Regulation of HIF-1alpha and VEGF by miR-20b tunes tumor cells to adapt to the alteration of oxygen concentration - PubMed (original) (raw)

Regulation of HIF-1alpha and VEGF by miR-20b tunes tumor cells to adapt to the alteration of oxygen concentration

Zhang Lei et al. PLoS One. 2009.

Abstract

The regulation of HIF-1alpha is considered to be realized by pVHL-mediated ubiquitin-26S proteasome pathway at a post-transcriptional level. The discovery of a class of small noncoding RNAs, called microRNAs, implies alternative mechanism of regulation of HIF-1alpha. Here, we show that miR-20b plays an important role in fine-tuning the adaptation of tumor cells to oxygen concentration. The inhibition of miR-20b increased the protein levels of HIF-1alpha and VEGF in normoxic tumor cells; the increase of miR-20b in hypoxic tumor cells, nevertheless, decreased the protein levels of HIF-1alpha and VEGF. By using luciferase reporter vector system, we confirmed that miR-20b directly targeted the 3'UTR of Hif1a and Vegfa. On the other hand, the forced overexpression of HIF-1alpha in normoxic tumor cells downregulated miR-20b expression. However, HIF-1alpha knockdown in hypoxic tumor cells caused the increase of miR-20b. The differential expression of miR-20b has important biological significance in tumor cells, either enhancing the growth or favoring the survival of tumor cells upon the oxygen supply. Thus, we identify a novel molecular regulation mechanism through which miR-20b regulates HIF-1alpha and VEGF and is regulated by HIF-1alpha so to keep tumor cells adapting to different oxygen concentrations.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Expression of miR-20b is reversely correlated with HIF-1α level in tumor cells.

(A) The expressions of miR-18a, 155, 20b and 199b in tumor cell lines were detected by RT-PCR in normoxia (20% oxygen) or hypoxia (1% oxygen). (B) The detection of miR-20b in tumor cell lines by real time RT-PCR. The miR-20b level in the hypoxia groups was designated as 1. (C and D) The expression of HIF-1α in tumor cell lines was detected by Western blot (C) or real time RT-PCR (D). (E) Expressions of miR-20b and HIF-1α in different tumor regions. 1×105 H22 tumor cells were inoculated subcutaneously into mice. When tumor size was >9×9 mm, the central and marginal regions of tumor tissues were used for miR-20b and HIF-1α detection by RT-PCR and Western blot, respectively. Data from three tumor tissues were presented in this figure.

Figure 2

Figure 2. HIF-1α is targeted by miR-20b.

(A) miR-20b inhibitor increased HIF-1α protein in normoxic H22 cells. miR-20b inhibitor or control oligonucleotide was transfected into normoxic H22 cells. 48 hr later, the cells were used to detected HIF-1α and VHL proteins by Western blot. (B) miR-20b decreased HIF-1α protein in hypoxic H22 cells. miR-20b or control oligonucleotide was transfected into hypoxic H22 cells. 48 hr later, the cells were used to detect HIF-1α and VHL proteins by Western blot. (C) miR-20b inhibitor or miR-20b did not affect the mRNA levels of HIF-1α. The cells of above (A) and (B) were used for real time RT-PCR to detect HIF-1α mRNA expression. (D) miR-20b targeted 3′-UTR of HIF-1α mRNA. The specificity of miR-20b to 3′-UTR of HIF-1α mRNA was identified as described in Methods. *, P<0.01, compared with HIF-1α 3′-UTR group.

Figure 3

Figure 3. miR-20b is downregulated by HIF-1α.

(A and B) HIF-1α decreased miR-20b expression in normoxic H22 cells. HIF-1α vector or mock vector was transfected into normoxic H22 cells. 72 hr later, the cells were used for HIF-1α analysis by Western blot (A) and miR-20b analysis by RT-PCR and real time RT-PCR (B). (C and D) HIF-1α siRNA was transfected into hypoxic H22 cells. 72 hr later, the cells were used for HIF-1α analysis by Western blot (C) and miR-20b analysis by RT-PCR and real time RT-PCR (D). (E and F) VHL siRNA was transfected into normoxic H22 cells. 72 hr later, the cells were used for VHL and HIF-1α analysis by Western blot (E) and miR-20b analysis by RT-PCR and real time RT-PCR (F).

Figure 4

Figure 4. VEGF is targeted by miR-20b.

(A) miR-20b inhibitor increased VEGF protein in normoxic H22 cells. miR-20b inhibitor or control oligonucleotide was transfected into normoxic H22 cells. 48 hr later, the cells were used to detected VEGF expression by Western blot. (B) miR-20b decreased VEGF protein in hypoxic H22 cells. miR-20b or control oligonucleotide was transfected into hypoxic H22 cells. 48 hr later, the cells were used to detect VEGF expression by Western blot. (C) miR-20b inhibitor or miR-20b did not affect the mRNA levels of VEGF. The cells of above (A) and (B) were used for real time RT-PCR to detect VEGF mRNA expression. (D) miR-20b targeted 3′-UTR of VEGF mRNA. The specificity of miR-20b to 3′-UTR of VEGF mRNA was identified as described in Methods. *, P<0.01, compared with VEGF 3′-UTR group.

Figure 5

Figure 5. Differential expression of miR-20b affects different aspects of tumor cells.

(A) miR-20b was required for H22 cell growth. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA or miR-20b or VHL siRNA or control oligonucleotide for 24 hr. Then the cells were seeded in 96-well plate (5×103 per well) for 48 h in normoxia. The proliferation assay was performed with MTT Cell Proliferation Kit (Roche Diagnostics, IN) according to the manufacturer's instructions. *, P<0.05, compared with control. (B) Downregulation of miR-20b enhanced the resistance to apotosis. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA for 24 hr. Then the cells (1×106) were irradiated by UVB (200 J/m2) or treated with mitomycin C (MMC, 10 µg/ml)for 12 h. The cells were stained with PE-Annexin V and 7-AAD for apoptotic analysis by flow cytometry. *, P<0.05, compared with control. (C) Analysis of the mRNA expressions of Bcl-2, Bcl-xL, Bax and Bad genes. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA for 72 hr. The cells were used for the analysis of gene expression by real time RT-PCR. *, P<0.05, compared with control. (D) Normoxic H22 cells were transfected with miR-20b for 24 hr. Then the cells were cultured under hypoxic condition for 12 h, and stained with PE-Annexin V and 7-AAD for apoptotic analysis by flow cytometry. *, P<0.05, compared with control.

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