| Implications and objective:The phenotypic modulation of pulmonary artery smooth muscle cells(PASMCs) is an important pathological feature ofhepatopulmonary syndrome(HPS). HPSis a serious complication of advanced liver disease characterised by intrapulmonary vascular dilatation(IPVD) and arterial hypoxemia. The reported incidence of HPS varies from 4% to 47% due to non-uniform diagnostic criteria, and the 2.5-year mortality rate following diagnosis with HPS reaches 41%. There is currently no effective pharmacological therapy for HPS, and orthotopic liver transplantation is considered to be the only reliable treatment. The main reason for the lack of viable therapeutic measures is that the exact pathogenesis of HPS has not been identified.Previous studies and our initial research have demonstrated that pulmonary vascular remodelling(PVR) may play an important role in the pathogenesis of HPS. In the pathogenesis of PVR, pulmonary artery smooth muscle cells(PASMCs) switch from a quiescent and contractile phenotype to a proliferative and synthetic phenotype, while endothelial cells differentiate into smooth muscle-like cells. These two features constitute the two basic pathophysiological components. The identification of molecular mechanisms underlying the phenotypic modulation of PASMCs could facilitate the development of improved treatments for HPS.Annexin A2(ANXA2), is a 36-kDa protein member of the annexin superfamily of calcium-dependent phospholipid-binding proteins. Previous studies have indicated that ANXA2 is an important regulatory protein in the process of proliferation, migration, skeletal formation and angiogenesis in a variety of malignant tumours, such as liver cancer, pancreatic cancer, breast cancer and lung cancer. We have recently demonstrated that ANXA2 plays an important role in the process of PASMC phenotypic switching.Micro RNAs(miRNAs) are small noncoding RNAs that post-transcriptionally regulate the expression of the genes that control multiple biological processes. In PASMCs, the stage-specific expression of certain mi RNAs suggests their participation in PASMC phenotypic switching, which might contribute to the pathogenesis of PVR. Therefore, we investigated whether some potential miRNAs implicated in PASMC phenotypic switching might regulate ANXA2 gene expression.Methods:Total five parts:1. HPS rat serum preparation and establishment of primary culture PASMCsThe HPS model was established in rats by chronic bile duct ligation(CBDL). Serum of HPS rats was obtained when the model was identified by pathological section andblood gas analysis. In addition, primary culture rat PASMCs was established by tissue-sticking method.2. Computational analysis; SM α-actin,ANXA2 and mi R-206detectingComputational analysis predicted that mi R-206 would target the 3′-UTR of ANXA2 m RNA. PASMCs were randomly divided into two groups: C group and HPS group. Group C was treated with normal rat serum as normal control, group HPS was treated with HPS rat serum. After treatment for 24, 48, 72 hours, the protein level of ANXA2 and SM α-actin were determined by western blotting, miR-206 and the mRNA level of ANXA2 were detected by qRT-PCR.3. Functional analysis of the mi R-206 on PASMCsPASMCs were transfected with different groups of oligo nucleotides, mi R-206 mimic, miR-206 inhibitor. The protein level of ANXA2 and SM α-actin were determined by western blotting, the m RNA levels of ANXA2 were detected by qRT-PCR, and the location of ANXA2, SM α-actin was detected by immunofluorescence.4. Dual-Luciferase Reporter AssayDouble-luciferase reporter assay was performed to ascertain whether the predicted miRNAs suppressed ANXA2 expression by directly targeting the predicted 3′-UTR site of the ANXA2 m RNA.5. Thymidine incorporation assay and CCK-8 assaymi R-206 mimic with or without ANXA2 expression plasmid were transfected into the PASMCs using the Lipofectamine2000 reagent. The changes of PASMCs proliferation induced by serum from rats with hepatopulmonary syndrome were detemined by Thymidine incorporation way and CCK-8 assay.Results:1. The effect of HPS rat serum on the expression levels of mi R-206 and ANXA2.Computational analysis predicted that mi R-206 would target the 3′-UTR of ANXA2 m RNA.In HPS rat serum-stimulated PASMCs, the expression of mi R-206 displayed an inverse correlation with ANXA2.2. miR-206 regulates the phenotypic modulation of PASMCs in vitro.PASMCs were transfected with different groups of miR-206 mimic and mi R-206 inhibitor. miR-206 mimic upregulated while mi R-206 inhibitor downregulated the protein levels of SM α-actin.3.miR-206 represses the ANXA2 gene expression by directly targeting the 3′-UTR of ANXA2.The miRNA functional analysis and luciferase reporter assay demonstrated that miR-206 effectively downregulated the expression of ANXA2 by binding to the 3′-UTR of the ANXA2 mRNA.4.miR-206/ANXA2 pathway mediates the effects of HPS rat serum-induced phenotypic modulation and proliferation of the PASMCs.mi R-206 effectively inhibited the HPS rat serum-induced phenotypic modulation and proliferation, while these effects were reversed in ANXA2-overexpressing PASMCs.Conclusions:This study demonstrates that mi R-206 inhibits the HPS rat serum-induced phenotypic modulation and proliferation in PASMCs by down-regulating ANXA2 gene expression. |
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