CircHIPK3 Regulates Lung Fibroblast-to-myofibroblast Transition By Functioning As A Competing Endogenous RNA

Background and objective: Myofibroblasts predominantly emerging through fibroblast-to-myofibroblast transition(FMT)are considered to be the key collagen-producing cells in pulmonary fibrosis.Circular RNAs(circ RNAs)are important players involved in many biological processes.circ HIPK3 has been identified as the one of the most abundant circ RNAs in human lung.In this study,we characterized the role of circ HIPK3 in pulmonary fibrosis.Methods: We first establised bleomycin induced pulmonary fibrosis mice model.And in vitro study,WI-38 fibroblast cells were induced into myofibroblasts through TGF-β1 treatment.We investigated circ HIPK3 expression pattern in bleomycin induced pulmonary fibrosis mice model and FMT-derived myofibroblasts by quantitative RT-PCR(q RT-PCR),sanger sequencing,FISH assay and etc.By conducting in vivo and in vitro loss-and gain-of-function assays,we delineate the functional role of circ HIPK3 in fibroblast.We then further investigated the molecular mechanism by which circ HIPK3 regulated FMT through bioinformatic analysis,RNA immunoprecipitation(RIP)assays,luciferase reporter assays and etc.Finally,the expression of circ HIPK3 in clinical pulmonary fibrosis samples was detected by real-time fluorescence quantitative PCR and FISH assay.Results: We revealed that circ HIPK3 is upregulated in bleomycin induced pulmonary fibrosis mice model.circ HIPK3 silencing could attenuate collagen deposition in BLM-induced pulmonary fibrosis mice model,as demonstrated by Masson’s trichrome and Sirius red staining.In accordance,α-SMA and Col-1 expression were significantly decreased in circ HIPK3 knockdown mice after bleomycin injury.circ HIPK3 knockdown alleviates fibroblast proliferation and FMT in vivo.Our vitro study revealed that circ HIPK3 is required for the activation of TGF-β1-induced FMT and TGF-β1-induced fibroblast proliferation.RIP assays showed that circ HIPK3 is enriched in Ago2-containing immunoprecipitates compared with control immunoglobulin G(Ig G)immunoprecipitates,which confirms the interaction between circ HIPK3 and Ago2 in WI-38 cells.Bioinformatics prediction using circ Net database and Circular RNA Interactome database shows that mi R-338-3p is a common mi RNA that potentially binds to circ HIPK3.FISH assays revealed that circ HIPK3 and mi R-338-3p are co-localized in the cytoplasm of TGF-β1 treated WI-38 cells.mi R-338-3p transfection could repress TGF-β1-induced protein expression of α-SMA and Col-1,whereas the repression was abrogated by circ HIPK3 overexpression.mi R-338-3p mimic transfection in the presence of TGF-β1 significantly inhibited SOX4 and COL1A1 expression.Moreover,the mi R-338-3p mediated repression of the two target genes was rescued by the circ HIPK3 overexpression.Luciferase report assay confirmed that SOX4 and COL1A1 are the downstream target genes of mi R-338-3p.q RT-PCR and FISH assay revealed significantly upregulated circ HIPK3 expression in IPF patient lung tissue samples.Conclusion: In this study,we identify that circ HIPK3 is upregulated in BLM-induced pulmonary fibrosis mice model,FMT-derived myofibroblasts and idiopathic pulmonary fibrosis patient lung samples.We show that circ HIPK3 silencing can ameliorate FMT and suppress fibroblast proliferation in vivo and vitro.We demonstrate that circ HIPK3 can function as an endogenous mi R-338-3p sponge and inhibit mi R-338-3p activity,thereby leading to increased SOX4 and COL1A1 expression in an Ago2-dependent manner.These observations strengthen our notion that circ HIPK3 intervention may represent a promising target for pulmonary fibrosis.