miR-21对肝癌细胞Keap-1/Nrf2通路的调控作用分析
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摘要
目的探讨miR-21对肝癌细胞Keap-1/Nrf2通路的调控作用。方法人肝癌细胞株HSMMC-7721随机分为三组,实验组、对照组分别转染miRNA-21模拟剂(mimics)、miRNA-21 NC,空白组不进行转染。采用MTT法检测细胞增殖,流式细胞仪实验检测细胞周期,Transwell小室实验检测细胞侵袭,Western blot检测蛋白表达。结果转染后36 h与48 h,实验组的miRNA-21相对表达量显著高于空白组和对照组(P <0. 05),实验组的细胞增殖率、侵袭率显著低于空白组和对照组(P <0. 05);转染后48 h,实验组的S期与G2/M期比率显著高于空白组和对照组,(P<0. 05),GO/G1期比率显著低于空白组和对照组,(P <0. 05),空白组和对照组对比差异无统计学意义(P> 0. 05)。转染后36 h与48 h,与空白组和对照组相比,实验组的Keap-1、Nrf2蛋白相对表达量显著下降(P <0. 05)。结论miRNA-21高表达能抑制Keap-1/Nrf2通路的激活,可调节肝癌细胞周期,从而抑制肝癌细胞的增殖与侵袭。
Objective To investigate the regulation of miR-21 on the Keap-1/Nrf2 pathway in hepatoma cells. Methods Human hepatoma cell line HSMMC-7721 were randomly divided into three groups. The experimental group and the control group were transfected with miRNA-21 mimics and miRNA-21 NC,respectively. The blank group were not transfected. Cell proliferation were detected by MTT assay. Flow cytometry was used to detect the cell cycle,cell invasion were detected by Transwell chamber assay,and protein expression were detected by Western blot. Results At 36 h and 48 h after transfection,the relative expression of miRNA-21 in the experimental group were significantly higher than that in the blank group and the control group( P < 0. 05). The cell proliferation rate of the experimental group were significantly lower( P< 0. 05). The invasive rates were significantly decreased( P < 0. 05); at 48 hours after transfection,the proportion of S and G2/M phases in the experimental group were significantly increased( P < 0. 05),and the proportion of GO/G1 phase were significantly decreased( P < 0. 05).There were no significant difference in the control group( P > 0. 05). At 36 h and 48 h after transfection,the relative expression of Keap-1 and Nrf2 protein in the experimental group were significantly lower than those in the blank group and the control group( P < 0. 05). Conclusion High expression of miRNA-21 can inhibit the activation of Keap-1/Nrf2 pathway,regulate the cell cycle of liver cancer,and inhibit the proliferation and invasion of liver cancer cells.
Objective To investigate the regulation of miR-21 on the Keap-1/Nrf2 pathway in hepatoma cells. Methods Human hepatoma cell line HSMMC-7721 were randomly divided into three groups. The experimental group and the control group were transfected with miRNA-21 mimics and miRNA-21 NC,respectively. The blank group were not transfected. Cell proliferation were detected by MTT assay. Flow cytometry was used to detect the cell cycle,cell invasion were detected by Transwell chamber assay,and protein expression were detected by Western blot. Results At 36 h and 48 h after transfection,the relative expression of miRNA-21 in the experimental group were significantly higher than that in the blank group and the control group( P < 0. 05). The cell proliferation rate of the experimental group were significantly lower( P< 0. 05). The invasive rates were significantly decreased( P < 0. 05); at 48 hours after transfection,the proportion of S and G2/M phases in the experimental group were significantly increased( P < 0. 05),and the proportion of GO/G1 phase were significantly decreased( P < 0. 05).There were no significant difference in the control group( P > 0. 05). At 36 h and 48 h after transfection,the relative expression of Keap-1 and Nrf2 protein in the experimental group were significantly lower than those in the blank group and the control group( P < 0. 05). Conclusion High expression of miRNA-21 can inhibit the activation of Keap-1/Nrf2 pathway,regulate the cell cycle of liver cancer,and inhibit the proliferation and invasion of liver cancer cells.
引文
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[11]吴丽媛,李偲,彭锐,等.miR-21介导的结肠癌细胞对5-氟尿嘧啶的耐药机制研究[J].中华医学遗传学杂志,2015,32(5):620-624.
[12]吴佳成,陆雅君,姜力.miR-21下调程序性死亡细胞4对抑制裸鼠肾细胞癌肿瘤转化和增殖的实验研究[J].疑难病杂志,2019,18(1):61-66.
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[14]杜敏,李志民,霍伟.DC-CIK免疫治疗联合化疗对晚期胃癌的效果及对Nrf2、ARE蛋白表达水平的影响[J].临床和实验医学杂志,2018,17(15):1627-1630.
[15]李晓冲,张秀英.小鼠nafld模型建立及nrf2缺失对肝脏中cyp2a5表达的影响[D].哈尔滨:东北农业大学,2013.
[2]范颖超,曹雅楠,吴亚红,等.长链非编码RNAAK057037在原发性肝癌患者组织和血液检测中的应用[J].国际检验医学杂志,2018,39(6):664-667.
[3]Ali FEM,Azouz AA,Bakr AG,et al.Hepatoprotective effects of diosmin and/or sildenafil against cholestatic liver cirrhosis:The role of Keap-1/Nrf-2 and P38-MAPK/NF-kappaB/i NOS signaling pathway[J].Food Chem Toxicol,2018,120:294-304.
[4]李维娜,何飞.miR-140表达与肿瘤发生发展关系的研究进展[J].解放军医学杂志,2018,43(6):523-529.
[5]Ali FEM,Bakr AG,Abo-Youssef AM,et al.Targeting Keap-1/Nrf-2 pathway and cytoglobin as a potential protective mechanism of diosmin and pentoxifylline against cholestatic liver cirrhosis[J].Life Sci,2018,207:50-60.
[6]Cai SA,Hou N,Zhao GJ,et al.Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats[J].Front Pharmacol,2018,9:540.
[7]李莹莹,詹丽芬,曾哲超,等.洛铂对肝癌HepG2细胞增殖和凋亡的作用及机制[J].临床肝胆病杂志,2018,34(4):784-788.
[8]胡静,周喜汉,胡高裕,等.苦参素联合顺铂对实验性肝癌miR-21、miR-122表达的影响[J].临床和实验医学杂志,2017,16(21):2084-2087.
[9]谢子英,赵亚刚,朱鸿武,等.急性胰腺炎患者血液miRNA-21表达水平与炎症反应相关性[J].实用医学杂志,2016,32(8):1292-1294.
[10]阿孜古力·阿不来提,彭媛媛,努尔麦麦提·叶尔逊,等.miR-21、miR-155在胰腺癌患者血浆中的表达及临床意义[J].临床和实验医学杂志,2018,17(13):1375-1378.
[11]吴丽媛,李偲,彭锐,等.miR-21介导的结肠癌细胞对5-氟尿嘧啶的耐药机制研究[J].中华医学遗传学杂志,2015,32(5):620-624.
[12]吴佳成,陆雅君,姜力.miR-21下调程序性死亡细胞4对抑制裸鼠肾细胞癌肿瘤转化和增殖的实验研究[J].疑难病杂志,2019,18(1):61-66.
[13]周卫,赵林静,高建芝.miRNAs在原发性肝癌中的表达及临床意义分析[J].中国医药导刊,2018,20(2):97-101.
[14]杜敏,李志民,霍伟.DC-CIK免疫治疗联合化疗对晚期胃癌的效果及对Nrf2、ARE蛋白表达水平的影响[J].临床和实验医学杂志,2018,17(15):1627-1630.
[15]李晓冲,张秀英.小鼠nafld模型建立及nrf2缺失对肝脏中cyp2a5表达的影响[D].哈尔滨:东北农业大学,2013.
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