| 钱倩宇,潘然,常开心,等.Let-7诱导自噬在酒精性肝病中的作用研究[J].浙江中医药大学学报,2019,43(5):474-480. |
| Let-7诱导自噬在酒精性肝病中的作用研究 |
| Let-7 Synergistically Regulate the Gene Expression in Alcoholic Liver Disease |
| DOI：10.16466/j.issn1005-5509.2019.05.021 |
| 中文关键词: 酒精性肝病 Let-7 lncRNA MicroRNA 自噬 |
| 英文关键词: alcoholic liver disease Let-7 lncRNA MicroRNA autophagy |
| 基金项目:浙江省自然科学基金(LR15H030003)；国家自然科学基金项目(81773981、81473393)；浙江省中医药科研基金计划资助(2019ZQ010) |
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| 中文摘要: |
| [目的]探讨lncRNA/let-7在酒精性肝病 (alcohol liver disease, ALD) 中的作用机制。[方法]通过喂食饲料含酒精的酒精喂养组 (alcohol feeding group, AF)，建立基于C57BL / 6小鼠的ALD动物模型。制备血液和肝脏组织样品。在ALD小鼠模型和H2O2处理的人肝癌细胞 (Human hepatocellular carcinoma, HepG2) 细胞氧化应激模型中测定6种let-7 miRNA候选物的表达水平。基于Targetscan 6.2和miRanda 3.3a的生物信息学分析用于预测可能与let-7相互作用的下游靶基因和长链非编码RNA (long non-coding RNA, lncRNA) 候选物。然后使用qRT-PCR在小鼠肝组织中定量预测的靶miRNA和lncRNA的水平。[结果] 1) AF组肝组织中let-7表达显著低于对照饲料(pair feeding, PF)组 (P<0.05) ，特别是let-7c、let-7d和let-7g表达水平显著降低 (P<0.05) 。2) 与对照组 (untreated, UT) 相比，H2O2处理的HepG2细胞中let-7表达也下调 (P<0.05) 。3) AF组中发现自噬相关基因4 (autophagy related gene 4, Atg4)、自噬相关基因1 (autophagy related gene 1, Atg1)、自噬基因Beclin、自噬微管相关蛋白1轻链3B (light chain 3B, LC3B) 等多种重要自噬生物标志物的表达水平均下调 (P<0.05) ，与慢性酒精摄入可抑制细胞自噬水平的理论相一致。4) 预测了3种调控let-7的潜在lncRNA候选物NONMMUT012132、NONMMUT019851和NONMMUT068425。其中，NONMMUT012132的表达在AF组中上调，而其他两个lncRNA被下调。[结论]本研究表明，由酒精消耗过量引起的肝组织氧化损伤可能导致let-7 miRNAs和细胞自噬活性的下调，lncRNA可能参与let-7活性水平的调节。 |
| 英文摘要: |
| [Objective] To investigate the mechanistic role of let-7 in the pathogenesis of alcohol liver disease(ALD).[Methods] An ALD animal model based on C57BL/6 mice was established by feeding an ethanol-containing(alcohol feeding group, AF) diet for 5 weeks. The blood and liver tissue samples were prepared. Hematoxylin and eosin staining, alanine aminotransferase activity and triglyceride assays were performed to verify liver injury and hepatic fat accumulation. Bioinformatics analysis based on Targetscan 6.2 and miRanda 3.3a was used to predict the downstream target genes and lncRNA candidates that could interact with let-7. The levels of the predicted target miRNAs and lncRNAs were then quantified in mouse liver tissues using qRT-PCR. [Results] 1) The liver tissues harvested from AF group showed markedly lower let-7 expression than those from PF group. In particular, let-7c, let-7d and let-7g levels declined most significantly. 2) Let-7 expression was also down-regulated in H2O2-treated HepG2 cells compared with the untreated control group(UT). 3) The expression levels of several important autophagy biomarkers, including autophagy related gene 4(Atg4), autophagy related gene 1(Atg1), beclin and light chain 3B(LC3B), were found to be down-regulated in AF group, which was consistent with the prevailing theory that chronic alcohol consumption could suppress cell autophagy activities.4) Three lncRNA candidates, denoted NONMMUT012132, NONMMUT019851 and NONMMUT068425, were predicted to be potentially able to target let-7. Among them, the expression of NONMMUT012132 was found to undergo up-regulation in AF group, whereas the other two lncRNAs were down-regulated. [Conclusion] Our study indicated that increased oxidative stress in liver tissues caused by excessive alcohol consumption could lead to down-regulation of let-7 miRNAs and cell autophagy activity. In addition, lncRNAs might be involved in the regulation of let-7 activity levels. These results could contribute to the development of effective therapeutics against let-7 related liver diseases. |
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