| Back ground:With the quickening tempo of life,aging population and changing dietary structure,the population of individuals suffering from chronic kidney disease（CKD）is increasing worldwide.As a global public health problem,CKD poses a great economic and medical burden to society and the healthcare system [1].If left untreated,CKD will eventually progress to end stage renal disease（ESRD）[2] and require renal replacement therapy such as kidney transplantation or dialysis to survive,which substantially increases the medical burden.Pathological features such as interstitial fibrosis,tubular atrophy and peripheral capillary sparseness are the main common hallmarks of CKD [3].Renal fibrosis is a common pathway in CKD of various etiologies and is the main pathological predecessor of end-stage renal disease[4].Renal fibrosis is a process described by hyperproliferation of fibroblasts and extracellular matrix（ECM）deposition,accompanied by tubular atrophy and inflammatory cell infiltration [5].Studies have shown that the fibrotic process is triggered and coordinated by crosstalk between multiple cell types,including renal tubular epithelium,mesenchymal cells,inflammatory cells,and endothelial cells [6].Epithelial-mesenchymal transition（EMT）,described as damaged renal tubular epithelial cells undergoing phenotypic changes and thus acquiring mesenchymal features,has been widely accepted as a typical mechanism of renal fibrosis and is implicated in the evolution of interstitial fibrosis in the kidney [7].In addition,molecular events including pathological features such as fibroblast proliferation,tubular injury,macrophage activation and endothelial cell depletion outline key events in the pathogenesis of renal fibrosis [8].Despite a large body of literature with in-depth and extensive mechanistic studies,the mechanism of renal interstitial fibrosis is still unclear at present,and there is still no effective treatment for renal fibrosis.Therefore,we are committed to exploring targets with potential therapeutic potential in order to contribute to reducing the burden of chronic kidney disease.circular RNAs（circ RNAs）are classified as a major category of homologous non-coding RNAs,and they are covalently closed loops produced by "reverse splicing" of precursor m RNAs [9].circ RNAs have become a new research hotspot in RNA biology,and most circ RNAs are stable and conserved across species.are stable and conserved in different species.Despite the previous view that circ RNAs are useless products of splicing of primary transcripts.With the constant evolution of high-throughput RNA sequencing technologies,emerging evidence suggests that circ RNAs can be involved in the pathophysiological processes of various diseases including cardiovascular diseases [10],neurodegenerative diseases [11],metabolic diseases [12],and tumors [9].circ RNAs play essential part in a large diversity of cellular processes ranging from cell apoptosis,proliferation,and senescence,and have been identified and demonstrated to provide new insights and potential therapeutic targets for disease diagnosis and treatment [13,14].Furthermore,as circ RNAs have been widely characterized functionally,they have emerged as novel therapeutics for fibrosis [15].xu et al [16] found that circ HIPK3 regulates pulmonary fibrosis by promoting a forkhead box transcription factor K2（FOXK2）-dependent glycolytic approach.In renal fibrosis,circ RNA010383 downregulation may promote the deterioration of renal fibrosis in diabetic nephropathy [17];Yi et al.found that circ RNA30032 could induce renal fibrosis by mediating the miR-96-5p/heparinbinding epidermal growth factor（HBEGF）/oncogene（KRAS）axis [18].circ-Ankib1 can promote tumorigenesis by controlling the expression of interferon-related genes and pro-inflammatory factors in sarcoma cells [19].circ-Ankib1 can also regulate Schwann cell proliferation after peripheral nerve injury [20].Bioinformatics suggests that circ-Ankib1 is downregulated in renal fibrosis [21].However,little is known about the role of circ-Ankib1 in renal disease.Here,our study focused for the first time on whether circ-Ankib1 has an anti-fibrotic role in the kidney.Micro RNAs（miRNAs）are approximately 22-nt long non-coding RNAs that control target gene expression through m RNA degradation or translational repression after transcription[22].Previous studies have shown that miR-449 a promotes cardiac fibrosis through the lysine oxidase 3（LOXL3）/target of rapamycin protein（m TOR）axis [23].We identified the presence of binding sites between circ-Ankib1 and miR-449 a by starbase software,predicting a potential interrelationship between the two.Furthermore,Mi R-449 a is able to target Bcl2 to regulate autophagy to promote lung fibrosis [24].Therefore,whether circ-Ankib1 is involved in renal fibrosis via miR-449 a is unknown.Krüppel-like factor 4（Klf4）is an evolutionarily conserved zinc finger protein that plays as a transcription factor involved in regulating cell proliferation,differentiation,and apoptosis,among other roles.It was shown that Klf4 expression was verified to be reduced in renal fibrosis in two in vivo models of unilateral ureteral obstruction,and that it can regulate renal fibrosis by suppressing inflammation,suggesting an antifibrotic role of Klf4 in the kidney [25,26].Macrophage migration inhibitory factor（MIF）and monocyte chemotactic protein-1（MCP-1）are important inflammatory cytokines associated with kidney disease,and Klf4 reduces inflammation by eliminating transforming growth factor-β1（TGF-β1）-induced production of MIF and MCP-1 in human renal tubular cells [27].Mitochondrial fusion protein 2（mitofusin2,Mfn2）is a transmembrane multifunctional protein located on the outer mitochondrial membrane and is involved in a large diversity of cellular processes.Downregulation of macrophage Mfn2 leads to macrophage polarization and conversion to a pro-fibrotic phenotype [28].It was found that pioglitazone exerts a nephroprotective effect by upregulating the expression of fusion proteins Opa-1 and Mfn2 and decreasing the fission protein Drp1 in a 5/6nephrectomy rat model [29].the role of the Klf4/Mfn2 axis in renal interstitial fibrosis has been documented [30].We predicted by bioinformatics that miR-449 a may directly bind to Klf4 m RNA,therefore,this study hypothesized that circ-Ankib1 may be involved in renal fibrosis via miR-449 a and that miR-449 a binds to the downstream transcription factor Klf4/Mfn2 to participate in renal fibrosis.In conclusion,we aimed to comprehensively explore the mechanism of circ-Ankib1 targeting miR-449a/Klf4/Mfn2 axis in inhibiting renal fibrosis formation and to provide insights into the discovery of potential therapeutic targets for renal fibrosis.Part I: Function of circ-Ankib1 in renal fibrosisObjective:Determining the role of circ-Ankib1 in renal fibrosis.Method:1.Firstly,polymerized primers of circ-Ankib1 that can be amplified were designed.The effect of RNase R on the expression of circ-Ankib1 was investigated in TCMK-1 cells.After securing permission for individual well-informed consent,fifteen untreated patients with CKD were selected to collect human kidney samples through renal puncture（CKD group）,and eighteen patients with renal cancer were elected as the controls.The levels of circ-Ankib1 in the two groups were compared relative to each other by Real-Time Quantitative Reverse Transcription PCR（qRTPCR）.The expressions of fibrosis and EMT related proteins （?）-SMA,collagen type IV（COL Ⅳ）,FN,N-cadherin and E-cadherin were determined by Western blotting in the kidney tissues of the two groups.2.TCMK-1 cells cultured in vitro were treated with different concentrations（1.25,2.5,5,10ng/ml）of TGF-β1.After 72 hours of induction,qRT-PCR was used to compare the level of circ-Ankib1 in TCMK-1 cells-.The expressions of fibrosis and EMT related proteins （?）-SMA,COL Ⅳ,FN,N-cadherin and E-cadherin were determined by Western blotting.3.The cultured TCMK-1 cells and primary renal tubular epithelial cells（TEC）were divided into the following four groups: normal control group（untreated TCMK-1 cells）;TGF-β1 Group（10ng/ ml TGF-β1 induced TCMK-1 cells）;TGF-β1+ pc DNA31.Group（10ng / ml TGF-β 1 induced transfection of pc DNA3.1 TCMK-1 cells）;TGF-β1+ pc DNA3.1 circ-Ankib1 group（10ng/ml TGF-β1 induced transfection of recombinant plasmid pc DNA3.1 circ-Ankib1 TCMK-1 cells）.The relative level of circ-Ankib1 in each group was detected by qRT-PCR.The levels of fibrosis and EMT related proteins （?）-SMA,COL Ⅳ,FN,N-cadherin and E-cadherin were determined by Western blotting.The α-SMA and COL Ⅳ levels in various group were determined by immunofluorescence staining.4.UUO mouse models were constructed,and mice were divided into shamoperated（Sham）group,UUO group,overexpression negative control UUO group（UUO+ oe-NC）,and overexpression circ-Ankib1 UUO group（UUO+oe-CircAnkib1）;qRT-PCR was performed to compare the relative Circ-Ankib1 levels in kidney tissues in these 4 groups;observation of Masson staining images of kidney tissues in the 4 groups;Western blotting to detect the levels of fibrosis and EMTrelated proteins α-SMA,COL Ⅳ,FN in the kidney tissues of each group,and immunohistochemical analysis to further demonstrate the expression of fibrosis and epithelial mesenchymal transition-related proteins α-SMA,COL Ⅳ,FN expression in the renal tissues of each group.Result:1.We designed specific primers for circ-Ankib1.Treatment of TCMK-1 cells with RNase R significantly inhibited the expression of Ankib1,while RNase R had no significant effect on the expression of circ-Ankib1.circ-Ankib1 was significantly downregulated in kidney tissues from CKD patients compared to normal kidney tissues.2.TGF-β1（10ng/ml）down-regulated the expression of circ-Ankib1 in TCMK-1 cells and significantly increased α-SMA,COL Ⅳ,FN protein levels.3.qRT-PCR showed that TGF-β1-induced reduction of circ-Ankib1 could be reversed by transfection of circ-Ankib1;by western blotting,we found that overexpression of circ-Ankib1 downregulated TGF-β1-induced α-SMA,COL Ⅳ,FN levels high expression.4.Reduced circ-Ankib1 levels were observed in UUO mice compared to normal mice;significantly increased levels of circ-Ankib1 in the kidneys of mice injected with circ-Ankib1 plasmid compared to oe-NC controls;Masson staining observed significant renal fibrosis in the kidney tissue of the UUO group compared to the sham-operated group,circ-Ankib1 overexpression greatly attenuated UUO-induced fibrosis;western blotting detected an increase in α-SMA,COL Ⅳ,and FN due to UUO,which was reversed by high circ-Ankib1 expression;immunohistochemical analysis had the same would result,indicating that renal tissue in the UUO group α-SMA,COL Ⅳ,FN increased,and circ-Ankib1 overexpression suppressed this change.Conclusion:circ-Ankib1 inhibits EMT and renal fibrosis.Part II: The role of miR-449a/Klf4 in renal fibrosis To clarify the role of miR-449 a /Klf4 interaction in renal fibrosis.Objective:To define the effect of miR-449 a /Klf4 interaction in renal fibrosis.Methods:1.qRT-PCR analysis of miR-449 a levels in TCMK-1 cells after 10ng/ml TGF-β1 treatment and in kidney tissues of CKD patients.2.TCMK-1 cells cultured in vitro and transfected with miR-NC and miR-449 a inhibitor,respectively.qRT-PCR administered to verify the level of renal fibrosis proteins in the two groups.3.qRT-PCR analysis of Klf4/Mfn2 levels in TCMK-1 cells after 10ng/ml TGF-β1 treatment.TCMK-1 cells cultured in vitro,transfected with oe-NC,oe-Klf4,respectively,and qRT-PCR to compare the levels of renal fibrosis proteins in the two groups.4.Biological information software and dual luciferase assay predicted the discovery of complementary binding sites between miR-449 a and Klf4;TCMK-1 cells cultured in vitro,transfected with miR-NC,miR-449 a inhibitor,and qRT-PCR compared the levels of Klf4/Mfn2 in both groups.5.Biological information software and dual luciferase assay predicted that Klf4 has a potential binding site with Mfn2;TCMK-1 cells cultured in vitro,transfected with oe-NC,oe-Klf4,sh-NC and sh-Klf4,respectively,and qRT-PCR compared the levels of Mfn2 in each group6.10ng/ml TGF-β1-induced TCMK-1 was cultured in vitro,and the cells were divided into five groups: normal control group（untreated TCMK-1 cells）;TGF-β1 group（10ng/ml TGF-β1-induced TCMK-1 cells）;TGF-β1+ miR-449 a inhibitor group（10ng/ml TGF-β1 induced transfected miR-449 a inhibitor TCMK-1 cells）;TGF-β1+ miR-449 a inhibitor + sh-NC group（10ng/ml TGF-β1 induced transfected miR-449 a inhibitor sh-NC TCMK-1 cells）;TGF-β1+miR-449 a inhibitor+sh-Mfn2 group（10ng/ml TGF-β1-induced transfected sh-Mfn2 TCMK-1 cells with miR-449 a inhibitor）.qRT-PCR and Western blotting were performed to detect the levels of fibrosis and EMT-related proteins α-SMA,COL Ⅳ,FN in each group and Klf4,Mfn2 protein levels.7.UUO mouse models were constructed and divided into four groups: shamoperated group,UUO group,antago miR-NC UUO group（UUO+miR-NC）,antago miR-449 a UUO group（UUO+miR-449 a inhibitor）.qRT-PCR was performed to detect miR-449 a inhibitor in the kidney tissue of mice injected with miR-449 a inhibitor.The relative levels of miR-449 a,Klf4,and Mfn2 in the kidney tissues of mice injected with miR-449 a inhibitor,Masson staining images of the kidney tissues of each group were observed,the levels of fibrosis and EMT-related proteins α-SMA,COL Ⅳ,FN and the levels of Klf4 and Mfn2 proteins in the kidney tissues of each group were detected by Western blotting,and immunohistochemistry The levels of EMT-related protein expression were verified by immunohistochemistry.Result:1.miR-449 a expression levels were upregulated in CKD patients compared to normal controls;TGF-β1 increased miR-449 a levels in TCMK-1 cells.2.The protein levels of （?）-SMA,FN,collagen IV was significantly decreased in TCMK-1 cells transfected with miR-449 a inhibitor.3.TGF-β1 decreased the levels of miR-449 a Klf4 and Mfn2 in TCMK-1 cells,and the protein levels of （?）-SMA,FN,and collagen IV in TCMK-1 cells transfected with oe-Klf4 were significantly decreased.4.miR-449 a mimics significantly reduced the luciferase activity of Klf4-WT,but had no effect on the activity of Klf4-MUT with mutations in the binding site;Klf4 was predicted to be a downstream m RNA of miR-449a;the levels of Klf4/Mfn2 were significantly increased in TCMK-1 cells transfected with miR-449 a inhibitor.5.Klf4 can transcriptionally activate Mfn2;overexpression of Klf4 elevates the m RNA level of Mfn2 in TCMK-1 cells,and conversely sh-Klf4 inhibition of Klf4 expression decreases the m RNA level of Mfn2.6.TGF-β1-induced upregulation of miR-449 a and downregulation of Klf4 and Mfn2 in TCMK-1 cells,transfection with miR-449 a inhibitor reversed the above changes induced by TGF-β1.Transfection with miR-449 a inhibitor of TGF-β1 induced again the inhibition of Mfn2 expression using sh-Mfn2 in TCMK-1 cells miR-449 a,Klf4 were not significantly changed and Mfn2 was significantly downregulated.western blotting results showed that TGF-β1-induced upregulation of （?）-SMA,COL Ⅳ,FN levels in TCMK-1 cells;transfection of miR-449 a inhibitor reversed TGF-β1-induced （?）-SMA,COL Ⅳ,FN protein level up-regulation,while in TCMK-1 cells transfected with miR-449 a inhibitor of TGF-β1-induced TCMK-1 cells,inhibition of Mfn2 expression using sh-Mfn2 up-regulated （?）-SMA,COL Ⅳ,FN level expression.7.miR-449 a expression was greatly reduced in mice injected with miR-449 a inhibitor;Masson staining observed that miR-449 a inhibitor group significantly attenuated UUO-induced renal fibrosis and injury;western blotting results showed that miR-449 a inhibitor group significantly inhibited UUO-induced increase in （?）-SMA,COL Ⅳ,and FN.In addition,the reduced Klf4 and Mfn2 levels in the UUO model could be significantly restored by miR-449 a inhibitor.Conclusion:miR-449 a targets Klf4/Mfn2 to promote fibrosis and EMT.Part III: Role of circ-Ankib1-targeted miR-449a/Klf4/Mfn2 axis in renal fibrosisObjective:To investigate whether circ-Ankib1 inhibits fibrosis by "sponging" miR-449 a and whether miR-449 a targets the downstream Klf4/Mfn2 axis involved in the renal fibrosis process.Method:1.Bioinformatic analysis to determine the complementary binding sites between circ-Ankib1 and miR-449 a.2.RNA Immunoprecipitation（RIP）assay was performed to observe the relative enrichment of circ-Ankib1 and miR-449 a.3.TCMK-1 cells cultured in vitro were divided into three groups: normal control group,oe-NC group（TCMK-1 cells transfected with oe-NC）,and oe-circ-Ankib1 group（TCMK-1 cells transfected with oe-circ-Ankib1）.qRT-PCR was performed to detect the relative circ-Ankib1 and miR-449 a in each group.levels.4.TCMK-1 cells cultured in vitro were divided into the following five groups: normal control group（untreated TCMK-1 cells）,TGF-β1 group（10ng/ml TGF-β1-induced TCMK-1 cells）,TGF-β1+ oe-circ-Ankib1 group（10ng/ml TGF-β1-induced transfection of recombinant plasmid pc DNA3.1 circ-Ankib1 in TCMK-1 cells）,TGF-β1+ oe-circ-Ankib1+ miR-NC group（10ng/ml TGF-β1-induced transfection of recombinant plasmid pc DNA3.1 circ-Ankib1 and mimics-NC in TCMK-1 cells）,TGF-β1+ oe-circ-Ankib1+ miR-449 a mimics group（10ng/ml TGF-β1-induced TCMK-1 cells transfected with recombinant plasmid pc DNA3.1 circ-Ankib1 and miR-449 a mimics）.qRT-PCR was performed to detect the cellular circ-Ankib1,miR-449 a,Klf4,Mfn2 m RNA levels.Western blotting was performed to detect the levels of fibrosis and EMT-related proteins α-SMA,COL Ⅳ,FN,and Klf4,Mfn2 proteins in each group of cells.5.UUO mouse model was constructed,and the mice were divided into shamoperated group,UUO group,UUO+oe-NC group,and UUO+oe-circ-Ankib1 group.qRT-PCR was performed to detect the relative levels of circ-Ankib1,miR-449 a,Klf4 and Mfn2 in each group,and Western blotting was performed to detect the levels of Klf4 and Mfn2 protein levels.Result:1.Bioinformatics analysis revealed complementary binding sites between circAnkib1 and miR-449 a.2.RNA immunoprecipitation revealed that immunoprecipitation of AGO2 antibody significantly enriched circ-Ankib1 and miR-449a;dual luciferase analysis observed that miR-449 a mimics greatly reduced the luciferase activity of circAnkib1-wt,while having no effect on mutant circ-Ankib1-MUT.3.Overexpression of circ-Ankib1 suppressed TGF-β1-induced miR-449 a expression in TCMK-1 cells.4.Overexpression of circ-Ankib1 in TCMK-1 cells decreased the expression of circ-Ankib1,Klf4,and Mfn2 and increased the expression of miR-449 a.Combined overexpression of miR-449 a and circ-Ankib1 upregulated the expression level of miR-449 a,decreased the expression of Klf4,Mfn2,and No significant effect on circAnkib1.overexpression of circ-Ankib1 in TCMK-1 cells reversed the TGF-β1-induced upregulation of α-SMA,COL Ⅳ,and FN proteins in TCMK-1 cells,and combined overexpression of miR-449 a and circ-Ankib1 again upregulated expression of α-SMA,COL Ⅳ,FN expression.5.An increase miR-449 a and a significantly decrease in circ-Ankib1,Klf4,and Mfn2 were observed in the UUO group were observed in the UUO set with respect to the sham-operated group.Overexpression of circ-Ankib1 suppressed the UUOinduced increase in miR-449 a and restored the expression of Klf4 and Mfn2.Conclusion:circ-Ankib1 inhibits renal fibrosis and EMT by targeting miR-449 a,and miR-449 a directly targets the Klf4/Mfn2 axis. |
PDF Full Text Request