The Role And Related Mechanisms Of CircHIPK3 In Chronic Tubulointerstitial Fibrosis

Objective Renal fibrosis is a pathological change,which is a gradual process of renal function from health to injury,then to damage,and finally to loss the function.The renal fibrosis includes renal tubular interstitial fibrosis and glomerulosclerosis.Its main pathological manifestation is the damage of intrinsic cells.In the later stage,a large amount of collagen deposition and accumulation appear,resulting in the gradual hardening of renal parenchyma and the formation of scars,until the kidney completely loses its organ function.The process of fibrosis and sclerosis of inherent cells in kidney is also the process of renal fibrosis.Renal fibrosis is a common pathological feature of chronic kidney disease(CKD).At present,the prevalence rate of CKD is about 10.8% in China.A considerable number of people will progress to end-stage renal disease and need renal replacement therapy,which has caused huge economic and social burden to our country.As the main pathological manifestation of end-stage renal disease,renal fibrosis is still lacking of effective treatment,which requires us to actively explore and strive to find the possible mechanism in the occurrence and development of fibrosis,to find the possible pathological process of delaying or even reversing fibrosis,and to provide new treatment methods for the treatment of renal disease.Circular RNA(circ RNA)is a special non coding RNA molecule,which is also a hot spot in the field of RNA.Different from the traditional linear RNA molecules,circ RNA is a kind of RNA molecules with closed loop structure.It has no 5 ’cap and 3’polyadenylate tail structure,and is not affected by RNA exonuclease.Its expression is more stable and difficult to degrade.It has been proved that circ RNA exists widely in a variety of eukaryotes and has important biological functions.Recent studies have shown that circ RNA plays an important regulatory role in disease through the interaction of miRNA associated with the disease.At the physiological level,endogenous circ RNAs play a "sponge" role by binding with miRNAs,and inhibit the expression of miRNAs.In addition,circ RNA is also involved in the regulation of gene transcription,regulation of protein translation and research as biomarkers.Our group has mainly studied circ RNAs and micro RNAs and accumulated rich experience.At the same time,the expression of circHIPK3 in cardiac and pulmonary fibrosis is significantly increased,and circHIPK3 plays an important role through different miRs,but how to express the circHIPK3 in renal fibrosis diseases is unknown.In order to further explore the development of circ RNA in renal fibrosis,we conducted the following studies.The purpose of this study was to investigate the expression of circHIPK3 in chronic tubulointerstitial disease and whether the fibrosis indexes were further aggravated after overexpression of circHIPK3.Whether circHIPK3 increased after renal tubular epithelial cells were stimulated by fibrosis factors and through which kind of miRNA to play its role.Methods: First,paraffin sections of chronic interstitial nephritis diagnosed by renal biopsy were selected,and renal tissue larger than 5 cm adjacent to the carcinoma were selected as normal control(NC),Fluorescence in situ hybridization(FISH)was used to detect the expression and localization of circHIPK3 and miR-30 a,and immunofluorescence was used to detect the expression of(Transforming growth factorβ1,TGF-β1),(Fibronectin,FN)and(Collagen 1,COL1).Semi quantitative analysed the correlation of the above indicators.Futhermore,16 week old C57BL/6J mice were intraperitoneally injected with folic acid(FA)(250 mg/kg)and the control group was injected with sodium bicarbonate of the same volume.The habits of mice were observed every day.After 30 days,serum and renal tissue samples(n = 6)were collected.The collected serum was used to detect creatinine,urea nitrogen,and part of renal tissue was used for paraffin section PAS(Periodic Acid-Schiff)and Masson staining to confirm the occurrence of renal fibrosis.RNA was extracted from mice kidney.The expressions of circHIPK3,miR-30 a,TGF-β1,FN and COL1 in kidney were detected.Western blot confirmed the expression of TGF-β1,FN and COL-1.And then we determine the correlation between circHIPK3,miR-30 a and TGF-β1.Finally,the expressions of miR-30 a,TGF-β1,FN and COL1 were detected by overexpression of circHIPK3 in HK-2 cells.HK-2 cells were stimulated with TGF-β1.The levels of FN,COL1,circHIPK3 and miR-30 a in HK-2 cells stimulated with and without TGF-β1 were detected.Results: 1.FISH and immunofluorescence showed that circHIPK3,miR-30 a and TGF-β1 were mainly expressed in cell cytoplasm and they were colocalized in the cytoplasm.The expression of circHIPK3 was higher and miR-30 a was lower in cTIN paraffin sections than in normal human renal tissues.The expression of TGF-β1,FN and COL1 was significantly increased,and there was statistical difference in semi quantitative analysis.2.After intraperitoneal injection of folic acid,PAS and Masson staining showed that compared with the NC group,the renal tubules atrophied,some tubular epithelial cells fell off,the space between renal tubules expanded,inflammatory cell infiltration and interstitial fibrosis was obvious in FA group.Western blot,q PCR and immunofluorescence were used to confirm the expression of TGF-β1,FN and COL1 increased significantly,which further confirmed the success of fibrosis model.Similar to human results,the colocalization of circHIPK3,miR-30 a and TGF-β1 were detected in the cytoplasm by FISH and immunofluorescence,and the three colocalization showed good consistency.In fibrotic mice,the expression of circHIPK3 was significantly increased,the expression of miR-30 a was decreased(P < 0.001),and the expression of TGF–β1 was increased.In addition,there was a significant negative correlation between circHIPK3 and miR-30 a,miR-30 a and TGF-β1.But other miRs associated with TGF-β1includes miR-29 a,miR-29 b,miR-146 a and miR-338,there was no significant difference in other indexes except miR-338(p=0.02).3.HK-2 cells cultured with 10% fetal bovine serum showed adherent growth,cobblestone like appearance,no lobulation and burrs.When the cells grew to 60-70%,the plasmids were used to transfect circHIPK3.The blank plasmids were selected in the control group,and the cells were collected for follow-up experiments after 48 hours.After transfection of circHIPK3 into HK-2 cells,q PCR confirmed that circHIPK3 increased about 5 times,miR-30 a decreased about half,and TGF-β1,FN and COL1 increased significantly compared with the control group.Western blot showed the same results.4.When HK-2 cells grew to 60-70%,250 pg/ml h TGF-β1 was applied to induced HK-2fibrosis and in the control group the cells were cultured in normal medium for 48 hours.Compared with the control group,the number of dead cells,burrs and lobulation of some cells increased in the experimental group.q PCR confirmed that circHIPK3 was significantly increased,miR-30 a decreased,FN and COL1 were significantly increased,and Western blot also confirmed the increase of fibrosis proteins.Conclusion:1.The expression of circHIPK3 increased and miR-30 a decreased in patients with chronic tubulointerstitial nephritis.Fibrogenic proteins TGF-β1,FN and COL1 increased significantly.It was suggested that circHIPK3 may play an important role in patients with chronic tubulointerstitial nephritis.2.To be similar with the patients,circHIPK3 showed similar results in the renal tissue of mice with high-dose FA induced tubulointerstitial fibrosis.In both protein and RNA levels,the expression of circHIPK3 increased,the expression of miR-30 a decreased,and the expression of TGF-β1,FN and COL1 increased.Moreover,circHIPK3 was negatively correlated with miR-30 a,and miR-30 a was negatively correlated with TGF-β1.3.Three channel staining was used to detect circHIPK3,miR-30 a and TGF-β1colocalization in the cytoplasm in HK-2 cells,indicating the interaction between the three molecules.In addition,after overexpression of circHIPK3 in HK-2 cells,it was found that miR-30 a was down regulated and TGF-β1,FN and COL1 were up regulated.These results suggested that circHIPK3 may play its sponging role in promoting fibrosis through miR-30 a.4.After HK-2 cells were treated with TGF-β1,the expression of circHIPK3 was increased,miR-30 a was down regulated,and the production of fibrogenic protein FN and COL1 were increased.It is suggested that TGF-β1 may play an important role in the pathogenesis.There may be a positive feedback loop between TGF-β1 and circHIPK3 sponging miR-30 a,which further aggravates tubulointerstitial fibrosis.