| Renal interstitial fibrosis is a common pathological change in many types of chronic kidney diseases(CKD)progression to end-stage renal disease(ESRD),patients who enter to ESRD will experience costly kidney replacement therapy,a significant reduction in the quality of life,shortening of survival time and other serious problems,so it is vital to find a new anti-fibrosis therapy.Chronic hypoxia is not only a common pathway of a variety of CKD progress to ESRD,more importantly,it is the most important factor in tubulointerstitial injury.Recent studies have shown that G2/M arrest in tubular epithelial cells can activate fibroblasts and promote the secretion of transcriptional growth factor-β1(TGF-β1),connective tissue growth factor(CTGF)、Ⅳcollagen α1(COL4A1)、α-smooth muscle actin(α-SMA),leading to renal fibrosis[5-7].Can renal tubular epithelial cells also occur cell cycle arrest and then promote fibrosis in hypoxic microenvironment?And what is the specific molecular mechanism that regulates this process? Are urgent questions to answer.Micro RNAs(mi RNAs),a type of endogenous non-coding RNA that regulates gene expression through the inhibition of target m RNA transcription and translation,or the promotion of m RNA degradation,is closely associated with renal fibrosis.Recent studies have shown that hypoxia could induce the expression of a number of mi RNAs to exert corresponding functions.In addition,some studies have suggested that several mi RNAs regulate the cell cycle.We previously used gene microarray to analyze the mi RNA expression ratio of HK2 cells under hypoxia,and we identified mi R-493 and demonstrated the increased expression of mi R-493 in HK2 cells under hypoxia、in a mouse model of unilateral ureteral obstruction(UUO)and patients with Ig A nephropathy(Ig AN).Furthermore,the bioinformatics prediction revealed that 3’-untranslated region(3’-UTR)of STMN1 had a potential binding site of mi R-493,and their expression was negatively correlated.STMN1 is a cell cycle regulator,encoding the protein stathmin1,a prominent microtubule regulator that regulates the stability of the microtubules and is essential for the proper formation of the mitotic spindle.In other words,STMN1 is involved in cell cycle regulation.We hypothesized that mi R-493 may promote renal interstitial fibrosis by inhibiting STMN1 to induce G2/M arrest in tubular cells in hypoxic microenvironment.This study will confirm this inference by in vitro and in vivo experiments.Objective 1)To investigate the role of chronic hypoxia in renal tubular epithelial cells G2/M phase arrest and renal interstitial fibrosis.2)To study the role of mi R-493 in renal tubular epithelial cells G2/M phase arrest and renal interstitial fibrosis induced by chronic hypoxia.3)To verify the role of mi R-493-STMN1 pathway in tubular cells G2/M phase arrest and renal interstitial fibrosis under hypoxic microenvironment.4)We inhibited the expression of mi R-493 and verify its role in reversing renal interstitial fibrosis in vivo.Methods 1)Flow cytometry,Western Blot and immunohistochemistry were used to detect cell cycle changes and renal interstitial fibrosis under hypoxic microenvironment.2)We detected the expression of mi R-493 by q RT-PCR and in situ hybridization under hypoxic microenvironment.Then,transfacted HK2 cells with mi R-493 mimic and inhibitor to investigate the role of mi R-493 in cell cycle changes and renal interstitial fibrosis by flow cytometry and Western Blot.3)To investigate the effect and mechanism of mi R-493 on STMN1 by q RT-PCR、Western Blot and luciferase reporter gene.Then,transfacted HK2 cells with STMN1-sh RNA to inhibit STMN1 and investigate the cell cycle changes and renal interstitial fibrosis by flow cytometry and Western Blot.4)We injected mice with mi R-493 sponge AAV to inhibit the expression of mi R-493 and verify its role in reversing renal interstitial fibrosis by q RT-PCR 、immunohistochemistry and Western Blot in vivo.Results 1)Chronic hypoxia mediate renal tubular epithelial cells G2/M arrest and renal interstitial fibrosis.We found that the percentage of G2/M phase was increased after hypoxia in HK2 cells by flow cytometry;Western Blot results showed that G2/M phase maker(cyclin B1/cyclin D1)increased with the prolongation of hypoxia,and the expression of CTGF、COL4A1、α-SMA also increased in HK2 cells subjected to hypoxia and UUO model.In the UUO model,the immunohistochemical results showed that the G2/M phase marker(PH3/Ki67)increased at 14 d after UUO,and Masson staining showed an increase in renal interstitial fibrosis,indicating that hypoxic microenvironment could mediate renal tubular epithelial cells G2/M phase arrest and renal interstitial fibrosis.2)mi R-493 is upregulated in tubular cells and could induce tubular cells G2/M arrest and renal interstitial fibrosis under hypoxia microenvironment.We tested the expression of mi R-493 in hypoxic HK2 cells and Ig AN via q RT-PCR,and it showed that mi R-493 is increased compared with control.Also,the expression of mi R-493 is increased at 14 d after UUO by situ hybridization.Then,we transfacted HK2 cells with mi R-493 mimic and inhibitor and found that mi R-493 mimic could promote tubular cells G2/M arrest and renal interstitial fibrosis.In contrast,mi R-493inhibitor could reverse this change.3)mi R-493 inhibited the expression of STMN1 at post-transcriptional level and mediated tubular epithelial cells G2/M arrest and renal interstitial fibrosis.First,we conducted a bioinformatics analysis and found that STMN1 is a target gene of mi R-493.Further,we confirmed that the mi R-493 could directly regulate STMN1 by luciferase reporter gene and detected m RNA and protein levels of STMN1 after transfection of mi R-493 mimic,indicating that mi R-493 regulated STMN1 at post-transcriptional level to inhibit its translation.Subsequently,we tested the expression of STMN1 in UUO model and found that STMN1 was decreased with the increase of obstruction time.Immunohistochemistry and Masson results showed that STMN1 decreased while the degree of interstitial fibrosis increased at 14 d after UUO.Transfected HK2 cells with STMN1-sh RNA to inhibit STMN1,we found that compared with control group,the expression of G2/M phase marker(cyclin B1/cyclin D1)increased while CTGF、COL4A1 and α-SMA also increased after transfection of STMN1-sh RNA.4)Intervention of mi R-493 could inhibit renal interstitial fibrosis in vivo.We inhibited mi R-493 with mmu-mi R-493-3p sponge AAV in mice,and found that mi R-493 sponge AAV reduced the expression of α-SMA and COL4A1 and enhanced STMN1 expression compared with the control,which indicated that targeting mi R-493 has a role in preventing renal interstitial fibrosis.Conclusion In summary,our data suggested that mi R-493 is upregulated under hypoxic microenvironment,and could induced tubular epithelial cells G2/M arrest and renal interstitial fibrosis through repressing downstream target STMN1.That is,mi R-493-STMN1 pathway mediates hypoxia-induced tubular epithelial cells G2/M arrest to promote renal interstitial fibrosis.Abrogating G2/M arrest and blocking the mi R-493-STMN1 pathway will provide novel insight into the future development of new anti-fibrosis therapy. |
