摘要
目的非酒精性脂肪性肝病(NAFLD)目前已逐渐成为威胁人类生活健康的最常见的肝病之一,然而其发病机制尚未完全阐释清楚,近期有研究表明在非酒精性脂肪肝病存在异常表达的piRNAs,其中piRNA-DQ723301可能通过靶向调控Smad2参与非酒精性脂肪肝病的发生发展,但其靶向作用及具体机制尚未被完全阐明。因此,本实验的目的旨在研究piRNA-DQ723301与Smad2是否存在靶向调控,从而参与NAFLD的发生发展,以不断完善piRNA在NAFLD发病机制中的作用奠定理论基础。方法1.设计含有piRNA-DQ723301结合位点的Smad23′-UTR PCR引物,通过双荧光素酶实验分析得出piRNA-DQ723301对Smad2的靶向调控作用。2.通过CCK8法检测,比较转染不同浓度piRNA-DQ723301模拟物及抑制剂48h后各组细胞的活力,通过比较得出较合适的浓度。3.Smad2 mRNA及蛋白、piRNA-DQ723301的表达情况通过Western-blot法、实时荧光定量PCR分析。并观察piRNA-DQ723301对非酒精性脂肪肝纤维化发病关键因子的影响。结果1.将构建的阴性对照序列、质粒、模拟物共同转染至293T细胞,针对荧光素酶活性进行检测。表明与对照组相比,piRNA-DQ723301以野生型质粒为载体时,酶活性能受到抑制;而以突变型质粒为载体时,荧光素酶酶活性则无明显改变,且piRNA-DQ723301类似物野生质粒组荧光素酶活性比piRNA-DQ723301类似物突变型质粒组组明显减少,差异有统计学意义(P<0.01)。2.CCK8法结果表明,小鼠原代肝星状细胞活力在转染100 nM、150 nM的模拟物、抑制剂后分别呈现出下降、增强趋势,转染50 nM的模拟物、抑制剂及各浓度的对照序列则基本不变。3.相较于未转染组,转染组piRNA-DQ723301表达量、Smad2 mRNA表达量、α-SMA mRNA在NC组、Mimic组、Inhibitor组分别呈现出基本不变、显著增高(P<0.001)、明显降低(P<0.001)的变化趋势。4.相较于未转染组,各转染组Smad2蛋白的表达量在NC组、Mimic组、Inhibitor组的变化趋势分别为基本不变、显著降低(P<0.001)、明显升高(P<0.001);Piwi蛋白的表达量在NC组、Mimic组、Inhibitor组的变化趋势分别为基本不变、Piwil3基本不变及Piwil1、Piwil2、Piwil4显著增高(P<0.01)、Piwil3基本不变及Piwil1、Piwil2、Piwil4降低(P<0.05)。与Inhibitor组相比,Mimic组Piwi表达量增加(P<0.01),Smad2表达量降低(P<0.001)。5.与未转染组比较,TGF-β1、α-SMA蛋白表达量在NC组、Mimic组的变化趋势分别为基本不变、显著降低(P<0.001),Inhibitor组则显著上升(P<0.001)。结论1.piRNA-DQ723301针对Smad2的表达具有靶向调节作用,能够抑制其表达;2.piRNA-DQ723301可靶向调控Smad2抑制肝星状细胞活力及功能;3.利用piRNA-DQ723301的靶向调控作用或可对肝纤维化的发生发展产生抑制甚至逆转作用。
Objective Non alcoholic fatty liver disease(NAFLD)has gradually become one of the most common liver diseases that threaten human health.However,its pathogenesis has not been fully elucidated.Recent studies have shown that piRNAs with abnormal expression exist in non-alcoholic fatty liver disease,among which piRNA-DQ723301 may participate in the occurrence and development of NAFLD by targeting Smad2,but its targeting effect and specific mechanism have not been fully elucidated.Therefore,the purpose of this experiment is to investigate whether piRNA-DQ723301 and Smad2 have targeted regulation and participate in the occurrence and development of NAFLD,in order to continuously improve the role of piRNA in the pathogenesis of NAFLD and lay a theoretical foundation.Methods 1:Design Smad23′-UTR PCR primers containing the binding site of piRNA-DQ723301,and analyze the targeted regulatory effect of piRNA-DQ723301 on Smad2 through dual luciferase assay.2.Using the CCK8 method,compare the viability of cells transfected with different concentrations of piRNA-DQ723301 mimetics and inhibitors for 48 hours,and determine the appropriate concentration through comparison.3.The expression of Smad2 mRNA and protein,as well as piRNA-DQ723301,was analyzed by Western blot and real-time fluorescence quantitative PCR.And observe the effect of piRNA-DQ723301 on key factors in the pathogenesis of non-alcoholic fatty liver fibrosis.Results 1.The constructed negative control sequence,plasmid,and mimic were co transfected into 293T cells for detection of luciferase activity.Compared with the control group,the enzyme activity of piRNA-DQ723301 can be inhibited when using wild-type plasmids as vectors;When using mutant plasmids as vectors,there was no significant change in luciferase activity,and the luciferase activity in the wild plasmid group of piRNA-DQ723301 analogs was significantly reduced compared to the mutant plasmid group of piRNA-DQ723301 analogs,with statistical significance(P<0.01).2.The results of CCK8 method indicate that the viability of mouse primary hepatic stellate cells showed a decreasing and increasing trend after transfection with 100nM and 150nM mimetics and inhibitors,respectively,while transfection with 50nM mimetics,inhibitors,and control sequences at various concentrations remained basically unchanged.3.Compared with the non transfected group,the expression levels of piRNA-DQ723301,Smad2 mRNA,andα-SMAmRNA in the transfected group showed a trend of basically unchanged,significantly increased(P<0.001),and significantly decreased(P<0.001)in the NC group,Mimic group,and inhibitor group,respectively.4.Compared with the non transfected group,the expression levels of Smad2 protein in the NC group,Mimic group,and inhibitor group showed a trend of basically unchanged,significantly decreased(P<0.001),and significantly increased in each transfected group(P<0.001);The expression levels of Piwi protein in the NC group,Mimic group,and Prohibitor group showed a trend of basically unchanged,Piwil3 basically unchanged,and Piwil1,Piwil2,and Piwil4 significantly increased(P<0.01),Piwil3 basically unchanged,and Piwil1,Piwil2,and Piwil4 decreased(P<0.05),respectively.Compared with the inhibitor group,the Mimic group showed an increase in Piwi expression(P<0.01)and a decrease in Smad2 expression(P<0.001).5.Compared with the non transfected group,the changes in the expression levels of TGF-β1 andα-SMA proteins in the NC group and Mimic group remained basically unchanged and significantly decreased(P<0.001),while the inhibitor group showed a significant increase(P<0.001).Conclusion 1.piRNA-DQ723301 has a targeted regulatory effect on the expression of Smad2 and can inhibit its expression;2.piRNA-DQ723301 can target and regulate Smad2 inhibition of hepatic stellate cell viability and function;3.The targeted regulatory effect of piRNA-DQ723301 may inhibit or even reverse the occurrence and development of liver fibrosis.
作者
杨雷雷
李昌平
刘超
宦徽
李沁鸿
YANG Leilei;LI Changping;LIU Chao;HUAN Hui;LI Qinhong(Department of Gastroenterology,Chengdu Office Hospital of the People′s Government of the Xizang Autonomous Region,Chengdu 610041,China;Department of Gastroenterology,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China;Health Management Center of West China Tianfu Hospital,Sichuan University,Chengdu 610213,China)
出处
《现代消化及介入诊疗》
2024年第10期1186-1192,共7页
Modern Interventional Diagnosis and Treatment in Gastroenterology
基金
西藏自治区自然科学基金项目(XZ202001ZR0073G)。
