中荧光素酶活性显著降低,mir-21mimic的转染对lrp6突变的3'-utr和lxra3’-utr的荧光素酶活性没有任何影响。结论:mir-21在nafld的体外细胞模型中调节脂类代谢,该作用通过抑制LRP6的表达来实现。LRP6是mi R-21的新靶标。mi R-21可能是通过靶向内源性LRP6治疗NAFLD的新策略。
[Abstract]:Objective: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by liver fat accumulation and abnormal lipid metabolism. With the global transformation of life style, the rapid increase of the population of NAFLD has become one of the most important public health problems in the 21 st century, which has brought great economic burden to society. In recent years, a large number of studies have shown that the microRNA (microRNA, miRNA) spectrum in NAFLD has changed, and the discovery of the new molecular mechanism targeted to the treatment of NAFLD and related lesions by miRNA has caused the attention of the researchers. It is reported that both miR-21 and low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) are involved in the development and development of NAFLD, but whether the relationship between miR-21 and miR-21 or miR-21 and LRP6 is unknown. In this study, an in vitro cell model of NAFLD was used to study the effects of mi R-21 on lipid synthesis and secretion and to determine the new target for miR-21 to play its role in NAFLD. Methods: (1) The in vitro cell model of NAFLD was established by using palmitic acid (PA) and oleic acid (OA) (1: 2) mixture (PA/ OA). The expression of miR-21 in HepG2 cells after the PA/ OA intervention was silenced by siRNA technology, and the miR-21 expression was upregulated with miR-21 mimoic or control (control) mimomic transfection of PA/ OA, and then miR-21 was monitored for cholesterol (Choleyl, C), cholesteryl ester (CE), triglyceride (Trilycoide, TG) and phospholipid (Phospholipid, (2) the expression of mir-21 and the expression of mir-21 were up-regulated and blocked by miR-21 momic or control mic, mir-21antagomir or conrol agomir, and the expression of lrp6 was detected; and (3) real-time fluorescence quantitative pcr and western blot were used to analyze and detect the genes involved in the lipid metabolism, include a liver x-receptor antagonist, an lxr antigen, a stearin-coenzyme a desaturase 1 (scd1), an ethylene-co-enzyme a-coacarbox1, acc1), and a sterol regulatory element binding protein 1 (srebp1), and the like, and the expression of lrp6 at the mrna and protein level; and (4) run the targetscan procedure, after which the lrp63 the 3 '-utr of the' untranslated area, utr 'or lrp6 mutation was cloned into the luciferase reporter gene vector, and the lxra3'-utr was cloned into the luciferase reporter gene vector as a negative control, followed by a double-luciferase reporter gene detection with the mir-21mmic or the control momic co-transfection of the pa/ oa, respectively, to study the effect of mir-21 expression level on lrp6. Results: (1) After the mir-21mmic transfection of pa/ oa, the hepg2 cells can increase the synthesis and secretion of lipids other than ce, including c, tg, pl, and increase the expression of mir-21 in real time, and the expression of mir-21 can induce the expression of the encoded fat-producing enzyme gene, including the expression of acc1, scd1, srefbp1, and lxr, and so on. (2) The expression of mrp6 in mrp6 in mrp6 was decreased in mrp6 after mir-21mmic transfected with pa/ oa. On the contrary, mir-21antagomir transfection resulted in an increase in the expression level of lrp6; (3) the runtescan procedure was run to show lrp6 as a potential target for mir-21. The luciferase activity was significantly reduced in both lrp63 '-utr and mir-21mmic co-transfected cells, and the transfection of mir-21mmic did not have any effect on the luciferase activity of the 3'-utr and lxra3 '-utr of the lrp6 mutation. Conclusion: Mir-21 regulates lipid metabolism in the in vitro cell model of nafld, which is achieved by inhibiting the expression of LRP6. LRP6 is a new target for mi R-21. The mi R-21 may be a new strategy for the treatment of NAFLD by targeting endogenous LRP6.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
本文编号：2424735
[Abstract]:Objective: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by liver fat accumulation and abnormal lipid metabolism. With the global transformation of life style, the rapid increase of the population of NAFLD has become one of the most important public health problems in the 21 st century, which has brought great economic burden to society. In recent years, a large number of studies have shown that the microRNA (microRNA, miRNA) spectrum in NAFLD has changed, and the discovery of the new molecular mechanism targeted to the treatment of NAFLD and related lesions by miRNA has caused the attention of the researchers. It is reported that both miR-21 and low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) are involved in the development and development of NAFLD, but whether the relationship between miR-21 and miR-21 or miR-21 and LRP6 is unknown. In this study, an in vitro cell model of NAFLD was used to study the effects of mi R-21 on lipid synthesis and secretion and to determine the new target for miR-21 to play its role in NAFLD. Methods: (1) The in vitro cell model of NAFLD was established by using palmitic acid (PA) and oleic acid (OA) (1: 2) mixture (PA/ OA). The expression of miR-21 in HepG2 cells after the PA/ OA intervention was silenced by siRNA technology, and the miR-21 expression was upregulated with miR-21 mimoic or control (control) mimomic transfection of PA/ OA, and then miR-21 was monitored for cholesterol (Choleyl, C), cholesteryl ester (CE), triglyceride (Trilycoide, TG) and phospholipid (Phospholipid, (2) the expression of mir-21 and the expression of mir-21 were up-regulated and blocked by miR-21 momic or control mic, mir-21antagomir or conrol agomir, and the expression of lrp6 was detected; and (3) real-time fluorescence quantitative pcr and western blot were used to analyze and detect the genes involved in the lipid metabolism, include a liver x-receptor antagonist, an lxr antigen, a stearin-coenzyme a desaturase 1 (scd1), an ethylene-co-enzyme a-coacarbox1, acc1), and a sterol regulatory element binding protein 1 (srebp1), and the like, and the expression of lrp6 at the mrna and protein level; and (4) run the targetscan procedure, after which the lrp63 the 3 '-utr of the' untranslated area, utr 'or lrp6 mutation was cloned into the luciferase reporter gene vector, and the lxra3'-utr was cloned into the luciferase reporter gene vector as a negative control, followed by a double-luciferase reporter gene detection with the mir-21mmic or the control momic co-transfection of the pa/ oa, respectively, to study the effect of mir-21 expression level on lrp6. Results: (1) After the mir-21mmic transfection of pa/ oa, the hepg2 cells can increase the synthesis and secretion of lipids other than ce, including c, tg, pl, and increase the expression of mir-21 in real time, and the expression of mir-21 can induce the expression of the encoded fat-producing enzyme gene, including the expression of acc1, scd1, srefbp1, and lxr, and so on. (2) The expression of mrp6 in mrp6 in mrp6 was decreased in mrp6 after mir-21mmic transfected with pa/ oa. On the contrary, mir-21antagomir transfection resulted in an increase in the expression level of lrp6; (3) the runtescan procedure was run to show lrp6 as a potential target for mir-21. The luciferase activity was significantly reduced in both lrp63 '-utr and mir-21mmic co-transfected cells, and the transfection of mir-21mmic did not have any effect on the luciferase activity of the 3'-utr and lxra3 '-utr of the lrp6 mutation. Conclusion: Mir-21 regulates lipid metabolism in the in vitro cell model of nafld, which is achieved by inhibiting the expression of LRP6. LRP6 is a new target for mi R-21. The mi R-21 may be a new strategy for the treatment of NAFLD by targeting endogenous LRP6.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
【参考文献】
相关期刊论文 前2条
1 Zhen-Ya Lu;Zhou Shao;Ya-Li Li;Muhuyati Wulasihan;Xin-Hua Chen;;Prevalence of and risk factors for non-alcoholic fatty liver disease in a Chinese population: An 8-year follow-up study[J];World Journal of Gastroenterology;2016年13期
2 范建高;;非酒精性脂肪性肝病诊疗指南[J];临床肝胆病杂志;2010年02期
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