Induction of microRNA-17-5p by p53 protects against renal ischemia-reperfusion injury by targeting death receptor 6
2016; Elsevier BV; Volume: 91; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2016.07.017
ISSN1523-1755
AutoresJielu Hao, Qingqing Wei, Shuqin Mei, Lin Li, Yunchao Su, Changlin Mei, Zheng Dong,
Tópico(s)Autophagy in Disease and Therapy
ResumoRenal ischemia-reperfusion injury is a leading cause of acute kidney injury; the pathogenesis of which remains poorly understood and effective therapies are still lacking. Here we tested whether microRNAs, identified as critical regulators of cell health and disease, are involved in this process. We found that miR-17-5p was significantly up-regulated during renal ischemia-reperfusion injury in mice and during hypoxia in cultured renal tubular cells. In cultured cells, miR-17-5p directly inhibited the expression of death receptor 6 (DR6) and attenuated apoptosis during hypoxia. Blockade of miR-17-5p abolished the suppression of DR6 and facilitated caspase activation and apoptosis. In vivo, an miR-17-5p mimic suppressed DR6 expression and protected against renal ischemia-reperfusion injury. We further verified that miR-17-5p induction during renal ischemia-reperfusion injury was dependent on p53. Inhibition of p53 with pifithrin-α or a dominant-negative mutant led to the repression of miR-17-5p expression under hypoxia in vitro. Moreover, miR-17-5p induction during renal ischemia-reperfusion injury was attenuated in proximal tubule p53 knockout mice, supporting the role of p53 in miR-17-5p induction in vivo. Thus, p53/miR-17-5p/DR6 is a new protective pathway in renal ischemia-reperfusion injury and may be targeted for the prevention and treatment of ischemic acute kidney injury. Renal ischemia-reperfusion injury is a leading cause of acute kidney injury; the pathogenesis of which remains poorly understood and effective therapies are still lacking. Here we tested whether microRNAs, identified as critical regulators of cell health and disease, are involved in this process. We found that miR-17-5p was significantly up-regulated during renal ischemia-reperfusion injury in mice and during hypoxia in cultured renal tubular cells. In cultured cells, miR-17-5p directly inhibited the expression of death receptor 6 (DR6) and attenuated apoptosis during hypoxia. Blockade of miR-17-5p abolished the suppression of DR6 and facilitated caspase activation and apoptosis. In vivo, an miR-17-5p mimic suppressed DR6 expression and protected against renal ischemia-reperfusion injury. We further verified that miR-17-5p induction during renal ischemia-reperfusion injury was dependent on p53. Inhibition of p53 with pifithrin-α or a dominant-negative mutant led to the repression of miR-17-5p expression under hypoxia in vitro. Moreover, miR-17-5p induction during renal ischemia-reperfusion injury was attenuated in proximal tubule p53 knockout mice, supporting the role of p53 in miR-17-5p induction in vivo. Thus, p53/miR-17-5p/DR6 is a new protective pathway in renal ischemia-reperfusion injury and may be targeted for the prevention and treatment of ischemic acute kidney injury. Renal ischemia-reperfusion injury (IRI) is one of the main causes of acute kidney injury.1Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar, 2Sharfuddin A.A. Molitoris B.A. Pathophysiology of ischemic acute kidney injury.Nat Rev Nephrol. 2011; 7: 189-200Crossref PubMed Scopus (525) Google Scholar For example, it is unavoidable in patients who undergo major cardiac events (such as infarction or surgery) or kidney transplantation. Renal IRI is associated with high rates of mortality and end-stage kidney failure and notably, even if patients recover from initial injury, renal IRI may have lasting effects including the development of chronic kidney disease.3Venkatachalam M.A. Weinberg J.M. Kriz W. Bidnai A.K. Failed tubule recovery, AKI-CKD transition, and kidney disease progression.J Am Soc Nephrol. 2015; 26: 1765-1776Crossref PubMed Scopus (414) Google Scholar For decades, numerous studies have investigated the molecular and cellular mechanisms of renal IRI and have suggested a variety of pathophysiological changes, including tubular or epithelial cell injury, microvascular dysfunction, and inflammation. However, the mechanistic understanding is incomplete and no therapies are available for effective treatment of IRI-associated kidney injury and renal failure. MicroRNAs (miRNAs) are recently discovered, critical regulators of gene expression that are approximately 21 to 25 nucleotides in length.4Mendell J.T. Olson E.N. MicroRNAs in stress signaling and human disease.Cell. 2012; 148: 1172-1187Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 5Lorenzen J.M. Haller H. Thum T. MicroRNAs as mediators and therapeutic targets in chronic kidney disease.Nat Rev Nephrol. 2011; 7: 286-294Crossref PubMed Scopus (179) Google Scholar, 6Bhatt K. Mi Q.S. Dong Z. MicroRNAs in kidneys: biogenesis, regulation, and pathophysiological roles.Am J Physiol Renal Physiol. 2011; 300: F602-F610Crossref PubMed Scopus (150) Google Scholar They target downstream genes by binding to the 3′ untranslated region (3′UTR) of mRNAs resulting in the repression of translation. Given this critical role, miRNAs not only have an effect on physiological cellular behavior, but they also contribute to the development of diseases in various pathological conditions. In kidneys, miRNAs play important roles in renal development and physiological maintenance and, as a result, the ablation of Dicer (a key enzyme for miRNA production) in specific kidney cell types led to renal dysfunction and renal diseases.7Harvey S.J. Jarad G. 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Borges G.R. et al.The microRNA-processing enzyme dicer maintains juxtaglomerular cells.J Am Soc Nephrol. 2010; 21: 460-467Crossref PubMed Scopus (124) Google Scholar In 2010, we established a conditional Dicer knockout mouse model, in which Dicer was ablated specifically from kidney proximal tubular cells by using phosphoenolpyruvate carboxykinase (PEPCK)-Cre.12Wei Q. Bhatt K. He H.Z. et al.Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.J Am Soc Nephrol. 2010; 21: 756-761Crossref PubMed Scopus (185) Google Scholar Because PEPCK-Cre is expressed for Dicer knockout at 2 to 3 weeks postnatally, these mice did not have renal development defects and showed normal renal function and histology under control conditions. However, these mice were remarkably resistant to renal IRI, suggesting an important role of miRNAs in the pathogenesis. By microarray analysis, we further identified 23 miRNAs with significant changes in expression during renal IRI.12Wei Q. Bhatt K. He H.Z. et al.Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.J Am Soc Nephrol. 2010; 21: 756-761Crossref PubMed Scopus (185) Google Scholar More recent studies have investigated the role and regulation of some of these miRNAs in renal IRI, including miR-494, -21, -126, -687, -150, and -489.13Hu H. Jiang W. Xi X. et al.MicroRNA-21 attenuates renal ischemia reperfusion injury via targeting caspase signaling in mice.Am J Nephrol. 2014; 40: 215-223Crossref PubMed Scopus (43) Google Scholar, 14Bijkerk R. van Solingen C. de Boer H.C. et al.Silencing of miRNA-126 in kidney ischemia reperfusion is associated with elevated SDF-1 levels and mobilization of Sca-1+/Lin- progenitor cells.Microrna. 2014; 3: 144-149Crossref PubMed Google Scholar, 15Bhatt K. Wei Q. Pabla N. et al.MicroRNA-687 induced by hypoxia-inducible factor-1 targets phosphatase and tensin homolog in renal ischemia-reperfusion injury.J Am Soc Nephrol. 2015; 26: 1588-1596Crossref PubMed Scopus (80) Google Scholar, 16Ranganathan P. Jayakumar C. Tang Y. et al.MicroRNA-150 deletion in mice protects kidney from myocardial infarction-induced acute kidney injury.Am J Physiol Renal Physiol. 2015; 309: F551-F558Crossref PubMed Scopus (49) Google Scholar, 17Wei Q. Liu Y. Liu P. et al.MicroRNA-489 induction by hypoxia-inducible factor-1 protects against ischemic kidney injury.J Am Soc Nephrol. 2016; 27: 2784-2796Crossref PubMed Scopus (65) Google Scholar Notably, whereas some of the miRNAs mediate kidney injury during IRI, others may play protective roles.18Wang I.K. Sun K.T. Tsai T.H. et al.MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury.Life Sci. 2015; 136: 133-141Crossref PubMed Scopus (42) Google Scholar, 19Bijkerk R. van Solingen C. de Boer H.C. et al.Hematopoietic microRNA-126 protects against renal ischemia/reperfusion injury by promoting vascular integrity.J Am Soc Nephrol. 2014; 25: 1710-1722Crossref PubMed Scopus (83) Google Scholar, 20Xu X. Kriegel A.J. Liu Y. et al.Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21.Kidney Int. 2012; 82: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Accordingly, investigation of these miRNAs may suggest therapeutic strategies for IRI-related kidney diseases. In this study, we specifically examined the role and regulation of miR-17-5p in renal IRI. We verified miR-17-5p induction during renal IRI. The induction was mediated by p53 and, following induction, this microRNA may repress death receptor 6 (DR6) to protect kidney cells and tissues from injury. Severe and moderate renal IRI was induced in mice respectively by 30 and 25 minutes of bilateral renal ischemia followed by reperfusion. MiR-17-5p in renal cortical tissues was determined by quantitative real-time polymerase chain reaction (PCR) using highly specific Taqman probes. In the severe IRI model, miR-17-5p was marginally induced after 12 hours of reperfusion (I30/12 h), but significantly induced after 48 hours of reperfusion (I30/48 h) (Figure 1a). To localize miR-17-5p induction, we conducted in situ hybridization. As shown in Figure 1b, miR-17-5p signal was weak in sham-control kidneys, but it was markedly induced in kidney cortical tissues after 30 minutes of ischemia and 48 hours of reperfusion, and the induction was mainly in proximal tubules. In the moderate IRI model, an increase of miR-17-5p was detected at 1 day of reperfusion (I25/1 d), and this increase was maintained during the whole observation period of a week (Figure 1c). In cultured rat kidney proximal tubular cells (RPTC), hypoxia (1% oxygen) induced marginal increases in miR-17-5p at 6 to 12 hours, but the increase became significant at 24 hours of hypoxic incubation (Figure 1d). These results demonstrated the miR-17-5p induction during renal IRI in kidney tissues and hypoxia in cultured tubular cells. To investigate the pathophysiological role of miR-17-5p, we initially examined the effect of anti-miR-17-5p locked nucleic acid (LNA) on hypoxic injury of RPTC cells. Hypoxic incubation resulted in a significant increase of apoptosis in RPTC transfected with scrambled sequence LNA (Figure 2a). Notably, transfection with anti-miR-17-5p LNA almost doubled the percentage of apoptotic cells during hypoxia treatment (Figure 2a and b). These morphological evaluations were verified by the measurement of caspase activity (Figure 2c). In contrast, the transfection of miR-17-5p mimic significantly reduced apoptosis during hypoxia as assessed by both morphological assays (Figure 3a and b) and immunoblot analysis of active caspase-3 (Figure 3c). Altogether, the results suggested that miR-17-5p induced in renal IRI may act as a protective factor against tubular cell injury and tissue damage.Figure 3MiR-17-5p mimic suppresses tubular cell apoptosis in hypoxia. Rat kidney proximal tubular cells were transfected with 200 nM miR-17-5p mimic or scrambled oligonucleotides, and then incubated in normoxia (control) or hypoxia (1% oxygen) for 48 hours. (a) Representative images show the morphology and nuclear staining of rat kidney proximal tubular cells. Bars = 200 μm. (b) Percentage of rat kidney proximal tubular cells apoptosis by morphology evaluation is shown. Data are expressed as mean ± SD (n = 4), *P < 0.05 versus normoxia control, #P < 0.05 versus scrambled transfection with hypoxia. (c) Immunoblot analysis of caspase-3 cleavage is shown. Whole cell lysate was collected from rat kidney proximal tubular cells with miR-17-5p mimic or scrambled oligonucleotides or reagent alone (mock) transfection under normoxia or hypoxia condition for immunoblotting. Caspase-3 cleavage was significantly inhibited by miR-17-5p transfection. β-actin was used as internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further examined the effect of miR-17-5p mimic in vivo. Mice were injected with miR-17-5p mimic or scrambled-sequence oligonucleotide (oligo), and then subjected to sham or bilateral renal IRI. Compared with the scrambled oligo group, the mice treated with miR-17-5p showed significantly lower blood urea nitrogen and serum creatinine (Figure 4a and b). We further examined renal tissue damage by hematoxylin and eosin staining. As shown in Figure 4d and e, the kidneys from the sham surgery group showed healthy histology. Notably, there was severe kidney injury with about 40% tubular damage in scrambled oligo–treated mice after 30 minutes of ischemia and 48 hours of reperfusion. In contrast, miR-17-5p mimic injection significantly reduced the tubular damage to about 20%. Following IRI, these mice also showed significantly less apoptosis (28/mm2) than the scrambled control mice (51/mm2) (Figure 4f and g). These results suggested that miR-17-5p may play a protective role in ischemic acute kidney injury. How does miR-17-5p protect? The key to this question is to identify the responsible target gene(s) of this microRNA. Depending on the cellular context, miR-17-5p may target a variety of downstream genes by binding to the 3′UTR of their mRNAs.21Marrone A.K. Stolz D.B. Bastacky S.I. et al.MicroRNA-17∼92 is required for nephrogenesis and renal function.J Am Soc Nephrol. 2014; 25: 1440-1452Crossref PubMed Scopus (54) Google Scholar We first searched 2 microRNA target analysis databases (miRanda and TargetScan), which predicted several hundred putative target genes of miR-17-5p. Among these genes, DR6, also known as tumor necrosis factor receptor superfamily member 21 (TNFRSF21), was consistently predicted as a candidate target of miR-17-5p. Further bioinformatics analysis showed that the miR-17-5p binding site in the 3′UTR of DR6 was conserved across species (Figure 5a). Experimentally, DR6 expression significantly decreased in kidney cortical tissues after 25 minutes of ischemia with 1 to 7 days of reperfusion (Figure 5b), whereas miR-17-5p was induced (Figure 1b). In RPTC cells, DR6 expression was suppressed specifically by the transfection of miR-17-5p mimic, whereas anti-miR-17-5p LNA increased DR6 expression (Figure 5c). In vivo, we treated mice with miR-17-5p mimic for 24 hours, which suppressed DR6 expression (Figure 5d). To further determine whether miR-17-5p directly targets the 3′UTR of DR6, we conducted miRNA Target Luciferase Reporter assay. To this end, we prepared a construct that contained the putative miR-17-5p targeting site of DR6 3′UTR sequence after the luciferase reporter gene (Luciferase-DR6-3′UTR). HEK cells were cotransfected with the Luciferase-DR6-3′-UTR construct or Luciferase-DR6-3′-empty vector along with miR-17-5p mimic or scrambled-sequence oligo. MiR-17-5p mimic significantly reduced luciferase expression in Luciferase-miR-17-DR6 transfected cells but not in the scrambled-sequence oligo (Figure 5e), indicating that the miR-17-5p directly targeted DR6-3′-UTR. Collectively, these results suggested that miR-17-5p may attenuate the ischemic renal cell death by targeting or repressing DR6. To test the role of DR6 in miR-17-5p–mediated renal protection, we transfected short hairpin RNAs (shRNAs) into RPTCs to knockdown DR6. Compared with scramble shRNA, DR6-shRNA transfection significantly decreased DR6 expression (Figure 6a). After 48 hours of hypoxia (1% oxygen) treatment, ∼16% of apoptosis occurred in scrambled oligo transfected cells, whereas <12% occurred in DR6 knockdown cells (Figure 6b). Representative recording of cell morphology further verified that less apoptosis developed following hypoxia in DR6-shRNA cells (Figure 6c). This result supported a proapoptotic role of DR6 in renal tubular cells during hypoxic injury in vitro and ischemic injury in vivo. This observation also suggested that miR-17-5p may protect kidney cells and tissues by repressing DR6. After showing DR6 as a downstream target gene of miR-17-5p (Figures 5 and 6), we would like to understand the upstream mechanism that accounts miR-17-5p induction in renal IRI. Hypoxia-inducible factor (HIF)-1 is considered the “master” transcription factor that regulates gene expression under conditions of hypoxia and ischemia.22Semenza G.L. Hypoxia-inducible factor 1 and cardiovascular disease.Annu Rev Physiol. 2014; 76: 39-56Crossref PubMed Scopus (385) Google Scholar In addition, HIF-1 has been reported to regulate miR-17 in leukemic cells.23He M. Wang Q.Y. Yin Q.Q. et al.HIF-1α downregulates miR-17/20a directly targeting p21 and STAT3: a role in myeloid leukemic cell differentiation.Cell Death Differ. 2013; 20: 408-418Crossref PubMed Scopus (80) Google Scholar Thus, we hypothesized that HIF-1 mediated miR-17-5p induction during renal IRI and hypoxia of RPTC cells. To test this possibility, we initially compared miR-17-5p induction by hypoxia in wild-type (WT) and HIF-1α-null mouse embryonic fibroblasts. As shown in Figure 7a, miR-17-5p expression increased in both WT and HIF-1α-null cells, negating a role of HIF-1 in hypoxic induction of miR-17-5p. To further test this in vivo, we took advantage of the proximal tubule-specific HIF-1α knockout mouse model that was established in our recent work.15Bhatt K. Wei Q. Pabla N. et al.MicroRNA-687 induced by hypoxia-inducible factor-1 targets phosphatase and tensin homolog in renal ischemia-reperfusion injury.J Am Soc Nephrol. 2015; 26: 1588-1596Crossref PubMed Scopus (80) Google Scholar, 17Wei Q. Liu Y. Liu P. et al.MicroRNA-489 induction by hypoxia-inducible factor-1 protects against ischemic kidney injury.J Am Soc Nephrol. 2016; 27: 2784-2796Crossref PubMed Scopus (65) Google Scholar The rationale was that miR-17-5p was mainly induced in kidney proximal tubules (Figure 1b). As shown in Figure 7b, miR-17-5p was induced in both WT and proximal tubule-HIF-1α-null mice, irrespective of the HIF-1 status. Thus it was concluded that HIF-1 was not the key transcription factor responsible for miR-17-5p induction by hypoxia in vitro and renal IRI in vivo. P53 plays an important role in renal IRI.24Kelly K.J. Plotkin Z. Vulgamott S.L. Dagher P.C. P53 mediates the apoptotic response to GTP depletion after renal ischemia-reperfusion: protective role of a p53 inhibitor.J Am Soc Nephrol. 2003; 14: 128-138Crossref PubMed Scopus (190) Google Scholar, 25Molitoris B.A. Dagher P.C. Sandoval R.M. et al.siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.J Am Soc Nephrol. 2009; 20: 1754-1764Crossref PubMed Scopus (257) Google Scholar Such a conclusion was further established recently by using proximal tubule-specific p53-knockout (PT-p53-KO) mouse models.26Zhang D. Liu Y. Wei Q. et al.Tubular p53 regulates multiple genes to mediate AKI.J Am Soc Nephrol. 2014; 25: 2278-2289Crossref PubMed Scopus (111) Google Scholar, 27Ying Y. Kim J. Westphal S.N. et al.Targeted deletion of p53 in the proximal tubule prevents ischemic renal injury.J Am Soc Nephrol. 2014; 25: 2707-2716Crossref PubMed Scopus (85) Google Scholar In these studies, we further identified multiple genes that are regulated by p53 in renal IRI and may contribute to the pathogenesis.26Zhang D. Liu Y. Wei Q. et al.Tubular p53 regulates multiple genes to mediate AKI.J Am Soc Nephrol. 2014; 25: 2278-2289Crossref PubMed Scopus (111) Google Scholar Is miR-17-5p subjected to p53 regulation? To address this, we first verified that p53 was induced or activated by hypoxia (1% oxygen) in vitro in RPTC cells. As shown in Figure 8a, both total and phosphorylated p53 were induced by 6 to 48 hours of hypoxia. We further used the JASPAR CORE 2016 database to conduct bioinformatics analysis of the promoter region of rat miR-17-5p gene. Interestingly, there appeared to be 2 miR-17 genes (miR-17-1 and miR-17-2) and both genes contained a putative p53-binding site in their promoter regions (Figure 8b). Experimentally, we analyzed the direct binding of p53 to the miR-17-1 and miR-17-2 promoter regions by chromatin immunoprecipitation assay. As a positive control, p21 (known p53 target gene) was used. As shown in Figure 8c, both predicted p53 binding sites showed significantly more p53 binding in hypoxia-treated RPTC cells. The results implicate that p53 may mediate miR-17-5p expression in hypoxia by direct transcriptional activation. To determine whether p53 plays a role in miR-17-5p induction during renal hypoxia and IRI, we initially tested the effect of pifithrin-α, a pharmacological inhibitor of p53. We first verified the inhibitory effect of pifithrin-α on p53 in RPTC cells (Figure 9a). Further real-time PCR analysis showed that pifithrin-α blocked miR-17-5p induction during hypoxia of RPTC cells (Figure 9b). To confirm this observation, we examined the stable RPTC cell line transfected with p53 dominant-negative mutant (DN-p53). As shown in Figure 9d, 24 hours of hypoxia treatment resulted in obviously miR-17-5p increase in WT cells, but the induction was clearly blocked in DN-p53 cells (Figure 9c and d). Finally, we determined whether miR-17-5p induction in renal IRI depends on p53. For this purpose, we used the conditional knockout mouse model from our recent study, which had specific p53 ablation from proximal tubules (PT-p53-KO).26Zhang D. Liu Y. Wei Q. et al.Tubular p53 regulates multiple genes to mediate AKI.J Am Soc Nephrol. 2014; 25: 2278-2289Crossref PubMed Scopus (111) Google Scholar As expected, compared with WT littermates (PT-p53-WT), PT-p53-KO mice showed marked lower p53 in kidney cortical tissues regardless of renal IRI (Figure 10a). Importantly, 30 minutes of bilateral renal ischemia with 48 hours of reperfusion led to 2.5-fold increase in miR-17-5p, which was reduced to <1.5 fold in PT-p53-KO tissues (Figure 10b). As in a previous study,26Zhang D. Liu Y. Wei Q. et al.Tubular p53 regulates multiple genes to mediate AKI.J Am Soc Nephrol. 2014; 25: 2278-2289Crossref PubMed Scopus (111) Google Scholar PT-p53-KO mice were partially protected from renal IRI as shown by the measurements of blood urea nitrogen and serum creatinine (Figure 10c and d). MiRNAs are important posttranscriptional regulators of gene expression that mediate various cellular activities and processes of homeostasis by repressing target genes. In the current study, we provide the first evidence of the regulation and functional role of miR-17-5p in ischemic acute kidney injury. MiR-17-5p was upregulated both in vivo in ischemia-reperfused kidneys and in vitro during hypoxic incubation of cultured renal proximal tubular cells. The miR-17-5p induction was shown to be mediated directly by p53 and not by HIF-1. Upon induction, miR-17-5p may play a cytoprotective role for renal cell survival via the suppression of DR6. MiR-17, encoded by the miR-17-92 family cluster, has been reported to increase in response to ischemia and hypoxia in the cardiovascular system.28Du W. Pan Z. Chen X. et al.By targeting Stat3 microRNA-17-5p promotes cardiomyocyte apoptosis in response to ischemia followed by reperfusion.Cell Physiol Biochem. 2014; 34: 955-965Crossref PubMed Scopus (68) Google Scholar, 29Zhou M. Cai J. Tang Y. Zhao Q. MiR-17-92 cluster is a novel regulatory gene of cardiac ischemic/reperfusion injury.Med Hypotheses. 2013; 81: 108-110Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar We and others15Bhatt K. Wei Q. 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To verify the induction of miR-17 and identify its functional form in ischemic acute kidney injury, we conducted quantitative real-time PCR using specific Taqman probes to miR-17-5a and -3a. The analysis revealed the induction of both miR-17-5p and miR-17-3p during renal IRI (Figure 1 and data not shown). Further functional study showed that inhibition of miR-17-5p by LNA increased apoptosis during hypoxic incubation (Figure 2), whereas miR-17-3p-LNA had no effect (not shown). Therefore, in the current study, we mainly focused on the regulation and function of miR-17-5p in kidney IRI. Functionally, anti-miR-17-5p-LNA increased apoptosis during hypoxic incubation (Figure 2), whereas miR-17-5p mimic suppressed apoptosis, supporting a protective role of miR-17-5p. Consistently, the majority of the studies in cardiovascular systems indicate that miR-17-5p may act as a prosurvival factor.28Du W. Pan Z. Chen X. et al.By targeting Stat3 microRNA-17-5p promotes cardiomyocyte apoptosis in response to ischemia followed by reperfusion.Cell Physiol Biochem. 2014; 34: 955-965Crossref PubMed Scopus (68) Google Scholar In various pathological conditions, miR-17-5p may target a variety of downstream genes, including those in the PTEN pathway, WNT/β-catenin pathway, PI3K/AKT pathway, and MAPK/ERK pathway.31Fang Y. Xu C. Fu Y. MicroRNA-17-5p induces drug resistance and invasion of ovarian carcinoma cells by targeting PTEN signaling.J Biol Res (Thessalon). 2015; 22: 12Crossref PubMed Scopus (21) Google Scholar, 32Yu F. Lu Z. Huang K. et al.MicroRNA-17-5p-activated Wnt/β-catenin pathway contributes to the progression of liver fibrosis.Oncotarget. 2016; 7: 81-93Crossref PubMed Google Scholar, 33Rao E. Jiang C. 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Death receptor signaling.J Cell Sci. 2005; 118: 265-267Crossref PubMed Scopus (364) Google Scholar When receptor-interacting protein and tumor necrosis factor receptor–associated death domain protein are recruited to the complex, DR6 along with DR3 may trigger nuclear factor κB signaling that promotes the expression of multiple survival genes.36Karin M. Lin A. NF-kappaB at the crossroads of life and death.Nat Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2445) Google Scholar On the contrary, when Fas-associated death domain, procaspase-8/10, and FLICE-inhibitory protein are recruited to form the complex, DR6 may activate the downstream death signaling.37Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1991) Google Scholar Recent studies suggested the upregulated endogenous DR6 during hypoxia could exacerbate neuritis damage and inhibition of DR6 could protect neurons and axons from injury after ischemic stroke.38Nikolaev A. McLaughlin T. O'Leary D.D. Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases.Nature. 2009; 457: 981-989Crossref PubMed Scopus (836) Google Scholar, 39Mi S. Lee X. Hu Y. et al.Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination.Nat Med. 2011; 17: 816-821Crossref PubMed Scopus (68) Google Scholar Consistently, in our study, shRNA knockdown of DR6 led to the protection of RPTC cells from apoptosis, suggesting that DR6 is prodeath in hypoxic renal tubular cells. Knockdown of DR6 also suppre
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