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Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer - PubMed

  • ️Mon Jan 01 2018

Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer

Neha Nagpal et al. Sci Rep. 2018.

Abstract

Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs (miR-100-5p, -191-5p, -193b-3p, -205-5p, -326, -422a and -425-5p) were found to be regulated by MED1. MED1 induces miR-191/425 cluster in an estrogen receptor-alpha (ER-α) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-α-dependent and ER-α-induced expression of these miRNAs is MED1-dependent. MED1 mediates induction of cell proliferation and migration and the genes associated with it (JUN, FOS, EGFR, VEGF, MMP1, and ERBB4) in breast cancer, which is abrogated when used together with miR-191-inhibition. Additionally, we show that MED1 also regulates the levels of direct miR-191 target genes such as SATB1, CDK6 and BDNF. Overall, the results show that MED1/ER-α/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

MED1 alterations in breast cancer patients. (A,B) Genetic and transcriptomic alterations of MED1 in breast cancer patients. MED1 copy number variations and expression alterations were found in breast cancer patients from TCGA database (A). Copy number variations of MED1 locus are correlated to variations in MED1 mRNA expression levels (B). (C) MED1 expression is significantly associated to survival in breast cancer patients. Survival data were obtained from TCGA data portal for two datasets of BC patients analysed with microarray (top graphs) or RNA sequencing (bottom graphs). Both datasets confirmed a significant positive association between MED1 expression and Overall Survival in ER+ BC patients.

Figure 2
Figure 2

MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression (A) or downregulation (B) was then checked by qRT-PCR and compared to that of their respective controls in MCF7 cells. (C,D) MED1 was overexpressed (C) or inhibited (D) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small RNA. The graphical data points represent mean

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S.D of at least three independent experiments (**P<0.05, *P<0.1). Error bars denote

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SD.

Figure 3
Figure 3

Effect of ER-α and MED1 on transcriptional regulation of miR-191/425 cluster. (A–C) MED1 or ER-α were overexpressed or inhibited and stem loop qRT-PCR was done to check the levels of miR-191 and miR-425 (A). Estrogen (E2) or vehicle (ethanol, Eth) treatment was given along with esiMED1 or tamoxifen (Tm) and the effect on miR-191/425 was observed using stem loop qRT-PCR. (B,C) The data was normalized to U6 small RNA (RNU6B) using 2−ΔCt method. (D) Levels of MED1 were transiently modulated using pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) in a panel of ER+ve/-ve breast cancer cell lines (MCF7, ZR75-1, MDA-MB-231) and effect on miR-191/425 levels was tested using stem-loop qRT PCR. Induced levels of both the cluster miRNAs (miR-191 and miR-425) were observed in response to MED1 overexpression in ER+ve cell lines (MCF7, ZR75-1) while opposite results were observed for ER-ve cell line (MDA-MB-231). Thereby confirming that MED1 mediated induction of miR-191/425 occurs in an ER-dependent manner. The graphical data points represent mean

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S.D of at least three independent experiments (**P<0.05). Error bars denote

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SD.

Figure 4
Figure 4

ERE present upstream of miR-191/425 cluster is responsive to ER-α or MED1 overexpression. (A) Diagram showing ERE consensus sequences in region upstream of pre-miR-191 using Promo 3.0 software. The locations of the two regions (ERE-A and ERE-B) used for luciferase promoter assays are marked. (B) Graph showing luciferase activity of ERE-A luciferase construct of miR-191/425 cluster in response to ER-α or MED1 overexpression. (C,D) Estrogen or ER-α mediated effect on luciferase activity of ERE-A construct when coupled with MED1 inhibition. Levels of MED1 were modulated (using esiMED1/esiCtrl) with/without ER-α overexpression or estrogen stimulation (10−9 M E2; estrogen or 10−9 M Eth; ethanol) and effect on luciferase activity of ERE-A (luciferase constructs of miR-191/425 cluster) was observed. Increase in luciferase activity in response to ER-α (C) or estrogen treatment (D) was not observed when estrogen stimulation was given along with inhibition of MED1. (E) Diagram showing wild type/mutated ERE-A region upstream of miR-191/425 cluster. (F,G) Graph showing promoter luciferase activity of ERE-A mut (luciferase constructs bearing mutated ERE site), in response to differential expression of ER-α and MED1 alone (F) or in combination (G). The graphical data points represent mean

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S.D of at least three independent experiments (**P<0.05). Error bars denote

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SD.

Figure 5
Figure 5

Recruitment of MED1/ER-α on the miR-191/425 promoter ERE. (A,B) qPCR (A) or semi-quantitaive (B) data of CHIP assay showing % input of bound chromatin on ERE-A element using antibody specific to MED1/ER-α. (C,D) qPCR (C) or semi-quantitative (D) data of CHIP assay shows recruitment of both ER-α and MED1 on ERE-A in response to ER-α inhibition using specific siRNA. (E) Estrogen dependent MED1 and ER-α interaction in MCF7 cells. MCF7 cells were starved for estrogen for 3-4 days followed by estrogen treatment (0–10 nM) for 45 min. The cells were lysed and probed for the interaction of ER-α and MED1 using co-immunoprecipitation assay. ER-α antibody was used for pull down and western blotting was done with MED1 specific antibody. (F,G) CHIP assay was performed using ER-α or MED1 antibodies in response to 10−8 M estrogen (E2) or ethanol (Eth) treatment for 2 hrs with/without inhibition of MED1. qPCR (F) or semi-quantitative (G) data shows PCR based quantification of bound chromatin (% input) on ERE-A (element present upstream of miR-191/425 cluster) in MCF7 cells. (**P<0.05). Error bars denote

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SD.

Figure 6
Figure 6

miR-191 acts as the downstream effecter of MED1 mediated cellular functions. (A–C) Functional implications of MED1 or miR-191 overexpression in breast cancer. MCF7 cells were transfected with MED1 or miR-191 or PC (control vector) and MTT assay was performed (48 hrs post transfection) to measure effect on cell survival. The results show that both MED1 and miR-191 promote cell survival (A). (B,C) Wound healing assay was performed to observe the effect of miR-191 and MED1 on cell migration. MCF7 cells were transfected with MED1 or miR-191 or control vector and gap or wound closure was observed (B) using Nikon microscope, 10X magnification and % gap closure was quantified using arbitrary scale (C). Graph shows that more healing (gap closure) was observed in response to both miR-191 and MED1 overexpression, hence both MED1 and miR-191 impart enhanced cell migratory capacity to the cells. (D–F) MED1 mediated cellular effects are miR-191 dependent. MCF7 cells were transiently transfected with MED1/PC (vector control) along with inhibition of miR-191(anti-miR-191)/Ctrl and effect on cell survival was looked for using MTT assay (D). Increase in cell survival due to MED1 overexpression was significantly reduced when miR-191 was inhibited along with MED1 overexpression (E). Wound healing assay was performed to confirm the effect of MED1 and miR-191 on cell migration. Levels of MED1 were transiently overexpressed along with/without miR-191 inhibition (using anti-miR-191/ctrl) and gap closure was observed after 24 hrs. Comparatively, lesser gap was filled when MED1 overexpression was coupled with miR-191 inhibition (F) when quantified using arbitrary scale thereby, confirming that miR-191 is a downstream effecter for MED1 mediated cellular migration. (G) qRT-PCR data showing transcript levels of JUN, FOS, EGFR, VEGF, MMP1 and ERBB4 on miR-191 or MED1 overexpression/inhibition. GAPDH has been used for normalization of qRT-PCR data. (H) qRT-PCR data showing transcript levels of JUN, FOS, EGFR, VEGF, MMP1 and ERBB4 on MED1 overexpression or miR-191 inhibition. (I) qRT-PCR data showing transcript levels of established miR-191-target genes (SATB1, CDK6 and BDNF) in response to both miR-191 or MED1 overexpression/inhibition. GAPDH has been used for normalization of qRT-PCR data. (J) qRT-PCR data showing transcript levels of established miR-191-target genes (SATB1, CDK6 and BDNF) on MED1 overexpression or miR-191 inhibition. (**P<0.05, (**P<0.05, * P<0.1). Error bars denote

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SD.

Figure 7
Figure 7

A proposed model detailing interplay of ER-α-MED1-miR-191/425 in breast cancer. A model for estrogen/ER-α induced MED1 mediated miR-191/425 regulation in breast cancer.

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