RESUMEN
Background: 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. Methods: A cellular hepatic fibrosis model was established by treating LX-2 cells with transforming growth factor ß (TGF-ß1). 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. Results: 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-ß1, 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-ß1. 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.
Asunto(s)
Atresia Biliar , Células Estrelladas Hepáticas , Cirrosis Hepática , MicroARNs , ARN Circular , Receptor Tipo II de Factor de Crecimiento Transformador beta , Humanos , Actinas/metabolismo , Actinas/genética , Apoptosis , Atresia Biliar/patología , Atresia Biliar/genética , Atresia Biliar/metabolismo , Línea Celular , Proliferación Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/patología , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
In order to examine new potential treatment options for the treatment of hepatoblastoma (HB), we identified the differential expression of five-candidate tumor suppressor miRNAs in HB and explored possible regulatory mechanisms of target miRNA molecule. By using real-time quantitative polymerase chain reaction (qPCR), we examined the expression of miRNAs in HB tissues and cells. The effect of has-miR-139-3p mimics on the invasion and migration ability was assessed by transwell assay and scratch-wound assay in HepG2 cells. Subsequently, we analyzed the target genes of miR-139-3p and their enrichment signaling pathways through bioinformatics. qPCR, Western-blot and dual-luciferase assays were further used to assess whether has-miR-139-3p targets Wnt5A. The results showed that hsa-miR-139-3p was significantly decreased in HB cells. Upregulation of hsa-miR-139-3p inhibited the invasive and migratory ability of HepG2. Bioinformatics analysis showed that hsa-miR-139-3p may target Wnt5A to regulate the WNT pathway, which was further confirmed by Western-blot and dual-luciferase assays. Overexpression of Wnt5A can reverse the miR-139-3p mimic-induced declines in the expression of WNT pathway-related proteins and restore the invasion and migration of HepG2. These data indicated that the hsa-miR-139-3p/Wnt5A axis inhibited HB metastasis, suggesting that miR-139-3p and Wnt5A may be potential targets for the treatment of HB.
