摘要
目的 探讨miR-21调控肝卵圆细胞活化和增殖过程的可能机制.方法 采用2-乙酰氨基芴（2-AAF）/部分肝切除法诱导SD大鼠肝卵圆细胞活化模型,分别于0、6、12、24、72、168 h处死动物,同时以单纯肝切除的相应时间点作为对照,分别提取各标本RNA逆转录后行SYBR Green实时荧光定量PCR,检测各组miR-21的表达,行两样本t检验,并用生物信息学方法分析其调控的miRNA转录分子和靶基因.实时荧光定量PCR检测其靶基因表达,Westem blot法检测其靶基因蛋白表达,进行两样本均值的t检验,以P＜ 0.05为差异有统计学意义. 结果 成功建立大鼠肝卵圆细胞活化模型,检测miR-21的扩增信号,miR-21在实验组表达12h开始升高,24 h达到峰值,随后开始降低,168 h再次升高;而对照组6h开始升高,24h后开始下降后基本恢复原水平.实验组与对照组表达比较,实验组在6 h miR-21的表达低于对照组,t=3.029,P=0.039,差异有统计学意义;而在24 h和168 h的表达高于对照组,t值分别为-3.433、-5.105,P值均＜0.05,差异有统计学意义.根据三个数据库的综合评分,结合相关文献,我们选取Smad7为研究的靶基因.检测两组Smad7mRNA表达,对照组中在6h略有下降,后开始升高,在24 h达到峰值后开始下降恢复原水平;在实验组中,6h升高后至24 h达到峰值,随后开始降低,168 h又略有升高.对照组Smad7蛋白表达从6h开始降低,到24 h达到最低后开始升高;实验组6h升高,随后下降,168 h达到最低.Smad7 mRNA表达量变化趋势与miR-21表达变化趋势基本一致,Smad7蛋白表达和miR-21表达变化趋势呈负相关. 结论 成功建立SYBR Green实时荧光定量PCR检测大鼠miR-21的方法,证实miR-21在肝卵圆细胞活化与增殖中起重要作用,Smad7作为miR-21的靶基因可能参与这一过程.
Objective To investigate the possible mechanisms of miR-21-mediated regulation of proliferation and activation of hepatic oval cells.Methods The 2-acetamidofluorene/partial hepatectomy （2-AAF/PH） method was applied to generate hepatic oval cell activation model in male Sprague-Dawley rats; after the 7 days of 2-AAF/PH or PH alone （control）,the rats were sacrificed at 0 h,6 h,12 h,24 h,72 h and 168 h.Expression of miR-21 was detected by real-time PCR and differences between groups were evaluated using the two-sample t-test.Differential transcription of miR-21 target genes was assessed bioinformatically,and with western blotting to detect changes in protein expression of the target gene.Results The rat hepatic oval cell activation model was successfully established.The 2-AAF/PH rats showed miR-21 expression beginning to increase at 12 h,peaking at 24 h,and decreasing thereafter until an increase at 168 h.For the control group,the miR-21 expression began to increase at 6 h,until 24 h when expression began steadily declining to reach the original level.Compared to the control group,the experimental group showed expression of miR-21 that was significantly less at 6 h （P =0.039,t =3.029） and significantly more at 24 h and 168 h （P =0.026,t =-3.433 andP =0.007,t =-5.105）.Among the predicted target genes of miR-21 were WW domain containing E3 ubiquitin protein ligase 1 （WWD）,Smad family member 7 （Smad7）,and polybromo-1 （Pbrm1）.Smad7 protein expression began to decrease at 6 h in the control group,until reaching its minimum at 24 h when it increased; in the experimental group,SMAD7 expression increased at 6 h,then began to decrease with the minimum detected at 168 hour.In the control group,the Smad7 mRNA expression decreased slightly at 6 h,then began to increase,reaching its peak at 24 h when the expression fell to the original level.In the experimental group,the Smad7 mRNA expression began to increase at 6 h and reached its peak at 24 h when it decreased; the expression was little more than its original level at 168 h.Smad7 protein expression was negatively correlated with miR-21,and Smad7 mRNA expression was positively correlated with miR-21 but negatively correlated with Smad7 protein expression.Conclusion miR-21 may play a vital role in the activation and proliferation of hepatic oval cells.As a target gene of miR-21,Smad7 might be involved in the process.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2014年第11期854-859,共6页
Chinese Journal of Hepatology
基金
国家自然科学基金（30700800）
浙江省自然科学基金（LYl2H03006）
温州市科委（Y20110078）
