MicroRNA-34a Is Induced via p53 during Cisplatin Nephrotoxicity and Contributes to Cell Survival
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MicroRNAs are small noncoding RNAs that are produced endogenously and have emerged as important regulators in pathophysiological conditions such as development and tumorigenesis. Very little is known about the regulation of microRNAs in renal diseases, including acute kidney injury (AKI). In this study, we examined the regulation of microRNA-34a (miR-34a) in experimental models of cisplatin-induced AKI and nephrotoxicity. By Northern blot and real-time polymerase chain reaction analyses, we detected an induction of miR-34a in vitro during cisplatin treatment of mouse proximal tubular cells and also in vivo during cisplatin nephrotoxicity in C57BL/6 mice. In cultured cells, miR-34a was induced within a few hours. In mice, miR-34a induction was detectable in renal tissues after 1 d of cisplatin treatment and increased to approximately four-fold of control at d 3. During cisplatin treatment, p53 was activated. Inhibition of p53 with pifithrin-α abrogated the induction of miR-34a during cisplatin treatment of proximal tubular cells. In vivo, miR-34a induction by cisplatin was abrogated in p53-deficient mice, a result that further confirms a role for p53 in miR-34a induction during cisplatin nephrotoxicity. Functionally, antagonism of miR-34a with specific antisense oligonucleotides increased cell death during cisplatin treatment. Collectively, the results suggest that miR-34a is induced via p53 during cisplatin nephrotoxicity and may play a cytoprotective role for cell survival.
Cisplatin, one of the most potent chemotherapy drugs, is also a well-documented nephrotoxin that induces acute kidney injury (AKI) in cancer patients. In kidneys, cisplatin accumulates in renal tubular cells, resulting in cell injury and death, contributing to the development of AKI (see [1,2] for reviews). Research during the last few years has identified several important signaling pathways that lead to tubular-cell apoptosis during cisplatin nephrotoxicity, including mitogen-activated protein kinase, p53 and others (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).
p53 is activated early after cisplatin treatment of renal tubular cells in vitro and in animals in vivo (10,15, 16, 17, 18, 19, 20, 21, 22). Pharmacological and genetic inhibition of p53 attenuates cisplatin-induced apoptosis in cultured tubular cells. An important finding was that cisplatin-induced nephrotoxicity is partially but significantly ameliorated in p53-deficient mice (22). Our recent work has further demonstrated a DNA damage response mediated by ATR-Chk2 in p53 activation and tubular cell apoptosis during cisplatin nephrotoxicity (19). After being activated, p53 can induce the expression of proapoptotic genes, such as PUMA-α and PIDD, to induce cell injury and death (18,20). On the other hand, p53 may also activate mechanisms for cell survival. For example, p21, a cell-cycle regulator with antiapoptotic activity, is induced during cisplatin nephrotoxicity via both p53-dependent and -independent mechanisms (23). In addition, p53 may contribute to the activation of autophagy, a cytoprotective mechanism for cell survival (24). As a result, the balance between the prodeath and prosurvival factors may determine if a cell can survive or be lethally injured during cisplatin nephrotoxicity.
MicroRNAs are small noncoding RNA molecules of 19–25 nucleotides, which regulate posttranscriptional gene regulation (25, 26, 27, 28). MicroRNAs bind to the 3′-untranslated regions of targetgene mRNAs, resulting in stalling of protein synthesis or mRNA degradation. MicroRNAs have emerged as critical regulators of a variety of physiological as well as pathological conditions (25, 26, 27, 28). In kidneys, specific microRNAs are highly expressed and play critical roles in signaling of transforming growth factor β during diabetic nephropathy (29, 30, 31). Moreover, depletion of microRNAs in podocytes by ablating Dicer, a key enzyme for microRNA production, results in a rapid progression to end-stage kidney disease, an effect that further demonstrates the involvement of microRNAs in renal development and physiology (32, 33, 34). Despite these phenomenal findings, little is known about microRNA regulation in AKI, including injury from cisplatin nephrotoxicity.
In 2007, several studies identified microRNA-34a (miR-34a) as a key target gene of p53 that is activated during DNA damage, and study results demonstrated that miR-34a might contribute to the ensuing apoptosis (35, 36, 37, 38, 39, 40). Because p53 is activated during cisplatin nephrotoxicity and contributes to renal cell injury and death, we investigated miR-34a expression and its role in relevant experimental models. We showed that miR-34a is induced during cisplatin treatment of renal proximal tubular cells in vitro and mice in vivo and that miR-34a induction under these experimental conditions is p53 dependent. Interestingly, we further found evidence for a protective role of miR-34a against cisplatin-induced apoptosis in renal cells.
Materials and Methods
Cell and Special Reagents
The mouse proximal tubular cell (BUMPT-306) line was originally obtained from W Lieberthal and JH Shwartz at Boston University (41). The cells were cultured in DMEM with 10% fetal bovine serum and 0.6% glutamine. The antibodies used were from the following sources: anti-phospho p53 (Ser-15) antibodies from Cell Signaling Technology (Danvers, MA, USA), anti-p21 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-puma-α antibody from J Yu at the University of Pittsburgh. Cisplatin and pifithrin-α were purchased from Sigma (St. Louis, MO, USA). Locked-nucleic-acid (LNA) oligonucleotides were purchased from Exiqon, Vedbaek, Denmark.
Cisplatin Treatment of BUMPT Cells
Confluent BUMPT proximal tubular cells were treated with 40 µmol/L cisplatin. After incubation for indicated time periods, the cells were morphologically examined for apoptosis or lysed to collect lysate for caspase assay or immunoblot analysis.
Mouse Model of Cisplatin Nephrotoxicity
Wild-type and p53-deficient C57BL/6 mice (male, 8–10 wks old) were intraperitoneally injected with one dose (30 mg/kg body weight) of cisplatin as described previously (22,42). The control animals were injected with saline. To monitor renal injury, blood urea nitrogen levels were determined using the assay kit from Biotron Diagnostics (Hemet, CA, USA). Whole kidney tissues were collected for immunoblot analysis or RNA extraction. All animal experiments were performed according to a protocol approved by the institutional animal care and use committee of the Charlie Norwood VA Medical Center.
Morphological Examination of Apoptosis and Measurement of Caspase Activity
Cells were stained with 10 µg/mL Hoechst 33342 to examine cellular and nuclear morphology by phase contrast and fluorescence microscopy as described previously (18,19). Typical apoptotic morphology was characterized by cellular shrinkage, formation of apoptotic bodies and nuclear condensation and fragmentation. Caspase activity was measured with an enzymatic assay by using the fluorogenic peptide DEVD.AFC, a substrate of executioner caspases including caspase-3, -6 and -7. Briefly, cells were lysed with 1% triton X-100 and the lysate was added to a caspase reaction mixture containing 50 µmol/L DEVD.AFC for 1 h of incubation. The fluorescence signal of AFC liberated by caspase activity was measured at excitation (360 nm) and emission (530 nm). Caspase activity was then calculated by using a standard curve of free AFC.
Analysis of mir-34a by Real-Time Polymerase Chain Reaction
Total RNA was extracted using the mirVana kit (Ambion, Austin, TX, USA). For real-time polymerase chain reaction (PCR), 40 ng of total RNA was reverse-transcribed into cDNA by using the miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was carried out using the Taqman miRNA assay kit (Applied Biosystems), which included sequence-specific primers for cDNA synthesis and Taqman probes for realtime PCR. Quantification was done using ΔCt values.
Northern Blot Analysis of miR-34a
For Northern blot analysis, total RNA was extracted by using the mirVana kit (Ambion) and run on 15% acrylamide-bisacrylamide (19:1) gel containing 7 mol/L urea in Tris-borate-EDTA buffer. The RNA was then transferred on to Hybond membrane (Amersham, Uppsala, Sweden) at 200 mA for 1 h, followed by ultraviolet crosslinking. The membrane was dried at 80°C for 1 h, followed by prehybridization for 1 h in Ultra-Hyb-Oligo hybridization buffer (Ambion). The radiolabeled probe (complementary of the mature microRNA sequence) was added to the prehybridization buffer and incubated overnight at 37°C and then washed in 2 × standard saline citrate (Ambion) buffer (0.1% sodium dodecyl sulfate [SDS]) and exposed to X-ray film at −70°C.
Transfection of LNA Oligonucleotides
BUMPT cells were plated at 50%–60% confluence. On the next day, the cells were transfected with 100 nmol/L of LNA oligonucleotides by use of Lipofectamine 2000 reagent. After an additional 1 d of culture, the cells were used for treatment.
We followed a standard protocol of immunoblot analysis. Briefly, equal amounts of protein samples were loaded and resolved on SDS-polyacrylamide gel electrophoresis gels. The proteins were then transferred onto a polyvinylidene-difluoride membrane, which was blocked with 5% bovine serum albumin and incubated in primary and secondary antibodies. The antigens on the blots were revealed by use of the ECL chemiluminescence kit from Pierce (Rockford, IL, USA).
Cisplatin-Induced Apoptosis and p53 Activation in BUMPT Cells
Induction of miR-34a during Cisplatin Treatment of BUMPT Cells
miR-34a Induction during Cisplatin Nephrotoxicity In Vivo
p53-Dependence of miR-34a Induction during Cisplatin Nephrotoxicity
Blockade of miR-34a Increases Cell Death during Cisplatin Treatment of BUMPT Cells
MicroRNAs have emerged as important regulators of various pathophysiological conditions (25, 26, 27, 28). By repressing gene expression, microRNAs regulate processes of cellular activity and homeostasis, including growth, proliferation, survival and death (25, 26, 27, 28). However, very little is known about the role and regulation of microRNAs in the development of renal diseases (29,44). The results of this study, in which we used in vitro cell culture as well as in vivo mouse models, demonstrated early miR-34a induction during cisplatin-induced nephrotoxicity. The induction of miR-34a was shown to be p53 dependent. Interestingly, our results also indicate that miR-34a induced during cisplatin nephrotoxicity may be a cytoprotective mechanism for cell survival.
A robust induction of miR-34a has been reported recently in response to DNA damage or genotoxic stress (35, 36, 37, 38, 39). Importantly, the results of these studies indicated that miR-34a is downstream of p53 and plays an important role in p53-mediated apoptosis and cell-cycle arrest. In experimental models of cisplatin nephrotoxicity, our recent work has revealed a rapid DNA damage response, which is centered on ATR and Chk2 (19). The activation of ATR and Chk2 leads to the phosphorylation and activation of p53, which induces the expression of genes that promote cell death by apoptosis (19). Such genes include PUMA-α and PIDD (18,20). Another interesting finding was that p53 might also induce the expression of genes that promote cell survival. For example, p21, a cell-cycle regulator with demonstrated antiapoptotic activity, is induced during cisplatin nephrotoxicity via both p53-dependent and -independent mechanisms (23). Our current results have demonstrated miR-34a induction during cisplatin nephrotoxicity. Notably, cisplatin induced miR-34a in both cultured proximal tubular (BUMPT) cells and renal tissues in C57BL/6 mice (see Figures 2 and 3). To our knowledge, this evidence is among the first reported for microRNA regulation in an experimental model of AKI. In this study, we observed miR-34a induction during cisplatin injury, and it was not clear whether and when miR-34a expression would return to the basal level. However, it is likely that the microRNA expression profile during renal recovery or regeneration is different from that of the injury phase.
We have further provided evidence that miR-34a induction by cisplatin is mediated by p53. We showed that in BUMPT cells, miR-34a induction by cisplatin was attenuated by pifithrin-α, a pharmacological inhibitor of p53 (Figure 4A). Furthermore, miR-34a induction during cisplatin nephrotoxicity was abrogated in p53-deficient mice (Figure 4C). In the in vivo model, 30 mg/kg cisplatin was used to induce kidney injury in C57/BL6 mice. This dose was also used by this and other laboratories in previous studies (22,42,43,45). It is noteworthy that cisplatin at 30 mg/kg induced apoptosis mainly in proximal and distal tubules in kidneys, and not in collecting tubules or glomeruli; some necrosis was induced and mainly localized in proximal tubules (42). Thus cisplatin at this dose does not induce widespread tissue damage, and the model is suitable for the study of cisplatin nephrotoxicity. These observations support the conclusion that miR-34a is a transcriptional target gene of p53. In fact, the promoter region of the miR-34a gene contains p53-binding sites (38). Thus, data suggest that binding of p53 to these sites may activate the transcription of miR-34a during cisplatin-induced injury in renal cells and tissues.
When miR-34a was first identified as a target gene of p53, it was shown to mediate the antiproliferative and proapoptotic effects of p53 (35, 36, 37, 38, 39). For example, Raver-Shapira et al. showed that antagonism of miR-34a could block etoposide-induced apoptosis in U2OS cells (37). In human colon cancer cells, Tazawa et al. showed that expression of miR-34a led to complete suppression of cell proliferation and induced senescence-like phenotypes (39). However, our current results do not support a proapoptotic role for miR-34a in cisplatin-induced injury in BUMPT cells. We showed that blockade of miR-34a with LNA-modified antisense oligonucleotides increased apoptosis during cisplatin treatment of BUMPT cells (Figures 5B, C). In addition, blockade of miR-34a decreased long-term survival of the cells following cisplatin treatment (Figure 5D). Of note, in this experiment we measured the amount of total cell proteins recovered, which may not be an accurate indication of cell survival. However, the results were supported measurement results for cell death or apoptosis (Figure 5B, C). Together, the results suggest that miR-34a induction during cisplatin treatment of BUMPT cells is not proapoptotic, but instead is cytoprotective. This observation was unexpected. However, a very recent study by Kato et al. demonstrated in Caenorhabditis elegans that loss-of-function mutation of miR-34a sensitized somatic cells to radiation injury, suggesting a cytoprotective role for miR-34a in these cells (46). In addition, in mammalian cell lines with different levels of miR-34a, there seemed to be an inverse correlation between miR-34a expression and cellular radiosensitivity, that is, cells with low miR-34a were more sensitive to irradiation-induced injury and death (46). Together, these results and those from our current study support a cytoprotective role for miR-34a under certain experimental conditions. It is currently unclear why miR-34a has been found to be proapoptotic in some studies, but anti-apoptotic in others. We speculate that miR-34a has multiple target genes, and the role played by miR-34a depends on which genes are under its regulation or suppression under various experimental conditions.
MicroRNAs regulate diverse cellular processes by repressing target gene expression. However, target identification is one of the main challenges in understanding the functional role of specific microRNAs. A microRNA can have multiple targets and the target genes of a microRNA can change under different experimental conditions. Several target genes have been suggested for miR-34a, but most of the targets are related to cell-cycle regulation. For example, cyclin E, CDK4, CDK6 and CDC25C were shown to be repressed by miR-34a, resulting in cell-cycle arrest at G1 and G2 phases (35, 36, 37, 38, 39). In terms of the regulation of cell death, Bommer et al. showed that Bcl-2 is a direct target of miR-34a (40). However, regulation of Bcl-2, a well-established antiapoptotic gene, cannot explain the cytoprotective role of miR-34a observed in our study, because repression of Bcl-2 should sensitize the cells to cisplatin injury. In our study, expression of either miR-34a or anti-miR-34a did not change the expression of Bcl-2 in BUMPT cells (data not shown), ruling out Bcl-2 as a target gene of miR- 34a in these cells. We speculate that miR-34a induced during cisplatin nephrotoxicity may regulate or repress a proapoptotic gene(s), leading to death resistance and cell survival. Future studies should identify such gene(s) for a better understanding of the cytoprotective role of miR-34a.
In conclusion, this study has demonstrated the first evidence for microRNA regulation in an experimental model of AKI. miR-34a is induced via p53 during cisplatin nephrotoxicity and may play a cytoprotective role for cell survival.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
The study was supported in part by the National Institutes of Health and Department of Veterans Affairs. K Bhatt was supported by a predoctoral fellowship from the American Heart Association.