miR-383-5p对大鼠肝细胞凋亡和增殖的影响
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摘要
为了研究miR-383-5p对大鼠肝细胞凋亡和增殖的影响,试验用不同浓度的miR-383-5p模拟物(mimics)或抑制剂(inhibitor)对大鼠肝细胞进行不同时间的瞬时转染,利用qRT-PCR方法检测miR-383-5p的相对表达量,筛选在肝细胞中过表达和干扰表达miR-383-5p的适宜条件;利用流式细胞仪研究miR-383-5p过表达和干扰表达对大鼠肝细胞凋亡的影响;利用WST-1细胞增殖及细胞毒性检测试剂盒检测miR-383-5p过表达和干扰表达对大鼠肝细胞增殖的影响。结果表明:大鼠肝细胞转染150 pmol mimics 24 h能够极显著提高miR-383-5p的相对表达量(P<0.01),转染100 pmol inhibitor 24 h能够极显著降低miR-383-5p的相对表达量(P<0.01)。在上述转染条件下,miR-383-5p过表达和干扰表达对肝细胞的凋亡均无显著影响(P>0.05),miR-383-5p过表达对肝细胞增殖无显著影响(P>0.05),但miR-383-5p干扰表达能够极显著促进肝细胞增殖(P<0.01)。说明miR-383-5p表达下调能够促进肝细胞增殖,或许可作为肝癌和肝细胞损伤相关疾病的辅助治疗方法。
In this study, different concentrations mimics or inhibitor of miR-383-5 p were used to transfect BRL-3 A cells for different time. The appropriate conditions for overexpression and interference expression miR-383-5 p in hepatocytes were screened by using qRT-PCR to detect the relative expression of miR-383-5 p. The effect of miR-383-5 p overexpression and interference expression on the apoptosis of rat hepatocytes were detected by flow cytometry.The effect of miR-383-5 p overexpression and interference expression on the proliferation of rat hepatocytes were detected by WST-1 cell proliferation kit. The results showed that the transfection of 24 h with 150 pmol mimics significantly improved the relative expression of miR-383-5 p(P<0.01),and transfection of 24 h with 100 pmol inhibitor significantly decreased the expression of miR-383-5 p(P<0.01). Under the above transfection conditions, overexpression and interference expression of miR-383-5 p in hepatocytes had no significant effect on rat hepatocyte apoptosis(P>0.05), overexpression of miR-383-5 p had no significant effect on rat hepatocyte proliferation(P>0.05),but the interference expression of miR-383-5 p significantly promoted the proliferation of rat hepatocytes(P<0.01). It suggests that the down regulation of miR-383-5 p can promote the proliferation of hepatocytes, and it can be used as an adjunctive therapy for liver cancer and liver cell injury related diseases.
In this study, different concentrations mimics or inhibitor of miR-383-5 p were used to transfect BRL-3 A cells for different time. The appropriate conditions for overexpression and interference expression miR-383-5 p in hepatocytes were screened by using qRT-PCR to detect the relative expression of miR-383-5 p. The effect of miR-383-5 p overexpression and interference expression on the apoptosis of rat hepatocytes were detected by flow cytometry.The effect of miR-383-5 p overexpression and interference expression on the proliferation of rat hepatocytes were detected by WST-1 cell proliferation kit. The results showed that the transfection of 24 h with 150 pmol mimics significantly improved the relative expression of miR-383-5 p(P<0.01),and transfection of 24 h with 100 pmol inhibitor significantly decreased the expression of miR-383-5 p(P<0.01). Under the above transfection conditions, overexpression and interference expression of miR-383-5 p in hepatocytes had no significant effect on rat hepatocyte apoptosis(P>0.05), overexpression of miR-383-5 p had no significant effect on rat hepatocyte proliferation(P>0.05),but the interference expression of miR-383-5 p significantly promoted the proliferation of rat hepatocytes(P<0.01). It suggests that the down regulation of miR-383-5 p can promote the proliferation of hepatocytes, and it can be used as an adjunctive therapy for liver cancer and liver cell injury related diseases.
引文
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[2] AMBROS V,CHEN X.The regulation of genes and genomes by small RNAs[J].Development,2007,134(9):1635-1641.
[3] 郑程远,邹晓,刘瑶,等.胎衣不下奶牛母体胎盘 miRNA-181a异常表达分析及验证[J].黑龙江畜牧兽医,2015(08上):21-24.
[4] DWIVEDI Y.Pathogenetic and therapeutic applications of microRNAs in major depressive disorder[J].Curr Neuropharmacol,2016,64:341-348.
[5] PRITCHARD C C,CHENG H H,TEWARI M.MicroRNA profiling:approaches and considerations[J].Nat Rev Genet,2012,13(5):358-369.
[6] 王晓玫,张石芬,成志强,等.上调microRNA-383对人髓母细胞瘤D341细胞系中PRDX3表达的影响[J].临床与实验病理学杂志,2012,28(4):422-428.
[7] ZHAO L,GU H,CHANG J,et al.MicroRNA-383 regulates the apoptosis of tumor cells through targetingGadd45g[J].PLoS One,2014,9(11):e110472.
[8] CHEN L,GUAN H,GU C,et al.MiR-383 inhibits hepatocellular carcinoma cell proliferation via targeting APRIL[J].Tumor Boil,2016,37(2):2497-2507.
[9] 崔盈,陈乐高.miR-383在结肠癌组织中的表达及其对结肠癌细胞株的作用[J].现代实用医学,2016,28(11):1454-1456.
[10] 王志琼.miR-150和miR-383沉默5SrRNA导致食管鳞癌细胞生长阻滞的研究[D].北京:北京化工大学,2015.
[11] 黄河龙.MicroRNA在男性不育中的功能研究[D].北京:中国科学技术大学,2015.
[12] 田卉.miRNA和遗传因素在男性不育发生中的功能研究[D].北京:中国科学技术大学,2014.
[13] 廖珂,柴志欣,张思源,等.miR-383在牦牛、犏牛睾丸组织中的差异表达及功能预测[J].中国畜牧兽医,2016,43(2):319-325.
[14] YIN M,LV M,YAO G,et al.Transactivation of microRNA-383 by steroidogenic factor-1 promotes estradiol release from mouse ovarian granulosa cells by targeting RBMS1[J].Mol Endocrinol,2012,26(7):1129-1143.
[15] 连杰.男性不育相关蛋白MLH1和miR-383在精子发生中的功能研究[D].北京:中国科学技术大学,2011.
[16] TIAN H,CAO Y X,ZHANG X S,et al.The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis[J].Cell Death Dis,2013 (4):e617.
[17] CHI S W,ZANG J B,MELE A,et al.Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps[J].Nature,2009,460(7254):479-486.
[18] 王丰,施一公.26S蛋白酶体的结构生物学研究进展[J].中国科学:生命科学,2014,44(10):965-974.
[19] 王晓,李果.泛素-26S蛋白酶体途径在凋亡中的作用[J].国外医学分子(生物学分册),2002,24(2):97-99.
[20] 郭文晋,甄莉,张婧馨,等.急性冷应激对大鼠肝脏miRNA的影响[J].中国兽医学报,2016,36(9):1582-1585.
[21] WHISNANT A W,BOGERD H P,FLORES O,et al.In-depth analysis of the interaction of HIV-1 with cellular microRNA bioge-nesis and effector mechanisms[J].MBio,2013,4(2):e000193.
[22] SCHMITZ C,PERRAUD A L,JOHNSON C O,et al.Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7[J].Cell,2003,114(2):191-200.
[23] DOROVKOV M V,RYAZANOV A G.Phosphorylation of annexin Ⅰ by TRPM7 channel-kinase[J].J Biol Chem,2004,279(49):50643-50646.
[24] CAI Z,JITKAEW S,ZHAO J,et al.Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis[J].Nat Cell Biol,2014,16(1):55-65.
[25] 倪训然,张黎明.TRM7通道与血管损伤关系的研究进展[J].医学综述,2016,22(20):3987-3990.
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