Research On The Regulation Mechanism Of Circ＿0001377 In Hepatic Fibrosis Through Competitive Binding Of Let-7a-5p

Background and PurposeHepatic fibrosis（HF）is a complex injury-repair response triggered by a variety of chronic liver injuries,including non-alcoholic steatohepatitis,non-alcoholic fatty liver disease,alcohol abuse,autoimmune hepatitis,viral hepatitis,biliary obstruction,and metabolic diseases which are caused by toxins such as copper and iron deposition,PCBs,carbon tetrachloride,and vinyl chloride.Excessive production and deposition of extracellular matrix（ECM）in the liver is a key process in the development of HF.This process is mainly triggered by the activation of the hepatic stellate cell（HSC）present in the perisinusoidal space（or subendothelial space of Disse,Disse cavity）.Intrahepatic cell necrosis and inflammatory stimuli can lead to activation and conversion of hepatic stellate cells to myofibroblast（MF）,producing α-smooth muscle actin（α-SMA）,releasing proinflammatory,pro-fibrotic and pro-cytokinesis factor and releasing large mounts of ECM,which would gradually replace the low-density basement membrane-like matrix in the Disse cavity with fibrotic collagen,resulting in abnormal deposition of ECM in the liver.Progressive HF,which eventually leads to cirrhosis,has a poor prognosis and a high mortality rate.With approximately 3 million or more patients suffering from chronic liver disease worldwide,the potential therapeutic receiver group for HF is enormous.Given that there is,as of now,no effective early diagnosis and treatment plan for HF,it is of great importance to further explore the molecular mechanism of its development to discover molecular targets with clinical translational value.However,at present,circular RNA（circRNA）is less investigated than other subjects in the field of HF and its related studies are still in an early stage,awaiting further exploration,which means that its function and regulatory mechanisms are still unclear.Based on the results of RNA-seq sequencing of preclinical HF tissues,we screened out circ＿0001377 which was significantly high expressed in the HF tissues.The expression of circ＿00013 77 was significantly increased after stimulation of TGF-β1 in LX-2.Twenty clinical human HF samples were collected and circ＿0001377 was highly expressed in clinical HF samples during qRT-PCR detection.The expression of circ＿0001377 increased with higher fibrosis staging level.Fluorescence in situ hybridization（FISH）showed circ＿0001377 was specifically expressed in the cytoplasm of HSC.The mouse HF model was induced by common bile duct ligation,and the homologous mmu>chr16₃2663738₃2671633₊was also increased in mouse HF.Overexpression of circ＿0001377 was found through function gain and function loss experiments can promote HSC activation,collagen secretion,and cell proliferation,migration and G1-S phase transformation;and after knock down circ＿0001377,HSC activation and collagen secretion were inhibited,cell proliferation and migration decreased,and apoptosis increased,suggesting circ＿0001377 may be a potential therapeutic target for HF.Through the reseach on its competing endogenous RNA（ceRNA）regulation mechanism,we found that circ＿0001377 had a direct binding relationship with let-7a-5p,and could regulate the expression of TGFBR1,Smad2 and Col1a2 which were the downstream target genes of let7a-5p.The fibrogenic effect of circ＿0001377 was partially canceled by let-7a-5p after its transfection into the stable circ＿0001377 overexpression strain,and the high expression levels of TGFBR1,Smad2 and Col1a2 were also partially canceled,suggesting that let-7a5p may inhibit HSC activation and HF induced by circ＿0001377.Based on the above experimental results,we speculate that circ＿0001377 may regulate the expression of TGFBR1,Smad2 and Col1a2 through competitive binding to let-7a-5p,so as to promote HSC activation and HF.The study was carried out in three parts:（1）identification of circ＿0001377 expression in human HSC and HF tissue;（2）in vitro experiments to explore the regulatory role of circ＿0001377 on HSC activation/HF development;（3）the ceRNA mechanism of circ＿0001377 in HF through competitive binding of let-7a-5p.Part Ⅰ:Identification of circ＿0001377 expression in human HSC and HF tissueMethod1.Based on the results of previous circRNA-seq sequencing in clinical human HF tissues,eight ecircRNA（FC>2,p<0.05）were initially selected for the study.Divergent primer or backsplice junction primer was designed and ecircRNA was validated by qRT-PCR assay in LX-2 cell line.2.Through sequence alignment,circRNA6354 obtained by the research group was confirmed to be hsa_circ＿0001377 included in the circBase database.The circularity of circRNA6354（circ＿0001377）was further confirmed by Sanger sequencing and RNase R digestion experiments.3.Twenty cases of clinical human HF tissues were collected.The expression of circ＿0001377 in HF tissues was verified by qRT-PCR assay.Cy3-labeled-circ＿0001377 probe was designed and constructed for the detection of circ＿0001377 in HF tissue through FISH assay.4.A mouse HF model was constructed by induction of common bile duct ligation.qRTPCR was used to detect the expression of homologous mmu>chr16₃2663738₃2671633₊in mouse HF tissues.Results1.Through qRT-PCR verification,five circRNA represented by circRNA6354（circ＿0001377）were successfully detected in human LX-2 cell line.The expression of circRNA6354（circ＿0001377）was significantly upregulated after activation of LX-2 cells with TGF-β1 stimulation.Initially,circRNA6354 was selected as the target for downstream study.2.Sequence alignment confirmed that the circRNA6354 obtained by our circRNA-seq sequencing was the hsa_circ＿0001377 included in the circbase database.Bioinformatics analysis showed that circ＿0001377 was formed of 8 exons of TNK2 and conserved among species.The junction region of circ＿0001377 was detected by Sanger sequencing.RNase R digestion experiments demonstrated that circ＿0001377 can significantly resist RNase R digestion.These resultes further confirmed the circularity structure of circ＿0001377.3.Twenty cases of clinical human HF tissues were collected.High expression of circ＿0001377 in HF tissues was confirmed by qRT-PCR reaction,and the expression of circ＿0001377 increased with increasing fibrosis staging level.FISH experiments confirmed that circ＿0001377 is specifically expressed in the cytoplasm of hepatic stellate cells.4.A mouse HF model was constructed by induction of common bile duct ligation.qRTPCR revealed that the expression of homologous mmu>chr16₃2663738₃2671633₊was elevated in mouse HF tissues.Conclusioncirc＿0001377 is specifically highly expressed in hepatic stellate cells,and its expression level can be increased with HSC cell activation;circ＿0001377 is highly expressed in HF tissues,and the expression of circ＿0001377 increases with increasing fibrosis stage level;circ＿0001377 is highly conserved between species.Expression of its homologue mmu>chr16₃2663738₃2671633₊is also elevated in murine HF tissue;circ＿0001377 may serve as a new biomarker of HF.Part Ⅱ:In vitro experiments to investigate the regulatory role of circ＿0001377 on HSC activation/HF developmentMethod1.circ＿0001377-overexpressed lentiviral vector and stable circ＿0001377-over-expression strain were constructed.2.Expression of HF indicator α-SMA and Collal were detected using qRT-PCR and western blot assays in stable circ＿00013 77-over-expression strain.cell proliferation,migration and cell cycle were detected by CCK8,transwell and cell cycle assays in the stable circ＿0001377-over-expression strain strain.3.Designed three siRNA interference targets based on the sequence information of circ＿0001377 interface region,and achieved the knockdown efficiency by qRT-PCR to obtain the best siRNA for downstream experiments.4.The expression of LX-2 HF indicators α-SMA and Collal were detected by qRT-PCR and western blot assay after knockdown of circ＿0001377.CCK8,transwell and Annexin V-APC cell apoptosis assays detected cell proliferation,migration and apoptosis after knockdown of circ＿0001377.Results1.The circ＿0001377 overexpressed lenti viral vector and stable circ＿0001377-overexpression strain were successfully constructed.The monoclonal cells with good identification results were cryopreserved and used for downstream functional assays.2.The results of qRT-PCR and western blot experiments showed that the expression of HF indicators,α-SMA and Col1a1,increased after overexpression of circ＿0001377 in LX-2 cells.CCK8,transwell and cell cycle assays showed that LX-2 cells had enhanced proliferation and migration ability and increased transformation of G1 to S Phase after overexpression of circ＿0001377.3.Three siRNA interference targets were designed based on the sequence information of circ＿0001377 interface region,and the siRNA2 with the best knockdown efficiency（82.10%）was obtained by qRT-PCR for downstream experiments.4.The results of qRT-PCR and western blot experiments showed that the expression of HF indicators α-SMA and Collal was reduced after knockdown of circ＿0001377.CCK8,transwell and Annexin V-APC apoptosis assays showed that LX-2 proliferation and migration decreased and apoptosis increased after knockdown of circ＿0001377.ConclusionThrough gain-of-function and loss-of-function experiments,we demonstrated that circ＿0001377 plays an important regulatory role in the process of HSC activation and HF.Overexpression of circ＿0001377 promoted HSC activation,cell proliferation,cell migration and G1-S transformation and while knockdown of circ＿0001377 resulted in inhibition of HSC activation,decreased cell proliferation and migration and increased the apoptosis.It is suggested that circ＿0001377 may be an important potential therapeutic target for HF.Part Ⅲ:The ceRNA mechanism of circ＿0001377 in HF through competitive binding of let-7a-5pMethod1.miRanda algorithm predicted miRNAs with binding sites to circ＿0001377.In vitro cytological assay was used to detect the regulatory function of let-7a-5p/let-7c-5p/let7f-5p/let-7g-5p initially.2.AGO-RIP and dual luciferase reporter experiments were conducted to the verify directly binding of circ＿0001377 with let-7a-5p.3.Preliminary screening of for possible target genes of let-7a-5p was performed by TargetScan prediction and qRT-PCR assay.The possible negative regulatory relationships between let-7a-5p and TGFBR1,Smad2 and Col1a2 were further explored by western blot,qRT-PCR and immunohistochemical staining.Biotin-coupled-let-7a-5p probe was designed for biotinylated miRNA capture assay to dectect directly binding of let-7a-5p and TGFBR1,Smad2 and Col1a2.4.In vitro cytological experiments were performed to investigate whether circ＿0001377 can promote HSC activation/HF by competitively binding let-7a-5p and regulating the expression of TGFBR1,Smad2 and Colla2.Results1.Through miRanda algorithm,combination score between hsa-let-7a-5p/hsa-let-7c5p/hsa-let-7f-5p/hsa-let-7g-5p and circ＿0001377 was the highest.Based on the in vitro cell function experiments,let-7a-5p was preliminarily selected as possible ceRNA of circ＿0001377 for downstream research.2.AGO-RIP and dual luciferase reporter experiments verified the direct binding of cire₀001377 with let-7a-5p.3.TGFBR1,Smad2 and Col1a2 were initially screened as possible target genes for let-7a5p by TargetScan prediction and qRT-PCR reaction.The western blot experiment,qRTPCR and immunohistochemical staining suggested a possible negative regulatory relationship between let-7a-5p and TGFBR1,Smad2 and Col1a2.Biotinylated miRNA capture assays revealed that TGFBR1,Smad2 and Col1a2 may be the direct targets of let-7a-5p in LX-2 cells.4.Western blot experiments confirmed that TGFBR1,Smad2 and Colla2 expression were significantly elevated in stable circ＿0001377-over-expression strain,while TGFBR1,Smad2 and Col1a2 expression was significantly decreased after knockdown of circ＿0001377 in LX-2 cells by siRNA.The intro experiments revealed after the transfection of let-7a-5p,cell activation,proliferation,migration and transformation of G1 to S Phase were partially canceled in stable circ＿0001377-over-expression strain,and the high expression levels of TGFBR1,Smad2 and Col1a2 were also partially canceled.Conclusion1.let-7a-5p and circ＿0001377 has direct binding relationship.2.TGFBR1,Smad2 and Col1a2 may be direct targets of let-7a-5p in LX-2 cells.3.circ＿0001377 may regulate the expression of TGFBR1,Smad2 and Col1a2 through competitive binding to let-7a-5p,so as to promote HSC activation and HF.