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. 2013 Mar 19;108(5):1133-42.
doi: 10.1038/bjc.2013.56. Epub 2013 Feb 28.

miR-210 is a target of hypoxia-inducible factors 1 and 2 in renal cancer, regulates ISCU and correlates with good prognosis

R I McCormick  1 , C Blick, J Ragoussis, J Schoedel, D R Mole, A C Young, P J Selby, R E Banks, A L Harris
Affiliations

miR-210 is a target of hypoxia-inducible factors 1 and 2 in renal cancer, regulates ISCU and correlates with good prognosis

R I McCormick et al. Br J Cancer. .

Abstract (VSports app下载)

Background: Clear cell renal cancer frequently harbours von Hippel-Lindau (VHL) gene mutations, leading to stabilisation of the hypoxia-inducible factors (HIFs) and expression of their target genes. We investigated HIF-1 and HIF-2 in the regulation of microRNA-210 (miR-210), and its clinical relevance in renal tumours. VSports手机版.

Methods: RCC4 and 786-O renal cancer cell lines transfected with either an empty vector or functional VHL and incubated in normoxia or hypoxia were examined for miR-210 expression. Hypoxia-inducible factor siRNAs were used to examine their regulation of miR-210. Seventy-one clear cell renal tumours were sequenced for VHL mutations. Expression of miR-210, VHL, CA9, ISCU and Ki-67 were determined by immunohistochemistry and qRT-PCR V体育安卓版. .

Results: In addition to HIF-1 regulating miR-210 in renal cancer, HIF-2 can regulate this microRNA in the absence of HIF-1. MicroRNA-210 is upregulated in renal cancer compared with normal renal cortex tissue. MicroRNA-210 correlates negatively with its gene target ISCU at the protein and mRNA level V体育ios版. MicroRNA-210 correlated with positive outcome variables and negatively with Ki-67. .

Conclusion: We provide further evidence of miR-210 activity in vivo, and show that high miR-210 expression is associated with better clinico-pathological prognostic factors VSports最新版本. .

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Figures

Figure 1
Figure 1
The hypoxic regulation of miR-210 in RCC4 cells. (A) Hypoxia-inducible factor-1α and -2α expression in RCC4 cells. RCC4 cells were transfected with an empty vector (VHL−) or vector coding for functional VHL (VHL+) and cultured in normoxia or 0.1% O2 for 24 h. Protein was immunoblotted on SDS–PAGE gels. (B) MicroRNA-210 is upregulated six-fold in hypoxia in RCC4 VHL+ cells, and constitutively expressed in VHL− cells. (C) MicroRNA-210 is upregulated in 786-O cells expressing HIF-1-only. 786-O VHL+ and VHL− cells were cultured in normoxia or 0.1% O2 for 24 h. Hypoxic upregulation of 2.5-fold was observed in VHL+ cells. VHL− cells showed constitutive upregulation of miR-210 in normoxia, with further upregulation in hypoxia. The degree of overexpression was lower than in RCC4 cells. *P<0.05, **P<0.01, unpaired t-test.
Figure 2
Figure 2
Hypoxia-inducible factor-2 mediates hypoxic miR-210 regulation in 786-O cells. (A) Validation of si-HIF-2α. 786-O VHL+ and VHL− cells were transfected with si-HIF-2α or a control molecule and incubated in normoxia or 0.1% O2 for 24 h. Hypoxia-inducible factor-2α was induced in hypoxia in the VHL+ cells, and constitutively expressed in the VHL− cells. Treatment with si-HIF-2α reduced expression to baseline. (B) MicroRNA-210 is regulated by HIF-2α in 786-O cells. 786-O cells were cultured as in the experiment in A. Hypoxic induction of miR-210 was almost completely reversed by si-HIF-2α in VHL+ cells. Constitutive overexpression of miR-210 in VHL− cells in normoxia was reduced to basal levels in VHL+ cells in normoxia. In hypoxia, si-HIF-2α only partially inhibited miR-210 expression. *P<0.05, **P<0.01, unpaired t-test.
Figure 3
Figure 3
CHIP-seq data for HIF-binding sites. A combination of chromatin immunoprecipitation of the HIF isoforms and high-throughput sequencing was used. 786-O cells showed strong HIF-2 binding at a HRE motif near the miR-210-coding sequence. MCF7 cells, which express functional HIF-1 and -2, demonstrate binding of both isoforms at the HRE near the miR-210-coding sequence.
Figure 4
Figure 4
Expression of mir-210 in renal cancer. (A) MicroRNA-210 expression was measured by qPCR in renal tumours and normal controls. MicroRNA-210 was overexpressed 9-fold in CCRCCs (N=43), 2.7-fold in papillary tumours (N=9), and was not upregulated in oncocytomas (N=4). *P<0.05 and ***P<0.001, Mann–Whitney test. (B) MicroRNA-210 expression in normal and CCRCC tissue in tumours with, and without, VHL mutation. In tumours with VHL mutations, there was a 16-fold upregulation of miR-210 compared with normal control tissue (N=49), ****P<0.0001.
Figure 5
Figure 5
Correlation of CA9 and VHL mRNA expression with miR-210 in renal tumours. MicroRNA-210 correlates positively with CA9 expression and negatively with VHL expression. Expression of mRNA in 43 tumours was measured by qRT–PCR. (A) CA9 correlates with miR-210 expression. R2=0.16, P<0.05, F-test. (B) VHL correlates negatively with miR-210 expression. R2=0.2, P<0.01, F-test (Fisher's linear regression).
Figure 6
Figure 6
Correlation of ISCU and miR-210 expression in renal tumours. ISCU is downregulated in tumours. (A) ISCU mRNA was downregulated two-fold in the tumours compared with the normal renal cortex. ***P<0.001, Mann–Whitney test. (B) ISCU protein expression was measured by IHC in renal TMAs, using a PIS system. Tumours expressed 40% less ISCU than the normal cortex. ***P<0.001, Unpaired t-test. (C) Tumours were separated into two groups, expressing low (PIS 0–3, N=23) or high (PIS 4–12, N=25) levels of ISCU protein. MicroRNA-210 was expressed 2.7-fold higher in the low ISCU-expressing group. ***P<0.001, Mann–Whitney test. (D) The CCRCCs expressed half the ISCU protein of the papillary tumours. *P<0.05, unpaired t-test.
Figure 7
Figure 7
MicroRNA-210 correlation with pathological and outcome variables. MicroRNA-210 is associated with lower tumour grade (A) and stage (B). (C) Patients grouped by CCRCC tumours of high grade (grade 1–2, N=24) or low grade (grade 3–4, N=49). MicroRNA-210 expression in high-grade tumours was 55% of that in the low-grade tumours. **P<0.01, Mann–Whitney test. (D) MicroRNA-210 expression in high-stage (stage 3–4, N=49) tumours was 74% of that in low-stage (stage 1–2, N=24) tumours. *P<0.05. (E) Patients were separated into three (N=23 for each group) groups based on miR-210 expression in their CCRCC tumours. High miR-210 associated with better survival. P=0.05, Gehan–Breslow–Wilcoxon test. (F) Tumours were separated into two groups based on high (N=20) and low (N=18) Ki-67 expression. MicroRNA-210 expression was 1.7-fold lower in the Ki-76-low group (P<0.05, Mann–Whitney test).
Figure 8
Figure 8
snoRNA controls and survival. (A–C) Survival of patients expressing snoRNA RNU44 (A), RNU48 (B) and RNU6B (C) genes >median or D) Patients expressing un-normalised miR-210 >median had improved survival post nephrectomy. P=0.005, log-rank test. Median survival miR-210 low 943 days, high 1959 days, HR 3.01, 95% CI 1.39–6.51.
Figure 9
Figure 9
Expression of snoRNAs related to clinical features. Whiskers=minimum to maximum, box 25th–75th percentile. (A) Controls in low- or high-grade tumours. Average CT in low grade=25.61 and high grade=25.99. (B) Control genes in low- or high-stage tumours. Average CT across all three controls in low stage=25.71 and high stage=25.77. (C) Controls in normal or tumour tissue. Average CT in normals=26.0 and tumours=25.7.
Figure 10
Figure 10
Expression of control genes in normal and CCRCC tissue. Whiskers=minimum to maximum, box 25th–75th percentile. Beta-actin CT in normal vs tumour=17.80 vs 17.02 (mean) and 17.73 vs 16.72 (median). GUSB CT in normal vs tumour=23.3 vs 21.15 (mean) and 23.12 vs 20.87 (median).
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