MiR-377靶向调控Egr1在乙肝肝纤维化病理机制中的作用
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摘要
目的:研究miR-377靶向调控早期生长反应因子(early growth response l,Egr1)在肝纤维化病理机制中的作用。方法:RT-qPCR检测肝纤维化患者血清中miR-377的表达;用miR靶基因数据库进行筛选,选择Egr1为miR-377的潜在靶基因;RT-qPCR和Western印迹法分别检测miR-377 mimics对Egr1的mRNA及蛋白表达的影响;CCK-8检测Ang Ⅱ诱导后HSC细胞和转染miR-377的HSC细胞增殖程度;RT-qPCR检测经AngⅡ和AngⅡ+miR-377刺激后与α-平滑肌动蛋白(α-smooth muscle actin,α-SM A),FSP1和Col lagenI的表达量。结果:MiR-377可能与肝纤维化的发生及发展有关;miR-377可以通过结合Egr1的3'-UTR直接抑制Egr1的mRNA翻译及其蛋白表达;miR-377可抑制肝细胞经AngⅡ诱导后的增殖活性;miR-377能抑制经AngⅡ刺激后α-SMA,FSP1和CollagenI的表达,在肝纤维化的进程中发挥抑制作用。结论:MiR-377通过抑制Egr1的表达抑制肝细胞的增殖和病理进程。
Objective: To study the role of miR-377 targeted regulation of EGR1 in the pathological mechanism of liver fibrosis. Methods: The expression of miR-377 in serum of patients with liver fibrosis were detected by RT-qPCR.Egr1 was selected as the potential target gene of miR-377 by screening with miR target gene database. The effect of miR-377 mimics on the mRNA and protein expression of Egr1 was detected by RT-qPCR and Western blot,respectively. CCK-8 was used to detect the proliferation of HSC cells and HSC cells transfected with miR-377 which were induced by AngⅡ. RT-qPCR was used to detect the expression of α-smooth muscle actin(α-SMA),FSP1 and Collagen I after AngⅡ and AngⅡ + miR-377 stimulated. Results: MiR-377 may be related to the occurrence and development of liver fibrosis. miR-377 can directly inhibit the mRNA translation and protein expression of Egr1 by binding 3'-URT of Egr1. miR-377 could inhibit the proliferation of liver cells induced by AngⅡ. miR-377 could inhibit the expression of α-SMA, FSP1 and Collagen I after AngⅡ stimulated and miR-377 played an inhibitory role in the process of liver fibrosis. Conclusion: MiR-377 inhibits the proliferation and pathological process of liver cells by inhibiting the expression of Egr1.
Objective: To study the role of miR-377 targeted regulation of EGR1 in the pathological mechanism of liver fibrosis. Methods: The expression of miR-377 in serum of patients with liver fibrosis were detected by RT-qPCR.Egr1 was selected as the potential target gene of miR-377 by screening with miR target gene database. The effect of miR-377 mimics on the mRNA and protein expression of Egr1 was detected by RT-qPCR and Western blot,respectively. CCK-8 was used to detect the proliferation of HSC cells and HSC cells transfected with miR-377 which were induced by AngⅡ. RT-qPCR was used to detect the expression of α-smooth muscle actin(α-SMA),FSP1 and Collagen I after AngⅡ and AngⅡ + miR-377 stimulated. Results: MiR-377 may be related to the occurrence and development of liver fibrosis. miR-377 can directly inhibit the mRNA translation and protein expression of Egr1 by binding 3'-URT of Egr1. miR-377 could inhibit the proliferation of liver cells induced by AngⅡ. miR-377 could inhibit the expression of α-SMA, FSP1 and Collagen I after AngⅡ stimulated and miR-377 played an inhibitory role in the process of liver fibrosis. Conclusion: MiR-377 inhibits the proliferation and pathological process of liver cells by inhibiting the expression of Egr1.
引文
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2.徐明洲.TIPSS治疗肝硬化门脉高压上消化道出血和顽固性腹水的体会[J].中国临床研究,2014,27(3):295-296.XU Mingzhou.Experience of TIPSS in the treatment of portal hypertension upper gastrointestinal bleeding and refractory ascites in cirrhosis[J].Chinese Journal of Clinical Research,2014,27(3):295-296.
3.占书箱,黄成,马陶陶,等.抑制p70S6K对HSC-T6增殖活化的作用研究[J].安徽医科大学学报,2014,49(8):1062-1067.ZHANG Shuxiang,HUANG Cheng,MA Taotao,et al.The study on the effect of silencing p70S6K on the proliferation and activation of HSC-T6[J].Acta Universitatis Medicinalis Anhui,2014,49(8):1062-1067.
4.Tsukamoto H,Zhu NL,Asahina K.Epigenetic cell fate regulation of hepatic stellate cells[J].Hepatol Res,2011,41(7):675-682.
5.Annalisa P,Furio P,Ilaria Z,et al.Anorganic bovine bone and a silicatebased synthetic bone activate different microRNAs[J].Oral Sci,200850(3):1343-4934.
6.Yao Y,Suo AL,Li ZF,et al.MicroRNA profiling of human gastric cancer[J].Mol Med Rep,2009,2(6):963-970.
7.Wang R,Ma Y,Yu D,et al.miR-377 functions as a tumor supperssor in human clear cell renal cell carcinoma by targeting ETSI[J].Biomed Pharmacother,2015,70:64-71.
8.Chang W,Liu M,Xu J,et al.MiR-377 inhibits the proliferation of pancreatic cancer by targeting Pim-3[J].Tumour Biol,2016,37(11):14813-14824.
9.Zhang J,Zhao M,Xue ZQ,et al.miR-377 inhibites timorous behaviors of non-small cell lung cancer through directly targeting CDK6[J].Eur Rev Med Pharmacol Sci,2016,20(21):4494-4499.
10.Maes OC,Sarojini H,Wang E.Stepwise up-regulation of microRNAexpression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts[J].J Cell Physiol,2009221(1):109-119.
11.Mesnage R,Aron M,Costanzo M,et al.Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure[J].Environ Health,2015,14(1):70.
12.中华医学会肝病分会,中华医学会感染病学分会.慢性乙型肝炎防治指南2010年更新版[J].中华实验和临床感染病杂志(电子版),2011,5(1):79-100.Liver disease branch of Chinese medical association,Chinese society of infectious diseases.The guideline of prevention and treatment for chronic hepatitis B(2010 version)[J].Chinese Journal of Experimental and Clinical Infectious Diseases.Electronic Version2011,5(1):79-100.
13.Atta HM.Reversibility and heritability of liver fibrosis:Implications for research and therapy[J].World J Gastroenterol,2015,21(17):5138-5148.
14.Popper H,Kent G.Fibrosis in chronic liver disease[J].Clin Gastroenterol,1975,4(2):315-332.
15.Laterza OF,Scott MG,Garrett-Engele PW,et al.Circulating miR-122as a potential biomarker of liver disease[J].Biomark Med,2013,7(2):205-210.
16.Ramachandran S,Ilias BH,Sarma NJ,et al.Hepatitis C virus induced miR200c down modulates FAP-1,a negative regulator of Src signaling and promotes hepatic fibrosis[J].PLoS One,2013,8(8):e70744.
17.Bandiera S,Pfeffer S,Baumert TF,et al.miR-122-a key factor and therapeutic target in liver disease[J].J Hepatol,2015,62(2):448-457.
18.Li Y,Hu F,Xue M,et al.Klotho down-regulates Egr-1 by inhibiting TGF-β1/Smad3 signaling in high glucose treated human mesangial cells[J].Biochem Biophys Res Commun,2017,487(2):216-222.
19.Guo X,Zhang H,Zhang Q,et al.Screening for and validation of a hepatic fibrosis-related pathway induced by insulin-like growth factorbinding protein-related protein 1[J].Eur J Gastroenterol Hepatol2016,28(7):762-772.
20.Li TZ,Kim SM,Hur W,et al.Elk-3 contributes to the progression of liver fibrosis by regulating the epithelial-mesenchymal transition[J]Gut Liver,2017,11(1):102-111.
21.Zhang Y,Bonzo JA,Gonzalez FJ,et al.Diurnal regulation of the early growth response 1(Egr-1)protein expression by hepatocyte nuclear factor 4alpha(HNF4alpha)and small heterodimer partner(SHP)cross-talk in liver fibrosis[J].J Biol Chem,2015,286(34):29635-29643.
22.Ibrahim S,Dayoub R,Melter M,et al.Antiapoptotic and antioxidative protein ALR in cholestatic liver diseases-do bile acids regulate ALRexpression via Egr1?[J].Z Gastroenterol,2015,53(12):A3_7.
23.Zhou Y,Jia X,Zhou M,et al.Egr-1 is involved in the inhibitory effect of leptin on PPARgamma expression in hepatic stellate cell in vitro[J]Life Sci,2009,84(15/16):544-551.