CircHIPK3 Facilitates Glycolysis Of Fibroblast To Promote Silica-induced Pulmonary Fibrosis

Introduction Long-term exposure to silica can cause a complex occupational pulmonary disease in workers termed silicosis,which is characterized by pulmonary fibrosis and dysfunction.Silicosis is one of the common types of occupational diseases in China.The process of silicosis is rapidly and irreversibly,however,existing drugs only slow disease progression,and there is a significant unmet medical need.At present,the clinical treatment of silicosis includes symptom control and supportive treatment,and the high cost causes a huge economic burden to the society.The initiation and development of silicosis is a complex process regulated by a variety of cells and cytokines.Understanding the pathological processes and regulatory factors of silicosis is an unmet need and an important step in the development of potential therapeutic targets and biomarkers,which has great medical value for the early diagnosis and treatment of silicosis.Myofibroblast is the primary cell type responsible for promoting silica-induced pulmonary fibrosis by producing extracellular matrix（ECM）.In the pathogenesis of pulmonary fibrosis,several cells can differentiate into myofibroblasts,including endothelial cells,epithelial cells,circulating fibroblasts,and lung-resident fibroblasts.Among them,the lung-resident fibroblast is the most important resource of myofibroblast,which can be activated by cytokines and mechanical stimulation.Upon activation,resting fibroblasts acquire a myofibroblast phenotype,which is characterized by the expression of contractile proteins,α-SMA,and enhanced release of the extracellular matrix.It is demonstrated that myofibroblasts can produce profibrotic cytokines and extracellular matrix as well as accelerate lung fibrosis,remodeling,and thus lung dysfunction.Transforming growth factor-β1（TGF-β1）is a critical pathway of fibroblast activation in physiologic and pathologic conditions and plays a central role in the persistent activation of fibroblasts in fibrotic diseases.Identification of central checkpoints and mechanisms of TGF-β1-induced fibroblast activation might offer potential therapies for fibrotic diseases.In recent years,the role of cellular metabolic dysregulation of cells in diseases has been revealed.Glycolysis reprogramming is an essential contributor to fibroblast activation and differentiation.During differentiation,myofibroblasts adopt a unique glycolytic program termed aerobic glycolysis.Compared with oxidative phosphorylation,efficiency in ATP production per molecule of glucose is much lower via glycolysis,however,the yield rate is much faster.Hence,glycolysis could meet the fast growth and proliferation demand of myofibroblasts.Besides,glycolysis also provides material guarantee for maintaining the proliferation and secretion activity of myofibroblasts,and the lactate produced during glycolysis can directly decrease the p H of the microenvironment to promote the activation of TGF-β1.Moreover,TGF-β1,the major growth factor involved in myofibroblast differentiation,has been proved to be a trigger of glycolytic reprogramming.As a result,glycolysis targeted therapies might represent new therapeutic strategies for pulmonary fibrosis reduction.Besides,a recent study demonstrated that the lactate produced by glycolysis of fibroblasts could be secreted into the extracellular to medicate the polarization of macrophages to pro-fibrotic phenotype,which further confirmed the important role of myofibroblast glycolysis in pulmonary fibrosis.Although existing studies have partially revealed the role of glycolysis in fibroblast activation and pulmonary fibrosis,the specific molecular mechanism is not yet fully understood.Accumulating studies have shown that many non-coding RNAs（nc RNAs）play an important role in initiation and progression of multiple diseases.Circular RNA （circ RNA）is a special subclass of endogenous non-coding RNAs with covalent and closed structures,thereby increasing the stability of them.Most circ RNAs are highly conserved across species and exhibit dynamic expression patterns in multiple physiology and pathological conditions.Therefore,circ RNAs are expected to be biomarkers and therapeutic targets of many diseases.Although,several recent studies have revealed that circ RNAs are also involved in the pathogenesis of fibrosis.Compared with the extensive studies of circ RNAs in cancers,there few studies on pulmonary fibrosis,especially silicosis,and the underlying mechanism has not yet been fully revealed.Circ HIPK3（hsa_circ₀000284）,a circ RNA derived from Exon2 of the HIPK3 gene,has been reported to be commonly expressed in multiple human tissues and enriched in the brain,liver,lung,and colon.Several studies suggest that circHIPK3 is significantly up-regulated in numerous tumor tissues and contributes to cell proliferation and migration.However,only a few studies have suggested that circHIPK3 is involved in the process of tissue fibrosis,and its role in silica-induced pulmonary fibrosis has not been reported.Our preliminary experimental results confirmed that circHIPK3 was upregulated in lung fibroblasts（MRC-5）following TGF-β1 treatment,companied with fibroblast activation.However,si RNA-medicated circHIPK3 silence blocked TGF-β1 induced fibroblast activation.Since circHIPK3 is derived from the exon of the gene and is mainly located in the cytoplasm of MRC-5,we speculate that it could act as a ce RNA to regulate fibroblast activation.Through bioinformatics analysis and experimental verification,we confirmed the interaction between miR-30a-3p and circHIPK3,and the expression of miR-30a-3p is regulated by circHIPK3.Therefore,we chose miR-30a-3p for further exploration,and a key regulator for glycolysis,FOXK2,was identified as a potential target of miR-30a-3p.In this study,we explored the function and molecular mechanism of circHIPK3 to regulate lung fibroblast activation and glycolysis and confirmed its function in silicosis-induced lung fibrosis at the animal level,which further revealing the pathogenesis of silicosis.It also provides a theoretical basis for further research on non-coding RNAs as biomarkers and therapeutic targets for silicosis.Objective We aimed to confirm the function of circHIPK3 in regulating fibroblast activation and further clarify the underlying molecular mechanism in vitro.Besides,we aimed to further determine the effect of miR-30a-3p and FOXK2 on fibroblast activation and glycolysis.The intervention effect of circHIPK3 silence and miR-30a-3p overexpression medicated by the adeno-associated virus was observed in silica-induced pulmonary fibrosis,further revealing the role and molecular mechanism of circHIPK3 regulating fibroblast activation in silicosis.Methods（1）We established TGF-β1 induced fibroblast activation cell models by using human embryo lung fibroblast MRC-5 cells.Western blot and immunostaining assays were used to detect the expression of pro-fibrotic markers and glycolytic enzymes.The proliferation of MRC-5 cells was examined by Ed U assay and MTT assay.RT-PCR assay was performed to determine the expression levels of circHIPK3 and miR-30a-3p.Glucose consumption,lactate production,glycolytic rate,and ATP production were performed with kits according to the manufacturer’s instructions.（2）The qRT-PCR assay was performed to detect the levels of circHIPK3 and miR-30a-3p.After RNase R or Actinomycin D treatment,the expression of circHIPK3 and HIPK3 was determined by qRT-PCR and RT-PCR assays.The location of circHIPK3 in MRC-5 cells was investigated by isolation of cytoplasmic and nuclear RNA assay and fluorescence in situ hybridization（FISH）assay.（3）The downstream miRNA of circHIPK3 was screened by bioinformatics analysis.Dual-luciferase reporter gene assay,RNA pull-down assay,and RNA immunoprecipitation（RIP）assay were used to verify the interaction between circHIPK3 and miR-30a-3p,miR-30a-3p and FOXK2.（4）Mouse primary lung fibroblasts were extracted and treated with TGF-β1.qRT-PCR was used to detect the expression levels of circHIPK3 and miR-30a-3p.Western blot was used to observe the effects of circHIPK3 si RNA and miR-30a-3p mimic on mouse fibroblast activation.（5）To build a silica-induced pulmonary fibrosis mouse model,mice were intratracheally administered a single installation with silica suspension.Then,AAV-medicated circHIPK3 silence and miR-30a-3p overexpression mouse model was established via intratracheally instilling with AAV-sh-circHIPK3 and AAV-pre-miR-30a-3p.After 3 weeks,mice were treated with Si O2 by using the above method.After the animal experiment is finished,lung tissues were harvested,and qRT-PCR was performed to detect the expression levels of circHIPK3 and miR-30a-3p.Then,HE staining was used to observe the pathological changes of lung tissue,and Western Blot was used to detect the expression of fibrosis-related molecules.Masson staining,Collagen I immunohistochemistry,and hydroxyproline content detection were performed to observe the deposition of collagen in lung tissue.（6）Total RNA from lung tissues of IPF patients and silicosis patients was extracted,and the expression levels of circHIPK3 and miR-30a-3p were detected by qRT-PCR.Results 1.Glycolysis is up-regulated during TGF-β1-induced pulmonary fibroblast activation Firstly,we established a fibroblast activation model in MRC-5 cells by stimulating with TGF-β1（0,1,2,5ng/m L）.It was confirmed that fibroblasts（MRC-5）were differentiation into myofibroblast following 5ng/ml TGF-β1 treatment for 48 h by detecting the expression of pro-fibrotic medicators and the cell proliferation ability.Therefore,we chose this concentration of TGF-β1 for subsequent experiments.Next,we found that the protein expression of glycolytic enzymes（HK2,PFKM,PKM2,and PDK1）was significantly increased in TGF-β1-stimulated MRC-5 cells,which was companied by the upregulation of lactate production and glucose consumption levels in the cultured medium.Moreover,2-Deoxy-d-glucose（2-DG）,an inhibitor of glycolysis,could decrease ECM accumulation,and cell viability induced by TGF-β1.Altogether,the data suggested that enhanced glycolysis may participate in fibroblast activation.2.circHIPK3 plays a key role in TGF-β1-derived fibroblast activation To observe the biological characteristics of circHIPK3,c DNA and g DNA were extracted separately from MRC-5 cells and subjected to nucleic acid electrophoresis detection.The results showed that circHIPK3 could be detected only in c DNA,as no products were detected in the extracted g DNA.Besides,the circHIPK3 and HIPK3 levels were detected after the exonuclease RNase R and actinomycin D treatment,and the results showed that circHIPK3 showed strong resistance to digestion by RNase R and was more stable than linear HIPK3 m RNA regarding actinomycin D treatment,indicating that circHIPK3 is a circular RNA formed by trans-splicing.Nuclear separation experiments showed that circHIPK3 was enriched in the cytoplasm,which is consistent with the results of fluorescence in situ hybridization（FISH）assay.Besides,circHIPK3 was elevated with TGF-β1 in a dose-dependent manner,suggesting the potential function of circHIPK3 during fibroblast activation.To investigate whether circHIPK3 is required for fibroblast activation,we performed loss-of-function experiments using small-interfering RNAs.Western blot analysis showed that circHIPK3 knockdown substantially inhibited the ECM protein production,and the staining of α-SMA in MRC-5 further confirmed the antifibrotic effects of circHIPK3 knockdown.Also,MTT assay and Ed U assay showed that loss of circHIPK3 significantly decreased the proliferation of MRC-5 cells.These data indicate circHIPK3 knockdown could reverse the myofibroblast phenotype.Interestingly,the production of lactate and the consumption of glucose were also reduced in circHIPK3 silence MRC-5 cells,indicating that circHIPK3 might be involved in the glycolysis of fibroblasts.3.circHIPK3 regulates fibroblast activation by directly binding with miR-30a-3p To verify the underlying molecular mechanism of circHIPK3,four databases（miRanda,miRDB,circ Bank,and RNAhybrid）were used to predict the potential target miRNAs of circHIPK3,and miR-30a-3p was found to be one of the potential targets of circHIPK3.We found miR-30a-3p was significantly down-regulated in IPF patients based on the microarray dataset GSE32538 and GSE27430.As opposed to circHIPK3,a significant reduction of miR-30a-3p was also observed in MRC-5 cells treated with TGF-β1.These data indicated the potential function of miR-30a-3p in regulating fibroblast activation and pulmonary fibrosis.To explore whether the expression level of miR-30a-3p is regulated by circHIPK3,we tested the expression level of miR-30a-3p in MRC-5 cells following circHIPK3 silence or overexpression.The results demonstrated that miR-30a-3p was upregulated after circHIPK3 knockdown,whereas circHIPK3 overexpression could decrease the expression of miR-30a-3p,indicating that miR-30a-3p medicated the pro-fibrotic effect of circHIPK3.According to bioinformatics analysis and the results of dual-luciferase reporter gene assay,Circ HIPK3 has two putative binding sites for miR-30a-3p,which was further confirmed by RIP and FISH assays.These data indicated that circHIPK3 could act as a sponge of miR-30a-3p.Similar to the effect of circHIPK3 silence,overexpression of miR-30a-3p could also block fibroblast activation.Moreover,the antifibrotic effect of si RNA-medicated circHIPK3 knockdown was abrogated by the miR-30a-3p inhibitor,whereas combined incubated with miR-30a-3p mimic abrogated the effect of circHIPK3 overexpression.These results revealed that miR-30a-3p mediated the function of circHIPK3 to regulate fibroblast activation.4.FOXK2 is a functional target of miR-30a-3p exerts its pro-fibrotic effect by regulating glycolysis Based on the bioinformatics analysis,FOXK2,which is considered a key transcription factor to regulate glycolysis,was deemed a potential target of miR-30a-3p.One putative miR-30a-3p-binding site within the 3′-UTR of human FOXK2 was predicted and the binding site is conserved in several species.The direct interaction between FOXK2 and miR-30a-3p was confirmed by a dual-luciferase reporter assay,an RNA pull-down assay,and a RIP assay.Contrary to the expression of miR-30a-3p,we observed that FOXK2 expression decreased in cells treated with miR-30a-3p mimics,whereas miR-30a-3p inhibitors could augment the expression of FOXK2 at protein levels.Then,we knocked down endogenous FOXK2 by transfecting si RNA and observed that FOXK2 knockdown could mimic the anti-fibrotic effects of miR-30a-3p.Besides,the fibrosis markers in miR-30a-3p-overexpression MRC-5 cells were reversed by FOXK2 overexpression.Taken together,these data indicated that miR-30a-3p exerts anti-fibrotic functions,at least partially,by directly targeting FOXK2.To investigate whether the FOXK2 plays its pro-fibrotic effect through the glycolytic reprogramming,we detected the key glycolysis-associated gene levels after FOXK2 manipulation.Western blot showed that silencing FOXK2 decreased the expression of HK2,PFKM,PKM,and PDK1 at protein levels,and the lactate production and glucose consumption were also reduced.However,plasmid-medicated FOXK2 overexpression exerted the opposite effect.Moreover,ectopic FOXK2 expression induced the pro-fibrotic protein production,whereas its effect was reversed by the inhibitor of glycolysis 2-DG.These data suggested that FOXK2 exerts its pro-fibrotic effect by regulating glycolysis of fibroblast.5.circHIPK3 and miR-30a-3p regulate silica-induced pulmonary fibrosis in vivo Circ HIPK3 and miR-30a-3p are highly conserved between humans and mice,and the results in mouse primary fibroblasts confirmed that circHIPK3 and miR-30a-3p could also regulate mouse lung fibroblast activation.Therefore,to verify the role of circHIPK3 and miR-30a-3p in pulmonary fibrosis in vivo,a silica-induced pulmonary fibrosis mouse model was established via intratracheal administration of silica suspension.Histologically,the normal alveolar architecture was destroyed and mature fibrotic nodules formed after silica exposure for 28 days.The finding that silica injury increased the expression of several pro-fibrotic mediators,including α-SMA,Collagen I,and Fibronectin further confirmed this observation.Meanwhile,hydroxyproline content and lactate content were also significantly up-regulated after silica treatment.Next,we detected circHIPK3 and miR-30a-3p expression levels in this model.We observed that circHIPK3 was up-regulated,and miR-30a-3p was markedly decreased in fibrotic lung tissues,which was consistent with our results in vitro and suggested the role of circHIPK3 and miR-30a-3p in regulating fibroblast activation.To confirm the role of circHIPK3 and miR-30a-3p during pulmonary fibrosis progression,we designed adeno-associated viral sh RNAs for circHIPK3 silencing and adeno-associated viral pre-miR-30 a for miR-30a-3p overexpression.After the animal experiment is finished,lung tissues were harvested,and qRT-PCR was performed to detect the expression levels of circHIPK3 and miR-30a-3p.As expected,AAV-pre-miR-30 a intratracheal injection significantly elevated miR-30a-3p levels and AAV-sh-circHIPK3 reduced lung circHIPK3 expression throughout the experiment.Circ HIPK3 silencing and miR-30a-3p overexpression exceedingly attenuated pulmonary fibrosis,according to the results of histological analysis,Western blot analysis of profibrotic proteins,and hydroxyproline analysis.Moreover,compared to the silica treatment group,the lactate content was also decreased following circHIPK3 knockdown or ectopic miR-30a-3p expression.Collectively,these data show the regulatory function of circHIPK3 and miR-30a-3p in silica-induced pulmonary fibrosis.6.Changes in the levels of circHIPK3 and miR-30a-3p in the lung tissues of silicosis and IPF patients To further verify our experimental results at the cellular and animal levels,we extracted total RNA from lung tissue samples of silicosis patients and IPF patients,and detected the expression levels of circHIPK3 and miR-30a-3p by qRT-PCR.The results showed that compared with the control group,the expression level of circHIPK3 in the lung tissues of IPF patients and silicosis patients was significantly increased,while the expression level of miR-30a-3p was significantly reduced.These data were consistent with our research results at the cell and animal levels and suggested that circHIPK3 and miR-30a-3p also have potential regulatory effects in你human pulmonary fibrosis.Conclusion In conclusion,our study investigated the expression and effects of circHIPK3 in pulmonary fibrosis and revealed a marked increase of circHIPK3 in fibroblasts exposed to TGF-β1 and the mouse lungs upon silica treatment.The silencing of circHIPK3 was sufficient to inhibit fibroblast activation and glycolysis in vitro and alleviates pulmonary fibrosis progression in vivo.Mechanistically,circHIPK3 could facilitate glycolysis and fibroblast activation as a sponge of miR-30a-3p,thus relieving its downstream target FOXK2 from miRNA-medicated translational repression.This study perfected the regulatory network of circHIPK3 in the initiation and progression of silicosis and confirmed the important role of circHIPK3 and its downstream molecules in fibroblast activation and silica-induced pulmonary fibrosis.We further revealed the underlying molecular mechanism of circHIPK3 to promote fibroblast activation by regulating glycolysis and suggested that interference with circHIPK3/miR-30a-3p/FOXK2 provides a potential strategy for the treatment of silicosis.