5-FU, n?=?4C8 Knockdown of miR-181a inhibits BIRC6/p53-dependent apoptosis pathway in 5-FU-induced nephrotoxicity To determine whether miR-181a inhibition attenuated 5-FU-induced apoptosis in kidney, renal apoptosis was examined using TUNEL assay
5-FU, n?=?4C8 Knockdown of miR-181a inhibits BIRC6/p53-dependent apoptosis pathway in 5-FU-induced nephrotoxicity To determine whether miR-181a inhibition attenuated 5-FU-induced apoptosis in kidney, renal apoptosis was examined using TUNEL assay. associated with reduced p53-mediated mitochondrial apoptosis induced by 5-FU. Moreover, miR-181a increased BIRC6 downstream gene p53 protein expression and transcriptional activity by reducing ubiquitin-mediated protein degradation. We found that miR-181a directly targeted 3-UTR of BIRC6 mRNA and negatively regulated BIRC6 expression. In vivo study, knockdown of miR-181a with adeno-associated computer virus harboring miR-181a-tough decoy attenuated 5-FU-induced renal cell apoptosis, inflammation and kidney injury. In conclusion, these results demonstrate that miR-181a increases p53 protein expression and transcriptional activity by targeting BIRC6 and promotes 5-FU-induced apoptosis in mesangial cells. Inhibition of miR-181a ameliorates 5-FU-induced nephrotoxicity, suggesting that miR-181a may be a novel therapeutic target for nephrotoxicity treatment during chemotherapy. Introduction 5-Fluorouracil (5-FU) is usually a potent antineoplastic agent widely used for the treatment of various malignancies because of its broad antitumor activity and synergistic action with other anticancer drugs1. However, unfortunately, 5-FU is Ifenprodil tartrate designed to act via misincorporation of its metabolites into DNA and inhibition of thymine synthesis, and thus may not affect only malignancy cells but also normal dividing cells of patients1C3. Consequently, it causes DNA damage, cell cycle termination, apoptosis and necrosis, and ultimately results in severe toxic effects and discontinuation of chemotherapy4. Of note, 5-FU gets catalyzed into dihydrouracil, which subsequently can be cleaved into -fluoro–alanine, ammonia, urea, and carbon dioxide in liver, leading to nephrotoxicity3,4. Mesangial cells play a critical role in maintaining the glomerular structural integrity and the function of the whole kidney, providing mesangial matrix homeostasis and regulating glomerular filtration5. It is reported that mesangial cell apoptosis can be observed during various chronic kidney diseases, including immunoglobulin A nephropathy, diabetic nephropathy, and lupus nephritis5C7. Moreover, the apoptosis of mesangial cells increases concomitantly with the severity of albuminuria and is directly involved in the pathogenesis of glomerulosclerosis8. These findings indicate that mesangial cell loss caused by apoptosis may contribute to the development of renal diseases. Importantly, mesangial cells are suggested to be susceptible to anticancer drugs such as 5-FU, and the apoptosis induced by 5-FU is considered to be associated with renal dysfunction4,9. However, the mechanisms of 5-FU-induced mesangial cell apoptosis are not fully comprehended. Baculoviral inhibition of apoptosis protein repeat made up of 6 (BIRC6), the largest member of the inhibition of apoptosis proteins (IAPs) family, contains a baculoviral IAP domain name and a C-terminal ubiquitin-conjugating (UBC) enzyme domain name10. BIRC6 like other IAPs promotes cell survival and inhibits apoptosis11. Another crucial regulator of cell apoptosis, p53, acts as a tumor suppressor by inducing cell apoptosis in most human tumors12. It has been reported that downregulation of p53 can ameliorate the cytotoxicity and nephrotoxicity induced by anticancer drugs4,13. Interestingly, p53 is a key downstream effector of BIRC6 and loss of BIRC6 triggers the upregulation of p5311,14. Ifenprodil tartrate BIRC6 directly catalyzes p53 ubiqutiylation and proteasome degradation, which in turn inhibits mitochondria-dependent apoptosis14. These findings, together with the identification of BIRC6 as an upstream regulator of p53 raise the possibility that BIRC6 may be a novel target for the treatment of nephrotoxicity of 5-FU. MicroRNAs (miRNAs) are small noncoding RNAs of 19C25 nucleotides in length that regulate gene expression by binding to the 3-untranslated regions (3-UTR) through mRNA translational repression or degradation15. Although previous studies showed that depletion of miRNAs by ablating Dicer, a key enzyme for miRNA maturation, results in a rapid progression to end-stage kidney disease16C18, only few miRNAs have been identified to be involved in renal dysfunction, such as 5-FU-induced nephrotoxicity19,20. In this study, we provided evidences that miR-181a regulates 5-FU-induced mesangial cell apoptosis through p53-dependent mitochondrial pathway by targeting BRIC6. Furthermore, we found that knockdown of miR-181a ameliorated 5-FU-induced nephrotoxicity. Materials and methods Materials and reagents Fetal bovine serum (FBS), penicillin, streptomycin, lipofectamine 2000, TRIzol reagent, and 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-benza-midazolocarbocyanin iodide (JC-1) were obtained from Invitrogen (Carlsbad, CA, USA). RIPA lysis buffer, LDH leakage assay kit, BCA kit, enhanced chemiluminescence (ECL) kit, and antibodies against CD68 and Ly-6G were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Unless otherwise indicated, all chemicals were purchased form Sigma-Aldrich (St. Louis, MO, USA). Cell culture The immortalized human mesangial cell (HMC) line was kindly provided by Dr. Fengxian Huang (Sun Yat-Sen University)21. HCT116 p53+/+ or HCT116 p53?/? cells were generous gifts from Dr. Ronggui Ifenprodil tartrate Hu (Chinese Academy of Sciences). Cells were cultured in RPMI Rabbit polyclonal to JOSD1 1640 medium made up of 10% FBS, 100?U/ml penicillin, and 100?g/ml streptomycin..