SB-525334下调肝星状细胞miR-19b和miR-29b的表达

肝脏 ›› 2019, Vol. 24 ›› Issue (9): 1011-1014.

SB-525334下调肝星状细胞miR-19b和miR-29b的表达

吴江华, 马俊骥, 郭昱, 郭平

050000 石家庄河北医科大学第二医院消化内科, 河北省消化病实验室, 河北省消化病研究所

收稿日期:2019-05-29 发布日期:2020-04-15
通讯作者: 马俊骥,Email: majunji2006@163.com
基金资助:
国家自然科学基金项目(81200311),河北省自然科学基金资助项目(H2015206431),河北省医学重点科技研究计划项目(20130184)

SB-525334 downregulates the expression of miR-19b and miR-29b in hepatic stellate cells

WU Jiang-hua, MA Jun-Ji, GUO Yu, GUO Ping

Department of Gastroenterology, the Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang 050000, Hebei Province, China

Received:2019-05-29 Published:2020-04-15
Contact: MA Jun-Ji, E-mail: majunji2006@163.com

摘要/Abstract

摘要： 目的了解TGF-β1 刺激HSC后SB-525334 对I 型胶原分泌、miR-19b和miR-29b表达的影响。方法用MTT法检测TGF-β1与SB-525334干预LX-2细胞的最适药物浓度、最佳作用时间。LX-2细胞干预分为4组,对照组、TGF-β1干预组、SB-525334干预组、TGF-β1和SB-525334共干预组。通过蛋白质印迹法分析I型胶原蛋白的表达。反转录实时定量聚合酶链反应(RT-qRCR)分析各组miR-19b和miR-29b的表达。结果 TGF-β1浓度为10 ng/mL时,HSC生存率最高,为132.0%。当SB-525334浓度为10 μmol/l时,其抑制率最强,为38.4%。TGF-β1干预组LX-2细胞I型胶原蛋白的表达量(82.80±4.39)%较对照组(17.89±4.27)%显著增高(P<0.01),TGF-β1和SB-525334共干预组LX-2细胞I型胶原蛋白表达量为(62.62±3.72)%低于TGF-β1组(82.80±4.39)%(P<0.05)。RT-qPCR结果显示SB-525334可下调miR-19b的表达,TGF-β1干预组、SB-525334干预组、以及TGF-β1和SB-525334共干预组LX-2细胞miR-19b相对表达量分别为0.62±0.07、0.28±0.06、0.64±0.20,较对照组明显降低(P<0.01),TGF-β1和SB-525334共干预组LX-2细胞miR-19b的相对表达量较SB-525334组高(P<0.05),TGF-β1和SB-525334共干预组与TGF-β1干预组LX-2细胞 miR-19b的相对表达量比较无明显差异(P > 0.05)。SB-525334 可下调miR-29b 的表达,TGF-β1干预组、SB-525334干预组、以及TGF-β1和SB-525334共干预组LX-2细胞miR-29b相对表达量分别为0.77±0.05、0.44±0.04、0.61±0.06,较对照组降低(P<0.05),TGF-β1和SB-525334共干预组LX-2细胞 miR-29b 的相对表达量较TGF-β1干预组降低(P<0.05),TGF-β1和SB-525334共干预组LX-2细胞miR-29b的相对表达量较SB-525334干预组升高(P<0.05)。结论 TGF-β1抑制剂SB-525334 可抑制HSC分泌I型胶原,并下调其miR-19b和miR-29b的表达。

关键词: SB-525334, 肝星状细胞, 转化生长因子β1, I型胶原, miR-19b, miR-29b

Abstract: Objective The hepatic stellate cell line LX-2 was selected to study the effect of SB-525334 on the secretion of type I collagen and the expression of micro-ribonucleic acid (miR)-19b and miR-29b after stimulation by transforming growth factor beta1 (TGF-β1), so as to evaluate the value of SB-525334 in the prevention and treatment of hepatic fibrosis.Methods Methyl thiazolyl tetrazolium (MTT) method was used to detect the optimal concentration and time of TGF-β1 and SB-525334 interfering with LX-2 cells. Then the LX-2 cells were divided into 4 groups, the control group, the TGF-β1 intervention group, the SB-525334 intervention group, and the co-intervention group. The expression of type I collagen, miR-19b and miR-29b was analyzed by western blot and reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR).Results Hepatic stellate cell MTT test showed that the highest survival rate was 132.0% when the concentration of TGF-β1 was 10 ng/mL. When the concentration of SB-525334 was 10 μmol/L, the inhibition rate was 38.4%. Western blot showed that SB-525334 inhibited type I collagen expression in LX-2 cells. The type I collagen expression of LX-2 cells in TGF-β1 intervention group was significantly higher than that in the control group ([82.80±4.39]% vs [17.89±4.27]%, P<0.01). The type I collagen expression of LX-2 cells in co-intervention group was lower than that in TGF-β1 intervention group ([62.62±3.72)% vs [82.80±4.39]%, P<0.05). RT-qPCR showed that SB-525334 downregulated miR-19b expression in LX-2 cells. The relative miR-19b expression of LX-2 cells in TGF-β1 intervention group, SB-525334 intervention group, and co-intervention group (0.62±0.07, 0.28±0.06, 0.64±0.20) was significantly lower than that in the control group (P<0.01). The relative miR-19b expression of LX-2 cells in co-intervention group was higher than that in SB-525334 group (P<0.05). There was no significant difference in the relative miR-19b expression of LX-2 cells between co-intervention and TGF-β1 intervention groups (P>0.05). Besides, SB-525334 downregulated miR-29b expression in LX-2 cells. The relative miR-29b expression of LX-2 cells in TGF-β1 intervention group, SB-525334 intervention group, and co-intervention group (0.77±0.05, 0.44±0.04, 0.61±0.06) was significantly lower than that in the control group (P<0.05). The relative miR-29b expression of LX-2 cells in co-intervention group was lower than that in TGF-β1 group (P<0.05). The relative miR-29b expression of LX-2 cells in co-intervention group was higher than that in SB-525334 group (P<0.05).Conclusion TGF-β1 inhibitor SB-525334 inhibited the secretion of type I collagen as well as downregulated the expression of miR-19b and miR-29b in hepatic stellate cells.

Key words: SB-525334, Hepatic stellate cells, Transforming growth factor beta1, Type I collagen, Micro-ribonucleic acid-19b, Micro-ribonucleic acid-29b

吴江华, 马俊骥, 郭昱, 郭平. SB-525334下调肝星状细胞miR-19b和miR-29b的表达[J]. 肝脏, 2019, 24(9): 1011-1014.

WU Jiang-hua, MA Jun-Ji, GUO Yu, GUO Ping. SB-525334 downregulates the expression of miR-19b and miR-29b in hepatic stellate cells[J]. Chinese Hepatolgy, 2019, 24(9): 1011-1014.