Differential Expression Of Circular RNAs And The Potential Regulatory Role Of Hsa_circ₀006508 In Idiopathic Pulmonary Fibrosis

Objective:Idiopathic pulmonary fibrosis?IPF?is a lethal fibrotic lung disease.There is no effective disease-modifying treatment largely due to the unclear pathogenesis.Circular RNAs?circRNAs?are a group of newly discovered non-coding RNA,which has been shown to play an important regulatory role in variety of diseases.However,the precise regulation mechanism of circRNAs in IPF progression is still unknown.In present study,we investigated the alterations of circRNAs' expression in venous blood from IPF.The significantly different circRNA was screened,and its functions and possible molecular mechanisms were further explored in cell experiments and in vivo.Those may provide new target for the diagnosis and treatment of IPF Methods:1.According to diagnostic criteria,six IPF patients and four healthy volunteers?older than 65?were recruited.The differentially expressed circRNA profiles in venous blood were investigated by high?throughput whole transcriptome sequencing and were analyzed by bioinformatics analysis.Then 20 patients with IPF and 20 healthy controls were selected according to the same standard,RT-qPCR was used to validate the expression of the significantly different circRNAs in venous blood.2.Cell experiments were carried out on two human lung fibroblast lines?MRC-5 and IMR-90?.Sanger sequencing was used to validate the cyclic structure of hsa_circ₀006508.The miRNA regulated by hsa_circ₀006508 and its downstream target proteins were predicted by bioinformatics.Hsa_circ₀006508 was knocked down by transfecting siRNA.The two cell lines were both divided into four group: Control group,TGF-?1group?cells were induced by TGF-?1?,TGF-?1+ si-NC group?cells were induced by TGF-?1 and transfected by negative siRNA?and TGF-?1+si-Circ group?cells were induced by TGF-?1 and transfected by siRNA?.The biological roles were measured by cell proliferation assays and wound healing assay.RT-qPCR was used to test the expression of hsa_circ₀006508 and miR-520a-5p.The luciferase reporter assay was performed toevaluate the interaction between hsa_circ₀006508 and miR-520a-5p,miR-520a-5p and TLR7.The expression of collage I and TLR7 was assessed by western blot.3.Nine weeks old male C57BL/6 mice were chosen to construct the pulmonary fibrosis mouse model induced by bleomycin in this study.We divided the mice into four groups: NC14 group?control group,samples were collected on the 14 th day?,NC28 group?control group,samples were collected on the 28 th day?,IPF14 group?bleomycin induced group,samples were collected on the 14 th day?and IPF28 group?bleomycin induced group,samples were collected on the 28 th day?.The injury of pulmonary structure in each group was observed by HE staining.The degree of pulmonary fibrosis was evaluated by Masson staining.The expression of hsa_circ₀006508's homologous gene in mouse lung tissue was detected by RT-qPCR.The expression of collage I and TLR7 in mouse lung tissue was assessed by western blot.Results:1.A total of 7869 differentially expressed circRNAs were identified in peripheral venous blood,of which 377 circRNAs?64 circRNAs were novel?showed statistically significant differences?fold change>2.0,P<0.05?between the two groups.Compared with normal controls,there were 286 upregulated circRNAs and 91 downregulated circRNAs in IPF group.GO analysis suggested that the differentially expressed circRNAs may be involved in histone methylation,negative regulation of type 2 immune response,formation of CCR4-NOT complex,formation of MHC protein complex,regulation of poly?A?specific ribonuclease activity,and SUMO binding.KEGG analysis showed the most enriched pathways regulated by the upregulated circRNAs,including protein processing in the endoplasmic reticulum,TNF signaling pathway,ErbB signaling pathway,and FoxO signaling pathway.The top five miRNAs regulated by each of the differentially expressed circRNAs were displayed by bioinformatics analysis.Five upregulated circRNAs were selected for the validation in more samples.We found that hsa_circ₀007334,hsa_circ₀001395,hsa_circ₀000972 and hsa_circ₀006508 showed the same change directions and statistical significance.2.The expression of the above four circRNAs was detected in two human lung fibroblast cell lines,MRC-5 and IMR-90,and hsa_circ₀006508 was abundantly expressed.We confirmed the head-to-tail splicing of hsa_circ₀006508 by Sanger sequencing.Compared to control group,the expression of hsa_circ₀006508 was significantlyincreased in pulmonary fibroblasts induced by TGF-?1.Using small interfering RNA?siRNA?to target the back-splicing sequence,we effectively knocked down hsa_circ₀006508 in both cell lines.Downregulation of hsa_circ₀006508 resulted in a reduction of collagen I,which was increased in TGF-?1 group.The results of MTT assay and wound healing assay revealed that TGF-?1 induced cell growth and migration,while knockdown of hsa_circ₀006508 attenuated the effect of TGF-?1.It was predicted that hsa_circ₀006508 could adsorb miR-520a-5p and regulate the expression of its downstream TLR7.The content of miR-520a-5p in TGF-?1 group was significantly lower than that in the control group?P < 0.01?,and miR-520a-5p was upregulated after knockdown of hsa_circ₀006508?compared with TGF-?1+ si-NC group,P< 0.01?.The double luciferase reporter assay verified that hsa_circ₀006508 could be used as miR-520a-5p sponge,and miR-520a-5p could combine with the mRNA3'-UTR of TLR7 directly.The expression of TLR7 was increased in TGF-?1 group?P<0.05?,and was significantly decreased in TGF-?1+ si-Circ group,compared with TGF-?1+si-NC group P<0.05.3.The results of HE and Masson staining in the lung tissues of mice showed that in the early stage of pulmonary fibrosis,the inflammatory reaction was obvious,with a large number of inflammatory cells infiltrating.In the latter stage,the alveolar structure was destroyed seriously,and there were a large number of fibroblasts and the extracellular matrix.It was revealed that mmu_circ₀003507 was the homologous gene of hsa_circ₀006508 in mice.The expression level of mmu_circ₀003507 was significantly upregulated in the lung tissues of bleomycin-induced mice,and was increased with the aggravation of fibrosis?P<0.05?.Western blot results showed that the expression levels of collagen I and TLR7 were significantly higher in IPF group than in control group?P<0.05?,and the increase was more significant in IPF28 group than in IPF14 group.Conclusions:1.Our study provides a venous blood circRNA profiling in IPF by high?throughput whole transcriptome sequencing,which may provide a database of circRNAs to study a novel class of biomarkers for IPF.2.The verification experiment with more samples shows that the expression of hsa_circ₀007334,hsa_circ₀001395,hsa_circ₀000972 and hsa_circ₀006508 is significantly increased in the blood of IPF.3.In cell experiments,we demonstrate that circ₀006508 is up-regulated in fibroblast lines induced by TGF-?1,and it promotes lung fibroblast proliferation,migration and the deposition of extracellular matrix as a sponge for miR-520a-5p to up-regulate the expression of TLR7.Hsa_circ₀006508 / mir-520a-5p /TLR7 pathway was first proposed as a possible new pathogenesis of IPF.4.In the bleomycin-induced pulmonary fibrosis mouse,mmu_circ₀003507?homologous gene of hsa_circ₀006508 in mice?plays a profibrotic role throughout the development of pulmonary fibrosis,and its expression level is closely related to the severity of the disease.It provides a theoretical possibility for hsa_circ₀006508 to become a new clinical diagnostic biomarker and therapeutic target for IPF.