Objective: To investigate the expression of miRNA associated with hepatic fibrosis induced by Schistosoma japonicum soluble egg antigen stimulation in mouse hepatocytes (AML12), so as to lay the foundation for clarifying the mechanism of schistosome infection leading to hepatic fibrosis.
Methods: The expressions of miR-122, miR-182, miR-23b, miR27b and KSRP in AML12 cells treated with SEA were measured by q-PCR. KSRP protein in cell lyses was measured by Western blotting. AML12 cells were transfected with miR-27b precursor or anti-miR-27b for 24 h, then q-PCR was adopted to determine KSRP mRNA, and KSRP protein was detected by Western blotting.
Results: The expressions of miR-182, miR-23b and miR27b were decreased and miR-122 was increased in AML12 cells following SEA treatment (all P < 0.05). An increase of mRNA and protein of KSRP expression was also observed in AML12 cells after SEA stimulation (both P < 0.05). In addition, KSRP mRNA expression was not changed significantly in AML12 cells transfected with anti-miR-27b or miR-27b precursor, and miR27b precursor reduced KSRP protein expression as compared with the control. In contrast, the expression of KSRP protein was increased in the anti-miR-27b group and decreased in the miR-27b precursor group.
Conclusions: After the stimulation of SEA, the expressions of a variety of liver fibrosis-related miRNAs and KSRP change in murine hepatocytes, including miR-27b. And miR-27b can regulate the expression of KSRP. These findings might lay a foundation for further study on the molecular mechanism of fibrosis induced by schistosome infection.
［摘要］目的 研究与肝纤维化相关miRNA (miR) 在日本血吸虫可溶性虫卵抗原刺激小鼠肝细胞 (AML12) 后的表达情 况, 为阐明血吸虫感染导致肝纤维化的机制奠定基础。方法 使用日本血吸虫可溶性虫卵抗原 (Soluble egg antigens, SEA) 刺激AML12后, 采用定量PCR检测AML12中的miR-122、miR-182、miR-23b、miR-27b及KH型剪切调控蛋白 (KH- type splicing regulatory protein, KSRP) 的mRNA 水平, 以Western blotting 检测KSRP 蛋白的表达变化; 分别用anti-miR- 27b、miR-27b precursor转染AML12后, 以定量PCR和Western blotting检测AML12中KSRP的mRNA和蛋白水平。结果 经SEA刺激后, AML12中miR-182、miR-23b及miR-27b mRNA水平下降 (P 均＜0.05), 而细胞中miR-122 mRNA与KSRP 的mRNA水平及蛋白表达水平均上调 (P 均＜0.05) 。此外, anti-miR-27b与miR-27b precursor转染组KSRP的mRNA水平 均无明显变化, 但anti-miR-27b 组细胞中KSRP 的蛋白表达增加, miR-27b precursor 组KSRP 的蛋白表达降低。结论 SEA刺激AML12导致诸多与肝纤维化相关的miRNA及KSRP的表达发生变化, 其中miR-27b可调控KSRP的表达, 这为 进一步研究血吸虫感染导致的肝纤维化的分子机制奠定了基础。.
Keywords: Hepatocytes; Liver fibrosis; Schistosoma japonicum; Soluble egg antigen (SEA); miRNA.