CircRNA₁5698 Promotes Folic Acid-induced Renal Fibrosis By Sponging MiR-185 To Increase TGF-β1

Objective: Renal interstitial fibrosis(RIF)is a common pathological manifestation of end-stage renal disease.In the end,patients need kidney replacement therapy,which places a heavy burden on society and families.Circular RNAs are a new class of noncoding RNAs,which are involved in the pathogenesis of various diseases.According to literature reports,circRNA＿15698 acted as the sponge of miR-185 and inhibited its activity to up-regulated transforming growth factor-β1(TGF-β1),further exacerbated the accumulation of extracellular matrix in mesangial cells and aggravating fibrosis.However,the role of circRNA＿15698 in folic acid(FA)induced-renal interstitial fibrosis(RIF)remains unclear.We aimed to investigate whether circ RNA＿15698 promotes FA-induced renal interstitial fibrosis and whether circRNA＿15698/miR-185/TGF-β1 pathway is involved in the pathogenesis for renal interstitial fibrosis.Methods: FA(250 mg/kg)was injected to CD1 male mice to induce RIF.After 48 hours,tail blood was collected,and serum was separated to detect serum creatinine and blood urea nitrogen as indicators of renal function.After 30 days,renal tissues was harvested after perfused by PBS to remove the effects of residual blood cells within renal tissues.1/2 kidney tissue was made into paraffin-embedded tissue blocks.Paraffin blocks were used for Periodic Acid-Schiff stain(PAS)to confirm renal morphological damage of renal interstitial fibrosis,Masson staining was used to analyze the degree of renal interstitial fibrosis,and immunofluorescent staining(IF)was used to analyze localization of phage infiltration marker CD68,profibrotic proteins including Fibronectin(Fn),collagen 1(Col1)and TGF-β1.The remaining kidney tissues were stored at-80 degrees for frozen RNA and protein extraction for molecular biological detection,and immunoblot detection of macrophage infiltration markers F4/80,Fn,Col1,TGF-β1 protein levels and semi-quantitative changes.qRT-PCR detected changes in RNA levels,including circRNA＿15698,miR-185,Tgfβ1,pro-inflammatory cytokines including interleukin-6(Il6)and tumor necrosis factor-α(Tnfα),Fn,Col1.Then we applied bioinformatics technologies CircInteractome and miRbase to predict whether there are binding sites between circRNA＿15698 and miR-185 as well as miR-185 and TGF-β1.Results: Blood creatinine [(87.130±8.373)μmol/L v.s.(30.28±3.760)μmol/L,p < 0.0001] and blood urea nitrogen [(12.970±2.068)mmol/L v.s.(6.438±0.902))mmol/L,p< 0.0001] increased significantly at 48 hours after FA injection.Loss of brush border,tubular atrophy,dysmorpholgy,scarred surface of renal cortex,and the infiltration of inflammatory cells showed in the kidneys of mice at day 30 after FA injection on PAS staining.Renal tubular injury scores were semi-quantitatively analyzed(1.817±0.649 v.s.0.217±0.075,p = 0.0001).Masson staining also showed renal tubular injury and renal interstitial fibrosis,and semi-quantitative analysis showed an increase in renal interstitial fibrosis(0.163±0.096 v.s.0.034±0.006,p = 0.009).The pro-inflammatory cytokines Il6(6.140±3.454 v.s.1.000±0.461,p = 0.005)and Tnfα(4.847±2.662 v.s.1.000±0.653,p = 0.006)were expressed elevated on qRT-PCR.Immunoblot showed that the level of macrophage marker F4/80 protein was increased(4.785±3.251 v.s.1.000±0.511,p = 0.018),and immunofluorescence staining showed an increase in CD68-positive cells.qRT-PCR showed that profibrotic genes Fn(3.244±2.251 v.s.1.000±0.336,P = 0.036)and Col1(12.830±11.720 v.s.1.000 ± 0.193,P = 0.033)were increased.Immunoblot showed that fibrotic protein FN(3.906±2.759 v.s.1.000±0.293,p = 0.028)and COL1(1.828±0.556 v.s.1.000±0.369,p = 0.0125)were highly expressed.Renal circRNA＿15698 expression was up-regulated(5.357±4.150 v.s.1.000±0.521,p = 0.029),miR-185 expression was down-regulated(0.710±0.181 v.s.1.000±0.213,p = 0.029),and TGF-β1 expression was up-regulated(4.919±2.447 v.s.1.000 ± 0.227,p = 0.003)on qRT-PCR.In addition,CircInteractome and miRbase predicted a binding site between circRNA＿15698 and miR-185 as well as miR-185 and TGF-β1.Conclusions: 1)In folic acid-induced renal interstitial fibrosis mice,circRNA＿15698 expression was up-regulated,miR-185 expression was down-regulated,and TGF-β1 was overproduced.2)The results of this study suggest that circRNA＿15698/miR-185/TGF-β1 may be involved in the mechanism of renal interstitial fibrosis and play an important role.Targeting circRNA＿15698/miR-185/TGF-β1 may be renal interstitial fibrosis treatment opens up new avenues.