■肝星状细胞(HSC)激活和肝纤维化介导的胆道闭锁(BA)发展,但是对潜在的分子机制知之甚少。本研究旨在探讨circRNAhsa_circ_0009096在HSC增殖和肝纤维化中的作用。
■用转化生长因子β(TGF-β1)处理LX-2细胞建立肝纤维化模型。进行RNaseR和放线菌素D测定以检测hsa_circ_0009096稳定性。hsa_circ_0009096,miR-370-3p,并使用逆转录-qPCR检测目标基因。使用双荧光素酶报告基因测定验证hsa_circ_0009096与miR-370-3p的直接结合。使用流式细胞术评估LX-2细胞的细胞周期进程和凋亡。α-平滑肌肌动蛋白(α-SMA),胶原蛋白1A1(COL1A1),使用免疫细胞化学和蛋白质印迹分析LX-2细胞中的TGFβ受体2(TGFBR2)蛋白水平。
■Hsa_circ_0009096对RNaseR和放线菌D消化的抗性高于UTRNmRNA。在TGF-β1处理的LX-2细胞中Hsa_circ_0009096表达显著增加,伴随着α-SMA和COL1A1表达升高。Hsa_circ_0009096siRNA在LX-2细胞中有效促进miR-370-3p并抑制TGFBR2表达,通过hsa_circ_0009096与miR-370-3p的直接缔合介导。Hsa_circ_0009096siRNA干扰细胞周期进程,促进细胞凋亡,TGF-β1处理的LX-2细胞中α-SMA和COL1A1的表达降低。MiR-370-3p抑制剂减轻了细胞周期进程的改变,凋亡,和α-SMA,由hsa_circ_0009096siRNA引起的LX-2细胞中的COL1A1和TGFBR2表达。总之,hsa_circ_0009096通过spongingmiR-370-3p加速TGFBR2表达促进BA发病过程中HSC增殖和肝纤维化。
UNASSIGNED: Hepatic stellate cell (HSC) activation and hepatic fibrosis mediated biliary atresia (BA) development, but the underlying molecular mechanisms are poorly understood. This study aimed to investigate the roles of circRNA hsa_circ_0009096 in the regulation of HSC proliferation and hepatic fibrosis.
UNASSIGNED: A cellular hepatic fibrosis model was established by treating LX-2 cells with transforming growth factor β (TGF-β1). RNaseR and actinomycin D assays were performed to detect hsa_circ_0009096 stability. Expression of hsa_circ_0009096, miR-370-3p, and target genes was detected using reverse transcription-qPCR. Direct binding of hsa_circ_0009096 to miR-370-3p was validated using dual luciferase reporter assay. Cell cycle progression and apoptosis of LX-2 cells were assessed using flow cytometry. The alpha-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), and TGF beta receptor 2 (
TGFBR2) protein levels in LX-2 cells were analyzed using immunocytochemistry and western blotting.
UNASSIGNED: Hsa_circ_0009096 exhibited more resistance to RNase R and actinomycinD digestion than UTRN mRNA. Hsa_circ_0009096 expression increased significantly in LX-2 cells treated with TGF-β1, accompanied by elevated α-SMA and COL1A1 expression. Hsa_circ_0009096 siRNAs effectively promoted miR-370-3p and suppressed
TGFBR2 expression in LX-2 cells, mediated by direct association of hsa_circ_0009096 with miR-370-3p. Hsa_circ_0009096 siRNA interfered with the cell cycle progression, promoted apoptosis, and reduced α-SMA and COL1A1 expression in LX-2 cells treated with TGF-β1. MiR-370-3p inhibitors mitigated the alterations in cell cycle progression, apoptosis, and α-SMA, COL1A1, and
TGFBR2 expression in LX-2 cells caused by hsa_circ_0009096 siRNA. In conclusion, hsa_circ_0009096 promoted HSC proliferation and hepatic fibrosis during BA pathogenesis by accelerating
TGFBR2 expression by sponging miR-370-3p.