HIF-inducible miR-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment - PubMed (original) (raw)
HIF-inducible miR-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment
Neha Nagpal et al. Sci Rep. 2015.
Abstract
The molecular mechanisms of hypoxia induced breast cell migration remain incompletely understood. Our results show that hypoxia through hypoxia-inducible factor (HIF) brings about a time-dependent increase in the level of an oncogenic microRNA, miR-191 in various breast cancer cell lines. miR-191 enhances breast cancer aggressiveness by promoting cell proliferation, migration and survival under hypoxia. We further established that miR-191 is a critical regulator of transforming growth factor beta (TGFβ)-signaling and promotes cell migration by inducing TGFβ2 expression under hypoxia through direct binding and indirectly by regulating levels of a RNA binding protein, human antigen R (HuR). The levels of several TGFβ pathway genes (like VEGFA, SMAD3, CTGF and BMP4) were found to be higher in miR-191 overexpressing cells. Lastly, anti-miR-191 treatment given to breast tumor spheroids led to drastic reduction in spheroid tumor volume. This stands as a first report of identification of a microRNA mediator that links hypoxia and the TGFβ signaling pathways, both of which are involved in regulation of breast cancer metastasis. Together, our results show a critical role of miR-191 in hypoxia-induced cancer progression and suggest that miR-191 inhibition may offer a novel therapy for hypoxic breast tumors.
Figures
Figure 1. Transcriptional regulation of miR-191 in hypoxic microenvironment.
(a). Stem loop qRT-PCR data showing relative level of miR-191 in breast cancer cell lines (MCF7, T47D, & MM231) exposed to different pO2 (0.2%, 0.5%, 1% & 21%) for 48 hrs. (b). Stem loop qRT-PCR data showing time dependent induction of miR-191 levels in response to 0.2% hypoxia treatment in MCF7, T47D, & MM231 cells. (c, d). Effect of HIF overexpression (HIF-1α or HIF-2α) or inhibition (shHIF1α or shHIF2α) in normoxic (c) or hypoxic (d) conditions on the levels of miR-191 in a panel of breast cancer cell lines (MCF7, T47D, & MM231). The graphical data points represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01, *^P > 0.05 < 0.1). Error bars denote ± SD.
Figure 2. Transcriptional regulation of miR-191 is HIF dependent.
(a). Picture Diagram showing six potential hypoxia response elements (HRE) upstream of pre-miR-191 sequence. (b, c). The functionality of the putative hypoxia response elements (H2 & H3) was confirmed through dual luciferase assay in response to hypoxia (b) or HIF stimulation/inhibition (c). Graph represents relative luciferase activity. (d). MCF7 cells were transfected with HA tagged constructs of HIF 1/2-α and exposed to hypoxia for 24 hr. CHIP assay was performed using antibodies against IgG or HA tag to validate the binding of HIF 1/2-α on the putative HREs in the promoter of miR-191. Bar graph represents fold enrichment of bound chromatin compared with input chromatin. (e, f). Cells were exposed to normoxia or hypoxia for 24 hr. CHIP assay was performed using antibodies against IgG or HIF-α. Bar graph represents fold enrichment of bound chromatin compared with input chromatin in MCF7 (e) and MM231 (f) cell lines. Ctrl refers to region lacking in HIF-binding sites. The graphical data points in b-f represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01, *^P > 0.05 < 0.1). Error bars denote ± SD.
Figure 3. miR-191 promotes breast cancer aggressiveness.
(a–f). Levels of miR-191 were modulated (overexpressed-Pre191 or inhibited-Anti191) in hypoxic microenvironment in a panel of breast cancer cell lines (MCF7, T47D & MM231) and various cellular effects were studied. Bar graphs represent effect of miR-191 level modulation on (a) Cell proliferation using MTT assay (b) Migration using wound healing assay (c) Migration using Boyden chamber assay (d) Invasion using Boyden chamber assay (e) Cell death using Trypan blue assay in reponse to chemotherapeutic drug (doxorubicin) or (f) Raditaion treatment.(gamma rays-2 Gray) (g). Soft Agar assay of cells transiently transfected with pre or anti-miR-191 with respective controls (n = 3) and cells were allowed to grow in hypoxic microenvironment. (h, i). Cell cycle analysis was performed with serum starvation synchronized MCF7 cells followed by differential modulation of miR-191 levels and exposure to hypoxic microenvironment. FACS analysis shows reduction in cells in G0/G1 phase while increase in cells in G2/M phase on miR-191 overexpression (h) while inhibition of miR-191 shows opposite (i). (j). The cells were transfected with miR-191 mimic or inhibitor with respective controls and exposed to hypoxia for 24 hrs. Cells were then stained with JC-1 for 15 min at RT. Mitochondrial membrane potential was measured as a ratio of red (JC-1 aggregates) to green (monomers) fluorescence intensity. Bar graph represents relative fold change as compared to control. The graphical data points represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01). Error bars denote ± SD.
Figure 4. miR-191 promotes TGFβ-signaling in hypoxic microenvironment.
(a, b). Bar graph represents relative luciferase activity of p3TP-Lux reporter plasmid on overexpression/inhibition of miR-191 under hypoxic (a) and normoxic conditions (b). (c–e). qRT-PCR data showing expression level of genes belonging to TGFβ-pathway in response to miR-191 overexpression/inhibition in hypoxic microenvironment in a panel of breast cancer cell lines- MCF7 (c), T47D (d), MM231 (e). The graphical data points in a-e represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01). Error bars denote ± SD.
Figure 5. TGFβ2 & HuR: bonafide targets of miR-191 in hypoxic microenvironment.
(a). The MCF7 cells with differential level of miR-191 were exposed to hypoxic microenvironment and analyzed for expression of TGFβ2 protein by western blotting. (b). Diagram showing wild type/mutated miR-191 binding site in TGFβ2-3′UTR. (c, d). Graph showing 3′UTR luciferase activity of TGFβ2-3′UTR luciferase constructs bearing wild type (c) or mutated (d) miR-191 binding site, in response to differential expression of miR-191 in hypoxic microenvironment. (e). The MCF7 cells with differential level of miR-191 were exposed to hypoxic microenvironment and analyzed for expression of HuR protein by western blotting. (f). Diagram showing miR-191 binding site (HuR B1, wild type as well as mutated) in the 3′UTR of HuR that were subsequently cloned and scored for luciferase activity. (g). qRT-PCR data showing that miR-191 overexpression (Pre191) leads to targeted downregulation of HuR at the transcript level while its inhibition (Anti191) leads to the opposite. (h). Bar graph showing relative luciferase activity of HuR-3′UTR luciferase construct in response to miR-191 level modulation. The graphical data points in c, d, g, h represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01). Error bars denote ± SD.
Figure 6. Targeted downregulation of HuR plays an important role in miR-191 mediated TGFβ2 induction in hypoxic microenvironment.
(a). Effect of HuR on the level of TGFβ2 transcript in hypoxic microenvironment. Overexpression of HuR led to reduction in level of TGFβ2 mRNA while Silencing the HuR (esiHuR) led to significant induction in level of TGFβ2 mRNA. (b). Interaction profile of HuR protein with TGFβ2 3′UTR. The cloned 3′UTR region of TGFβ2 encompassing the binding sites of miR-191 and HuR is highlighted. The region was considered for determining the combined effect of both miR-191 and HuR in the regulation of TGFβ2. (c). qRT-PCR data showing regulation of TGFβ2 levels in MCF7 cells in response to overexpression of miR-191 or HuR or both. (d, e). Graph showing luciferase activity of TGFβ2-wild type (d) or mutated 3′UTR (e) in response to overexpression of miR-191 or HuR or both in MCF7 cells. Results show that miR-191 mediated induction of TGFβ2 was partially reduced in presence of HuR overexpression. The graphical data points in a & c-e represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01, *^P > 0.05 < 0.1). Error bars denote ± SD.
Figure 7. TGFβ2 is a critical mediator of miR-191 induced breast cancer migration under hypoxia.
(a, b). Effect of miR-191 and TGFβ2 on migration under hypoxia. (a, b) The cells were transfected with control miRNA or miR-191mimic along with TGFβ2 silencing using esiTGFβ2 and migration was quantitated using Boyden chamber assay (a) or wound healing assay (b). The results from the three cell lines show that miR-191 mediated increase in migration is abolished with the TGFβ2 silencing. (c, d) The cells were transfected with control miRNA or anti-miR-191 along with TGFβ2 overexpression and migration was quantitated using Boyden chamber assay (c) or wound healing assay (d). The results show that the decrease in migration by inhibition of miR-191 was reduced when TGFβ2 was coexpressed. (e). miR-191 induced migration is SMAD3 dependent as reduction in migration was observed on miR-191 overexpression along with the inhibition of SMAD3. (f). A proposed model detailing hypoxic regulation of miR-191 along with its functional impact on HIF & TGFβ-pathways and further implication in regulation of migration of hypoxic breast cancer. The graphical data points in a-e represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01). Error bars denote ± SD.
Figure 8. Effects of anti-miR-191 treatment in breast cancer cell 3D tumor spheroids.
(a). MTT assay results showing difference in cell proliferation in 3D tumor spheroids generated from cells with differential level of miR-191. (b). qRT-PCR data showing TGFβ2 transcript level in the 3D tumor spheroids generated from cells with differential miR-191 levels. Significant induction in the level of transcripts was observed (at day 5/day 1) in the multicellular spheroids generated from cells with transient overexpression of miR-191. (c). Graph showing difference in volume of the tumor spheroids in response to anti-miR-191 treatment as compared to that of the control miRNA. (d). Graph showing results of MTT assay as a measure of cell proliferation of MCF7 spheroids in response to anti-miR-191 treatment. The graphical data points in a–d represent mean ± S.D of at least three independent experiments. (*P < 0.05, **P < 0.01). Error bars denote ± SD.
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