miR-21靶向內(nèi)源性脂蛋白受體相關(guān)蛋白6突變?cè)诜蔷凭灾拘愿尾“l(fā)病中的作用
發(fā)布時(shí)間：2019-02-16 18:47
【摘要】：目的:非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)是以肝臟脂肪蓄積和異常脂質(zhì)代謝為特征的慢性肝臟疾病。隨著生活方式的全球轉(zhuǎn)型,NAFLD患者人口急速上升,現(xiàn)已成為21世紀(jì)全球重要的公共健康問(wèn)題之一,給社會(huì)帶來(lái)了巨大的經(jīng)濟(jì)負(fù)擔(dān)。近年來(lái),大量研究已經(jīng)顯示NAFLD中微小RNA(microRNA,miRNA)譜發(fā)生改變,探索通過(guò)miRNA靶向治療NAFLD及相關(guān)病變的新興分子機(jī)制引起了研究者的注意。有研究報(bào)道,miR-21及低密度脂蛋白(Low-density lipoprotein,LDL)受體相關(guān)蛋白6(LDL receptor-related protein 6,LRP6)均參與NAFLD的發(fā)生、發(fā)展,但miR-21是否可以作為其治療靶標(biāo)以及miR-21與LRP6之間的關(guān)系均是未知的。在本研究中,使用NAFLD的體外細(xì)胞模型進(jìn)行實(shí)驗(yàn),以研究mi R-21對(duì)脂質(zhì)合成和分泌的影響,并確定miR-21對(duì)NAFLD發(fā)揮其作用的新靶標(biāo)。方法:(1)使用軟脂酸(Palmitic acid,PA)和油酸(Oleic acid,OA)(1:2)混合物(PA/OA)干預(yù)Hep G2細(xì)胞建立NAFLD體外細(xì)胞模型。用siRNA技術(shù)沉默PA/OA干預(yù)后HepG2細(xì)胞中的miR-21表達(dá),用miR-21 mimic或?qū)φ?control)mimic轉(zhuǎn)染PA/OA干預(yù)后Hep G2細(xì)胞使miR-21表達(dá)上調(diào),然后監(jiān)測(cè)miR-21對(duì)膽固醇(Cholesterol,C),膽固醇酯(Cholesteryl ester,CE),甘油三酯(Triglyceride,TG)和磷脂(Phospholipid,PL)合成和分泌的影響;(2)miR-21 mimic或controlmimic,mir-21antagomir或controlantagomir轉(zhuǎn)染pa/oa干預(yù)后hepg2細(xì)胞分別上調(diào)和阻斷mir-21的表達(dá),檢測(cè)lrp6表達(dá);(3)實(shí)時(shí)熒光定量pcr和蛋白免疫印跡(westernblot)分析檢測(cè)參與脂質(zhì)代謝的基因,包括肝臟x受體α(liverxreceptorα,lxrα),硬脂酰輔酶a去飽和酶1(stearoyl-coadesaturase1,scd1),乙酰輔酶a羧化酶1(acetyl-coacarboxylase1,acc1),和固醇調(diào)節(jié)元件結(jié)合蛋白1(sterolregulatoryelementbindingprotein1,srebp1)等,以及l(fā)rp6在mrna和蛋白質(zhì)水平的表達(dá);(4)運(yùn)行targetscan程序,之后將lrp63’非翻譯區(qū)(untranslatedarea,utr)或lrp6突變的3'-utr克隆到熒光素酶報(bào)告基因載體中,lxra3’-utr克隆到熒光素酶報(bào)告基因載體作為陰性對(duì)照,之后分別與mir-21mimic或controlmimic共轉(zhuǎn)染pa/oa干預(yù)后hepg2細(xì)胞進(jìn)行雙熒光素酶報(bào)告基因檢測(cè),探討mir-21表達(dá)水平對(duì)lrp6的影響。結(jié)果:(1)mir-21mimic轉(zhuǎn)染pa/oa干預(yù)后hepg2細(xì)胞可以使除了ce之外的脂類,包括c,tg,pl合成和分泌增加,實(shí)時(shí)熒光定量pcr顯示mir-21表達(dá)上調(diào)可以誘導(dǎo)編碼脂肪生成酶基因,包括acc1,scd1,srebp1和lxrα等的表達(dá)增加;(2)mir-21mimic轉(zhuǎn)染pa/oa干預(yù)后的hepg2細(xì)胞,實(shí)時(shí)熒光定量pcr和westernblot顯示lrp6在mrna和蛋白質(zhì)水平表達(dá)降低。反之,mir-21antagomir轉(zhuǎn)染卻使lrp6的表達(dá)水平增加;(3)運(yùn)行targetscan程序,顯示lrp6為mir-21的潛在靶標(biāo)。雙熒光素酶報(bào)告基因檢測(cè)顯示,lrp63'-utr和mir-21mimic共轉(zhuǎn)染的細(xì)胞中熒光素酶活性顯著降低,mir-21mimic的轉(zhuǎn)染對(duì)lrp6突變的3'-utr和lxra3’-utr的熒光素酶活性沒(méi)有任何影響。結(jié)論:mir-21在nafld的體外細(xì)胞模型中調(diào)節(jié)脂類代謝,該作用通過(guò)抑制LRP6的表達(dá)來(lái)實(shí)現(xiàn)。LRP6是mi R-21的新靶標(biāo)。mi R-21可能是通過(guò)靶向內(nèi)源性LRP6治療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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
本文編號(hào)：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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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診療指南[J];臨床肝膽病雜志;2010年02期
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