Functions And Mechanisms Of CircCREBBP/miR1291/LEFTY2 Axis In Hepatic Fibrosis

Purpose: Hepatic fibrosis(HF)is a common pathologic change of persistent liver injury,the sustaining development of which causes liver cirrhosis,so far as to primary hepatocellular carcinoma.However,early screening and diagnostic methods as well as effective preventing means are in short.To solve this existing preventing problem,we considered pathophysiological process of activation and proliferation of hepatic stellate cells as the crux issue.CircularRNA(circRNA),a new-type noncodingRNA,was suggested momentous functions in various diseases,thus gaining insights as a potential disease marker.As yet,there has been few report focusing on circRNA in HF related disease though.Our research prepared to detect that if the circRNA expressed differentially in tissues of HB patients with high throughput sequencing.Combining with bioinformatics analysis,we would discuss the regulations of circRNA on targeted genes and activation and proliferation of hepatic stellate cells,ulteriorly deduce its role and conceivable mechanism during the progression of HB.Method: Collecting 17 patient who had hepatocellular carcinoma(HCC)related HB to provide liver tissues,randomly select 4 of the samples to take high-throughput sequencing.As the control group,collecting 11 hepatic hemangioma patients for the samples,randomly select 3 to take high-throughput sequencing.Then build a circRNAs database,applying Gene Ontology(GO)function classification,enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis to the differentially expressed circRNA.Choose 7 of these circRNAs,conduct clinical verifications in 13 HCC related HB samples,and samples from 8 hemangioma patients in the control group.By the means of qRT-PCR and western blot(WB),our study detected the results of overexpressed hsa_circ₀007637 in the aspects of gene and protein expression of ?-SMA and COL1 in human Hepatic stellate cells line(LX2 cell)pretreated by TGF-?1,LX2 Cyclin D1,C-Myc and CDK4 expression,and gene and protein expression of microRNA1291 and LEFTY2.We overexpressed mmu_circ₀006288 in liver of adenovirus mice,examined expression of miR1291,LEFTY2 mRNA and LEFTY2 protein.Finally we used qRT-PCR,WB and immunohistochemical method to monitor the influences of expression and overexpression of LEFTY2 on Smad3 phosphorylation in clinical samples,then verified the outcome of overexpression and block of miR-1291 on ?-SMA,COL1 a and LEFTY 2.Results: By means of high-throughput sequencing,we detected 11136 corcRNAs in total,103 of which have apparent differential expression.Compared with the control group,there was 18 circRNAs' expression being up-regulated in HF group,while other 85 was lower expressed.GO analysis prompted circRNAs relating with biolaial regulation and active proliferation of liver diseases.KEGG analysis reveal that,via participating signal transduction,material transportation and catabolism,mediating the hydropretein molecular pathway,etc,circRNAs may influence the progression of HF.Verification of 7 discrepant circRNAs byReal-time fluorescence quantification(qRTPCR)was corresponded to the up-regulation of hsa_circ₀003502 in HF,and downregulation of hsa_circ₀072309,hsa_circ₀023919,hsa_circ₀008267,hsa_circ₀007637,hsa_circ₀006117 and hsa_circ₀003441.Circ CREBBP presented lower expression in HB tissues,mice HB tissues and TGF-?1 stimulated LX2 cells.The mice liver overexpressed mmu_circ₀006288,while degraded miR-1291 and upgraded LEFTY2.The expression of hsa_circ₀007637 in LX2 cells markedly down-regulated gene and protein expression of ?-SMA,COL1 a,likewise LX2 Cyclin D1,C-Myc and CDK4 protein.Its regulating function to miR-1291 and LEFTY2 was corresponding to the results overexpression experiment in mice liver.Dual-luciferase report experiment discovered the targeted binding of miR-1291 with hsa_circ₀007637 and LEFTY2.Further testing indicated high expression of miR-1291 in HB tissues,mice HB tissues and TGF-?1 stimulated LX2 cells.The overexpression of miR-1291 may signally increase the expression levels of ?-SMA and COL1 a gene and protein in LX2 cells,while lead to obvious decline of LEFTY2,which was in contrast with the trends of miR-1291 blocked group and hsa_circ₀007637 overexpression group.Also finding through dual-luciferase report experiment that,miR-1291 could targeted combine with LEFTY2,which expressed dramatically lower in HF group than the control group.Overexpression of LEFTY2 would memorably decrease expression of ?-SMA,COL1 a and Smad3 in LX2 cells,which fit the trend of blocking miR-1291.Conclusion: Our research firstly suggested that hsa_circ₀007637 was apparently low expression in HB patients' tissues.Overexpression of hsa_circ₀007637 would suppress the expression of LX2 activation and proliferation factors,then restrained LX2 cell cycle procedure,thus reduced LX2 cell proliferation.Hsa_circ₀007637 may get involved in HSCs activation and proliferation by regulating LEFTY2 expression through adsorbing miR-1291.The targeted gene LEFTY2 regulated HSCs activation and related TGF-?1/Smad3 pathway,finally formed a completed work chain of Circ CREBBP regulating the expression of miR1291/LEFTY2/Smad3 axis-intracellular signal transduction-HSCs activation and proliferation,providing alternative target spots for diagnosis and treatment of clinical fibrosis.