Zhang Liting, Gao Jing, Zhou Dan, Wang Xiaojun, Li Junfeng, Wang Juan, Chen Hong, Xie Xiaodong, Chen Tuo
State Key Laboratory of Cryospheric Science, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, China.
University of Chinese Academy of Sciences, Beijing, China.
Ann Transl Med. 2021 Sep;9(18):1451. doi: 10.21037/atm-21-4215.
MicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs.
HSC-T6 cells were treated with cobalt chloride (CoCl), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1α (HIF-1α), collagen type I, transforming growth factor-β1 (TGF-β1), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1α, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 µmol/L CoCl medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.
With the increase of CoCl concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1α, collagen I, TGF-β1, and Smad7, and the protein expressions levels of HIF-1α, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 µmol/L CoCl medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.
Our results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.
微小RNA（miRNA）在肝星状细胞（HSCs）激活和肝纤维化过程中发挥重要作用。本研究旨在探讨缺氧对大鼠肝星状细胞中mRNA和miRNA差异表达的影响。
用氯化钴（CoCl）处理肝星状细胞系T6（HSC-T6）细胞，采用CCK-8法检测HSC-T6细胞活性。通过逆转录定量聚合酶链反应（RT-qPCR）检测缺氧诱导因子-1α（HIF-1α）、Ⅰ型胶原、转化生长因子-β1（TGF-β1）和Smad7的mRNA表达水平。采用蛋白质免疫印迹法检测HIF-1α、Bax、Bcl-2和半胱天冬酶-3（caspase-3）的蛋白表达水平。我们使用基础培养基和400 μmol/L CoCl培养基处理HSC-T6细胞48小时。48小时后收集细胞提取RNA。进行转录组测序以研究差异表达的miRNA和mRNA（变化倍数>2；P<0.05）。进行生物信息学分析以预测差异表达的miRNA和mRNA的功能。此外，我们使用RT-qPCR检测mRNA和miRNA的表达以确认测序的准确性。
随着CoCl浓度的增加，HSC-T6细胞活性降低（P<0.05）。与对照组相比，HIF-1α、Ⅰ型胶原、TGF-β1和Smad7的mRNA表达水平，以及HIF-1α、Bax、caspase-3的蛋白表达水平和Bcl-2/Bax比值均升高（P<0.05），而Bcl-2的表达降低。与对照基础培养基组相比，在400 μmol/L CoCl培养基组中共有54个miRNA（20个上调和34个下调）和1423个mRNA（685个上调和738个下调）差异表达。进一步的生物信息学分析表明，差异表达的mRNA和miRNA主要富集于细胞外基质的合成。此外，我们使用RT-qPCR检测mRNA和miRNA的表达以确认测序的准确性。
我们的研究结果展示了大鼠缺氧诱导的HSC-T6细胞中mRNA和miRNA的图谱、信号通路和共表达网络。这些发现可能为肝星状细胞激活和肝纤维化的早期诊断和治疗提供新的见解。
