Abstract
Inactivation of the von-Hippel Lindau (VHL) tumor suppressor gene occurs in 90% of human clear cell renal cell carcinomas (ccRCC), and leads to the stable expression of the hypoxia inducible factors HIF1α and HIF2α. The constitutive expression of HIF1α in a majority of VHL-deficient tumors is counterintuitive, given that HIF1α functions as a tumor suppressor in ccRCC, whereas HIF2α clearly enhances tumor growth. We demonstrate here that miR-30c-2-3p and miR-30a-3p specifically bind and inhibit expression of HIF2α transcripts, and that the locus encoding miR-30c-2-3p and miR-30a-3p is selectively repressed in “H1H2” VHL-deficient tumors expressing both HIF1α and HIF2α proteins. Inhibiting miR-30a-3p expression increases HIF2α levels in H1H2 ccRCC cells, and promotes cellular proliferation, angiogenesis, and xenograft tumor growth. Our results indicate that miR-30c-2-3p and miR-30a-3p repression enhances HIF2α expression, and suggest a mechanism whereby the tumor suppressive effects of constitutive HIF1α expression are attenuated in VHL-deficient H1H2 tumors.
Introduction
Clear cell renal cell carcinoma (ccRCC) is the most commonly diagnosed form of kidney cancer and accounts for the majority of renal cancer deaths. A characteristic feature of nearly 90% of sporadic ccRCCs is mutation or silencing of the von Hippel-Lindau (VHL) tumor suppressor gene (1). pVHL, the protein encoded by VHL is a critical component of a ubiquitin ligase complex that regulates hypoxia inducible factor (HIF) accumulation (1). HIFs are heterodimeric transcription factors consisting of α and β subunits (2), and HIFα subunits (HIF1α and HIF2α) are hydroxylated by prolyl hydroxylase (PHD) enzymes, resulting in pVHL recognition, polyubiquitination, and proteasomal degradation (3). However, under low oxygen (O2) conditions, HIFα subunits are stabilized, bind the β subunit (ARNT), and activate numerous genes involved in metabolism, angiogenesis, proliferation, cellular motility/invasion and extracellular matrix remodeling (4). Although HIF1α and HIF2α have overlapping functions, recent studies have illustrated remarkably distinct roles for each α isoform in both normal physiology and disease (4).
We have previously stratified >200 VHL-deficient ccRCC tumors into 2 subtypes: one group (~60% of all cases) expressing both HIF1α and HIF2α (designated “H1H2”) and another 30% expressing HIF2α exclusively (designated “H2”) (5, 6). Whereas HIF2α promotes tumorigenesis (7), HIF1α has been clearly shown to be a tumor suppressor (8–10) in this disease. This differential HIFα expression pattern raises an important question: if HIF1α is a tumor suppressor, how do H1H2 ccRCCs evade the inhibitory activities of HIF1α? Delineating the mechanisms by which H1H2 ccRCCs overcome the tumor suppressive effects of HIF1α could identify novel molecular targets for future ccRCC treatment.
Integrated genomic analyses revealed that H1H2 tumors display enhanced mitogen-activated protein kinase and mTOR signaling, while H2 ccRCCs exhibit increased c-Myc activity (5, 6). In addition, an important role for miRNAs in ccRCC progression has been suggested for (11, 12). miRNAs are small, non-coding RNAs (~22 nucleotides) representing 1–2% of the eukaryotic transcriptome that constitute important post-transcriptional regulators of > 30% of mammalian genes (13). miRNAs can act as either oncogenes or tumor suppressors (13) and because each miRNA potentially regulates numerous genes, they represent powerful discovery tools to identify novel pathways impacting cancer. To investigate the role of miRNAs in regulating the progression of distinct ccRCC subtypes, we performed miRNA microarray studies on H1H2 and H2 ccRCCs, along with matched normal control tissue samples. We observed preferential repression of multiple miR-30 family members in H1H2 tumors when compared to adjacent kidney tissues, whereas the repression was less pronounced in H2 tumors. Moreover, we further demonstrate that repression of these miRNAs contributes to higher HIF2α levels in H1H2 tumors, apparently as a compensatory mechanism to circumvent the stable expression of HIF1α. Since HIF2α plays a key oncogenic role in ccRCCs, identification of miRNAs that specifically target HIF2α is of great importance with potential therapeutic implications for kidney cancer.
Results
miR-30c-2-3p and miR-30a-3p are repressed in H1H2 tumors in a VHL-dependent manner
To identify miRNAs whose differential expression might promote the selective growth and progression of H1H2 or H2 ccRCCs, we performed microarray analysis of RNA from H1H2 (n=5) and H2 (n=8) tumors, as well as adjacent normal kidney tissue. Significant differences in miRNA levels were observed between tumors and their respective control samples (Figure 1A). As expected, levels of the previously identified hypoxia-regulated miR-210 (14) were elevated in both H1H2 and H2 subtypes. We then focused on miRNAs that were differentially expressed in each ccRCC subtype, and chose miR-30c-2-3p for further analysis, as its expression was significantly more repressed in H1H2 than in H2 tumors when normalized to adjacent normal kidney RNA (Figure 1A, arrow; B). MIR30C2 maps to human chromosome 6q13, and is closely linked to MIR30A (Figure S1A). Intriguingly, miR-30a-3p expression was also repressed in H1H2 tumors relative to H2 tumors (Figure 1C, S1B, arrows), suggesting common regulation of the genomic locus. Importantly, TCGA data analysis revealed that miR-30c-2-3p and miR-30a-3p are significantly repressed in numerous ccRCCs (n=437) when compared to normal tissue samples (n=68) (Figure 1D, E). Moreover, correlation analysis using TCGA data indicated that both miR-30c-2-3p and miR-30a-3p are significantly co-regulated in ccRCCs (Figure S1C; n=437).
Figure 1. miR-30c-2-3p and miR-30a-3p are significantly repressed in H1H2 subtypes, and positively regulated by pVHL.
A, Heat map showing miRNAs that are differentially expressed at least four fold at a false discovery rate below 5% in either H2 Tumor vs H2 Normal (n=8) or H1H2 Tumor vs H1H2 Normal (n=5) samples. miR-30c-2-3p (arrow) and miR-210 (red highlighted) expression in H1H2 and H2 tumors compared to normal tissue samples. B, C, Quantitative PCR analysis showing miR-30c-2-3p and miR-30a-3p levels in H1H2 and H2 tumors vs matched controls. D, E, Relative expression of miR-30c-2-3p and miR-30a-3p between ccRCCs and normal renal tissue samples (TCGA). F, Levels of miR-30c-2-3p and miR-30a-3p in RCC10 cells upon re-expression of pVHL. Western blot of HIFs validating the re-introduction of pVHL in RCC10 cells. The data presented here is the average of three biological replicates, unless specified. For all statistical analyses, (*) p<0.05, (**) p<0.005 and (***) p<0.0005. Data are presented as mean±SEM.
As both HIF1α and HIF2α are constitutively expressed in H1H2 ccRCCs, we first investigated whether HIFs regulate the expression of miR-30c-2-3p and miR-30a-3p. Inhibition of either HIFα subunit using shRNA (Figure S2A, B) or siRNA (Figure S2C, D) techniques demonstrated that miR-30c-2-3p and miR-30a-3p were not regulated by HIF. However, since both miRNAs are repressed in VHL-deficient ccRCCs, we re-introduced pVHL into RCC10 cells, and observed significant de-repression of miR-30c-2-3p and miR-30a-3p (Figure 1F). Altogether, these studies indicate that the preferential inhibition of miR-30c-2-3p and miR-30a-3p observed in H1H2 tumors is pVHL-dependent, but HIF-independent.
miR-30c-2-3p /30a-3p repress HIF2α expression in H1H2 ccRCCs
We employed bioinformatic tools (15) (DianaMicroT) to identify specific molecular targets of miR-30c-2-3p and miR-30a-3p. Interestingly, both miRNAs were predicted to bind HIF2α transcripts, which we tested by fusing the HIF2α 3’ UTR to a standard luciferase reporter gene construct. Mutating miR-30c-2-3p or miR-30a-3p seed sequences in the HIF2α 3’ UTR was sufficient to block miR-30c-2-3p/30a-3p -dependent regulation of luciferase activity (Figure 2A, B, S3A). We selected RCC4, RCC10 and UMRC2 ccRCC cell lines for further functional analyses, as they stably express both HIF1α and HIF2α. Ectopic expression of miR-30c-2-3p (miR-30c-2-3p EE) in RCC4 and RCC10 cells decreased HIF2α mRNA expression (Figure 2C; S3B), whereas miR-30c-2-3p inhibition (miR-30c-2-3p INH) increased HIF2α transcript levels (Figure 2D). HIF2α protein levels were similarly reduced by ectopic expression of miR-30c-2-3p, and increased by miR-30c-2-3p inhibition in both RCC4 and RCC10 cells (Figure 2E), with consequent effects on the expression of HIF2α-regulated target genes, including VEGF, GLUT1 and TGF-α (Figure 2F, S3C, D). To confirm that miR-30a-3p also regulates HIF2α, we stably inhibited miR-30a-3p, and found elevated HIF2α abundance in RCC4 and UMRC2 cells (Figure 2G, S3E). In each of these studies, expression levels of HIF1α, and its transcriptional target phosphoglycerate kinase 1 [PGK1], were not altered by miR-30c-2-3p or miR-30a-3p (Figure 2C-G, S3B-E). Finally, our analysis of paired ccRCC tumor samples (TCGA data) revealed significant negative correlation between HIF2α or HIF2α targets (VEGF, GLUT1, TGF-α) vs miR-30c-2-3p/miR-30a-3p levels in renal tumors (Figure S3F, G, H).
Figure 2. Regulation and expression of HIF2α in ccRCC subtypes.
A, Schematic diagram depicting miR-30c-2-3p (H1) and miR-30a-3p (H2) binding sites in HIF2α 3’UTR. B, RCC4 cells transfected with pMIR-REPORT with intact or mutated seed sequences for miR-30c-2-3p and miR-30a-3p binding in HIF2α 3’UTR were tested for luciferase activity in the presence of miR-30c-2-3p and miR-30a-3p mimics, respectively. C, D, Expression of HIF1α and HIF2α transcripts upon stable ectopic expression (EE) and inhibition (INH) of miR-30c-2-3p in RCC4 cells. E, Western blot showing the abundance of HIF1α and HIF2α protein after stable ectopic expression or inhibition of miR-30c-2-3p in RCC10 and RCC4 cells. β-tubulin was used as a loading control. The quantified value of protein expression represents the average of two experiments. F, Transcript levels of HIF1α and HIF2α regulated target genes after stable ectopic expression of miR-30c-2-3p in RCC4 cells. G, Western blot showing the levels of HIF1α and HIF2α protein after stable inhibition of miR-30a-3p in RCC4 and UMRC2 cells. The quantified value of protein expression represents the average of two experiments. β-tubulin was used as a loading control. H, HIF2α expression in ccRCC patient and normal renal tissue samples (TCGA). I, J, Relative expression of HIF2α between H1H2 and H2 ccRCC subtypes from TCGA and U. Pennsylvania human ccRCC patient samples. For all statistical analyses, (*) p<0.05, (**) p<0.005 and (***) p<0.0005. The data presented here is the average of three biological replicates, unless specified. Data are presented as mean±SEM.
As stated above, recent studies have confirmed that HIF1α acts as a tumor suppressor in ccRCCs (10); however, 60% of ccRCCs constitutively express HIF1α (5, 6). We hypothesized that repression of miR-30c-2-3p and miR-30a-3p ameliorates the suppressive effects of HIF1α by elevating relative HIF2α expression in VHL-deficient ccRCCs. To test this hypothesis, we first analyzed the basal expression of HIF2α in clinical ccRCC samples using TCGA data, and observed significantly increased HIF2α levels relative to normal renal tissue (Figure 2H). Since miR-30c-2-3p and miR-30a-3p are preferentially repressed in H1H2 tumors, we investigated whether HIF2α mRNA levels are significantly elevated in H1H2 tumors, compared to H2 tumors. To first identify H1H2 and H2 tumors among the TCGA ccRCC data set, we first performed copy number (CN) analysis to determine the status of VHL and HIF1α. Based on this approach (see Methods), approximately 77% (373/470) of TCGA ccRCC samples were inferred to have VHL loss. Of these, approximately 30% also displayed a CN value for HIF1α of <1.6, signifying gene deletion (Figure S3I). These samples were confirmed as having hemizygous HIF1α deletions by independent analyses using cBioPortal for Cancer Genomics (MSKCC). Based on these criteria, we designated VHL-deficient ccRCC samples with CN<1.6 at HIF1α as the “H2” subtype (n=110/362; Figure S3I), whereas VHL-deficient tumors expressing both HIFα isoforms were designated as the “H1H2” group (n=252/362). It should be emphasized that this stratification is based solely on VHL mutations/deletions and HIF1α deletions, and does not include other forms of genomic alteration that could reduce expression, such as aberrant DNA methylation or altered chromatin structure. Despite this limitation, our classification system was consistent with the stratification based on HIF1α and HIF2α immunohistochemical staining, where approximately one-third of VHL- deficient ccRCCs were defined as H2 tumors (5, 6). Importantly, our analysis of the stratified TCGA ccRCC samples revealed significantly higher HIF2α transcript levels in H1H2 tumors, relative to H2 ccRCCs (Figure 2I). In addition, we measured HIF2α transcript levels in U. Pennsylvania (UPENN) ccRCC samples that were previously stratified into H1H2 and H2 subtypes by immunohistochemical staining for HIF1α and HIF2α (5, 6). Consistent with our results using the TCGA data set, the UPENN ccRCC H1H2 tumors displayed higher HIF2α expression levels than the H2 tumors (Figure 2J; n=11 each), supporting our hypothesis that elevating HIF2α expression is a general mechanism whereby H1H2 ccRCCs compensate for the tumor suppressive activities of HIF1α.
HIF2α is a critical target of miR-30c-2-3p in ccRCC
Our data suggest that increased HIF2α protein levels is a primary functional consequence of miR-30c-2-3p/miR-30a-3p repression in H1H2 ccRCCs. To test this hypothesis directly, we transiently expressed miR-30c-2-3p/miR-30a-3p in the UMRC2 ccRCC cell line. Ectopically expressed miR-30c-2-3p inhibited UMRC2 proliferation during in vitro cell culture; however, no proliferative effect was observed with increased miR-30a-3p expression (Figure S4). Similarly, miR-30c-2-3p expression reduced the ability of UMRC2 cells to form colonies in vitro (Figures 3A, B, S5A and S6A). This effect was amplified when cells were first exposed to serum- and glucose-free conditions for 48 hours, followed by growth under serum- and glucose-replete conditions for 12 days (Figure 3A, B). Finally, the anchorage-independent growth of UMRC2 cells was compromised by ectopic expression of miR-30c-2-3p (Figure 3C). Similar results were obtained for the “H2” ccRCC cell lines 786-0 and 769-P (Figure S6B, C). To determine the HIF2α-dependence of these responses, a HIF2α open reading frame (Figure S5C) was introduced into UMRC2 cells expressing either miR-30c-2-3p or a miR-SCR control construct. We observed that ectopic HIF2α expression significantly reversed the miR-30c-2-3p-mediated phenotypes in these assays (Figure 3A-D). Collectively, these studies specifically illustrate that HIF2α is an important target of miR-30c-2-3p in ccRCC cells, and suggest that miR-30c-2-3p repression enhances HIF2α activity in H1H2 tumors, at the expense of HIF1α.
Figure 3. Role of HIF2α in miR-30c-2-3p mediated effects on ccRCC cell growth.
A, UMRC2 cells ectopically expressing miR-SCR or miR-30c-2-3p were transduced with Empty ORF or HIF2α ORF. Colony forming assays were performed under replete (10% FBS, 5 mM glucose) or SG (no FBS and glucose) medium for 48 hrs, and then grown under replete medium for 12 days. Colonies were stained with crystal violet. B, Data demonstrating the quantification of colonies grown under replete and SG conditions after 12 days. C, D, Representative images and quantification of soft agar colony assays performed with UMRC2 cells expressing miR-SCR or miR-30c-2-3p after transduction with Empty ORF or HIF2α ORF plasmids. Scale bar is 1 mm. The data presented here is the average of three biological replicates. For all statistical analyses, (*) p<0.05, (**) p<0.005 and (***) p<0.0005. Data are presented as mean±SEM.
Modulation of miR-30c-2-3p and miR-30a-3p expression affects tumor growth and predicts human ccRCC patient survival
Based on our in vitro results, we first analyzed whether increased expression of miR-30c-2-3p and miR-30a-3p impacts in vivo tumor growth. As UMRC2 cells reproducibly generate subcutaneous xenograft tumors (10), we injected immunocompromised (Nu/Nu) mice subcutaneously with UMRC2 cells ectopically expressing miR-30c-2-3p or miR-30a-3p or a scrambled control. Increased expression of either miR-30c-2-3p or miR-30a-3p significantly inhibited xenograft tumor growth when compared to the control group (Figures 4A; S5A, B). Therefore, we postulated that miR-30c-2-3p or miR-30a-3p inhibition would enhance ccRCC tumor growth in vivo. However, as noted above, the abundance of these miRNAs is already significantly repressed in ccRCC cells, making it potentially difficult to further reduce their expression. Given that basal miR-30a-3p levels are higher than miR-30c-2-3p in UMRC2 cells, we focused on the functional outcome of inhibiting miR-30a-3p in these cells. We injected immunocompromised (Nu/Nu) mice subcutaneously with UMRC2 cells expressing either a miR-30a-3p antagomir (miR-30a-3p INH) or a scrambled control (miR-SCR INH) (Figures 4B and S7A, B). Importantly, tumors derived from the miR-30a-3p INH cells were significantly larger than controls (Figures 4B and S7A, B). Histological analyses also revealed mild to moderate degenerative changes in tumors from the miR-SCR INH group, a feature not observed in miR-30a-3p INH tumors (Figure S7C). Immunohistochemical staining for Ki67 demonstrated increased cell proliferation in miR-30a-3p INH tumors compared to miR-SCR INH tumors (Figure 4C). Of note, the effect of miR-30a-3p on cell proliferation is very similar to HIF2α, where increased miR-30a-3p expression (Figure S4) or HIF2α inhibition did not affect cell growth in vitro, but inhibited tumor growth in vivo (7). Although no significant difference in tumor blood vessel area was observed between the two cohorts, as reflected by the number of CD31+ endothelial cells (Figure 4D,G), vessel coverage by smooth muscle actin-expressing (SMA+) pericytes was significantly increased in miR-30a-3p INH tumors (Figure 4D,E), suggesting that these vessels are more mature (Figure 4F). Furthermore, we observed increased HIF2α protein expression in miR-30a-3p inhibited tumors compared to the miR-SCR INH group (Figure 4H), suggesting that elevated HIF2α levels contribute directly to the tumor angiogenesis and cell proliferation phenotypes in miR-30a-3p INH tumors. These results are also consistent with previous reports, indicating that HIF2α expression is positively associated with ccRCC cell proliferation and tumor angiogenesis (16).
Figure 4. Tumor suppressive role of miR-30c-2-3p and miR-30a-3p in ccRCC tumor growth.
A, Growth curve of xenografts subcutaneously implanted with UMRC2 cells after ectopic expression of miR-30c-2-3p or miR-30a-3p. UMRC2 cells transduced with miR-SCR EE are used as the control. (n=8) B, Xenograft umor growth with UMRC2 cells after inhibition of miR-30a-3p, and miR-SCR INH are used as the control. (n=5) C, Ki67+ proliferating cells in UMRC2 miR-SCR INH or miR-30a-3p INH xenograft tumors. Scale bar is 20 µm. D, CD31 and SMA immunofluorescence in UMRC2 miR-SCR INH or miR-30a-3p INH xenograft tumor sections. Scale bar is 20 µm. E, Quantification of SMA+ area indicative of pericyte coverage of blood vessels in UMRC2 miR-SCR INH or miR-30a-3p INH sections. F, Percentage of SMA+ area coverage of total blood vessel area (CD31+) in UMRC2 miR-SCR INH or miR-30a-3p INH tumors. G, Data demonstrating CD31+blood vessel area in UMRC2 miR-SCR INH or miR-30a-3p INH tumor sections. H, Western blot showing HIF2α abundance in UMRC2 miR-SCR INH or miR-30a-3p INH xenograft tumors (n=3 each). I, J, Kaplan-Meier survival curve based on high and low miR-30c-2-3p or miR-30a-3p expression in ccRCC patients (TCGA). “miR-30c-2-3p low” (n=143) represents the bottom one-third of the cases whereas “miR-30c-2-3p high” (n=286) is designated as the top two-third of the samples. Log rank test was used to assess statistical significance. Similar classification was used for performing survival analysis based on miR-30a-3p levels. K, Proposed model illustrating the role of miR-30c-2-3p and miR-30a-3p in modulating HIF2α levels in ccRCC tumors. For all statistical analyses, (*) p<0.05, (**) p<0.005 and (***) p<0.0005. Data are presented as mean±SEM.
Finally, we next determined whether miR-30c-2-3p and miR-30a-3p expression levels correlate with human ccRCCC patient survival. Reduced expression of either miR-30c-2-3p or miR-30a-3p was significantly associated with poor prognosis in ccRCC patients (Figure 4I, J), suggesting that their repression is important for ccRCC growth and/or progression. Collectively, our studies illustrate a novel compensatory mechanism whereby tumor cells alter miRNA expression to increase the abundance of an oncoprotein, HIF2α.
Discussion
Biallelic VHL inactivation leads to an increased abundance of HIF1α and HIF2α in ccRCCs, and compelling evidence suggests that HIF2α, rather than HIF1α, promotes pVHL-deficient ccRCC tumorigenesis (17). However, nearly two-thirds of VHL-mutant ccRCCs express HIF1α (5, 6), prompting us to investigate the molecular mechanisms overcoming the previously characterized tumor suppressive activity of HIF1α in these tumors. Although the role of various miRNAs has been documented in ccRCCs (12), the major goal of our study was to identify miRNAs that are preferentially altered in H1H2 versus H2 subtypes. This analysis gave us a unique opportunity to probe novel molecular pathways that are distinct between the two ccRCC groups. We demonstrate here that HIF2α expression is elevated in H1H2 tumors, and propose that preferential repression of miR-30c-2-3p and miR-30a-3p in the H1H2 subclass contributes significantly to this phenotype.
miR-30c-2-3p and miR-30a-3p are located on chromosome 6q13, and our data suggest that their expression is pVHL dependent, but HIF independent. Similar to our results, miR-204 is also repressed in VHL-deficient ccRCCs, and positively regulated by pVHL independent of HIF activity (11). Although pVHL stimulates miR-204, miR-30c-2-3p and miR-30a-3p expression, it is likely that additional intermediate factors are involved. pVHL clearly impacts the activity of multiple transcription factors, including nuclear factor kappa B (NF-κB) (18) and β-catenin (19). Of note, GATA3 regulates miR-30c-2-3p transcription in breast tumor cells (20), and GATA3 expression is decreased in all stages of ccRCC (see Figure S8), due to epigenetic silencing (21). Therefore, it is plausible that loss of GATA3 in ccRCCs is responsible for reduced miR-30c-2-3p and miR-30a-3p levels in VHL-deficient ccRCCs.
In addition to the modulation of HIF2α levels by miRNA repression, alternative mechanisms favoring enhanced HIF2α activity have been described previously. Factor inhibiting HIF1 (FIH1) is an oxygen-dependent enzyme that hydroxylates an asparaginyl residue in its C-terminal transactivation domain (CTAD), preventing the recruitment of co-activator proteins required for transcriptional activity (22). However, HIF2α is less sensitive to FIH1- mediated inhibition than HIF1α (23), resulting in a relative increase in HIF2α target gene expression. Additionally, HIF1α undergoes preferential proteasomal degradation in the absence of pVHL, possibly via distinct ubiquitin ligases or direct interaction with the proteasome (24). These findings clearly suggest that HIF2α abundance is critical for ccRCC tumorigenesis, and miR-30c-2-3p and miR-30a-3p repression represents a novel means whereby tumor cells antagonize the tumor suppressive role of HIF1α (Figure 4K).
A more general role for miRNAs has been demonstrated in numerous cancers (including ccRCCs), suggesting that the development of miRNA-based therapies could ultimately be beneficial for patient care. Systemic miR-26a delivery using adeno-associated virus inhibits the progression of hepatocellular carcinoma in mice, illustrating the potential utility of these biomolecules in disease treatment (25). Although some progress has been made in miRNA delivery, the use of synthetic miRNAs for renal tumor therapy is still in its infancy. Since miR-30c-2-3p and miR-30a-3p directly regulate HIF2α, which acts as an oncoprotein in ccRCC, these miRNAs represent novel therapeutic agents in the future. In conclusion, we demonstrate a novel molecular mechanism by which HIF2α levels are elevated to compensate for the inhibitory effects of HIF1α in VHL-deficient H1H2 ccRCCs.
Methods
Cell culture
The human RCC cell lines RCC4 and 786-0 were obtained from American Type Culture Collection. RCC10, UMRC2 and 769-P cells were a kind gift from Dr. W.G. Kaelin, Dana-Farber Cancer Institute, Harvard Medical School, MA. These cells were cultured in DMEM containing 10% FBS and antibiotics. All cell lines were verified for VHL and HIFα expression status using qPCR and Western blot analysis within the last six months.
ccRCC patient samples
For performing microarray analysis, frozen tumor samples were obtained from the Hospital of the University of Pennsylvania, Philadelphia, PA. Samples were embedded in optimum cutting temperatures and sectioned for RNA extraction. The protocols used were approved by the Institutional Review Board of University of Pennsylvania.
Other information on methods is described in the Supplemental Data.
Supplementary Material
Significance.
HIF1α is constitutively expressed in a majority of VHL-deficient ccRCCs, despite its tumor suppressor activity in these malignancies. This study demonstrates that repression of miR-30c-2-3p/miR-30a-3p increases HIF2α levels to promote tumor growth, thereby ameliorating the inhibitory effects of HIF1α in ccRCCs.
Acknowledgements
We thank Hongwei Yu for histological preparations, Paul Hallberg for his assistance with cell sorting and the Simon lab for helpful discussions and comments. This work was funded by the Howard Hughes Medical Institute and NIH grant (CA104838). M.C.S is an investigator of the Howard Hughes Medical Institute.
Footnotes
Conflict of interest: The authors declare no competing financial interests
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