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Results: 8

1.
Figure 2

Figure 2. Characterization of ERα, ERK2, and pSer5 RNA Pol II recruitment to the regulatory regions of miRNA genes. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

ERα (panel A) and ERK2 (panel B) recruitment to the overlapping binding sites of hsa-miR-196a2, hsa-miR-135a2, hsa-miR-944, and hsa-miR-101-1 genes and recruitment of phospho-serine5 RNA Polymerase II to the transcription start site (TSS) of the respective miRNA genes (panel C) after cell treatment with 10 nM E2 for 0, 45, or 120 min.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
2.
Figure 4

Figure 4. Anti-miR-196a* reduces expression of hsa-miR-196a2* and increases expression of TP63 and proliferation of MCF-7 and BT474 cells. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) MCF-7 cells were transfected with Anti-miR-196a* or negative control (Ctrl) and treated with 0.1% ethanol (veh) or 10 nM E2 for 24 h. Total RNA was harvested and expression level of hsa-miR-196a2* was determined using Taqman probe based quantitative PCR. (B) Impact of anti-miR-196a* on the expression of TP63 in MCF-7 cells treated with 0.1% ethanol (veh) or 10 nM E2 for 24 h. * p-value < 0.05. (C) Impact of anti-miR-196a* on MCF-7 cell proliferation. Cells were transfected with 20 nM anti-miR-196a* and then were treated with control 0.1% ethanol (veh) or 10 nM E2 for 4 days. Cell proliferation was assessed using WST-1 reagent. ***, p-value < 0.001; ****, p-value < 0.0001. (D) Impact of anti-miR-196a* on BT474 cell proliferation. Cells were transfected with 20 nM anti-miR-196a* and then were treated with control 0.1% ethanol (veh) or 10 nM E2 for 4 days. Cell proliferation was assessed using WST-1 reagent. *, p-value <0.1; ***, p-value < 0.001; ****, p-value < 0.0001.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
3.
Figure 1

Figure 1. Association of ERα and ERK2 binding sites with miRNA gene promoters and impact of ERα or ERK2 knockdown on miRNA regulation by estradiol (E2). From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) ERα and ERK2 binding sites are shown near four E2 and ERK2 regulated miRNA genes. Shown are the binding sites at the hsa-miR-196a2, hsa-miR-135a2, hsa-miR-944 and hsa-miR-101-1 locus. (B) Impact of ERα or ERK2 knockdown on expression of these miRNAs that harbor overlapping ERα and ERK2 binding sites. MCF-7 cells were transfected with siCtrl, siERα or siERK2 and treated with 0.1% ethanol (veh) or 10 nM E2 for 6 h and expression levels of each miRNA were determined from our miRNA microarray analysis. miRs are arranged from left to right based on magnitude of regulation by E2.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
4.
Figure 5

Figure 5. Expression of pre-miR-196a* greatly reduces TP63 expression and MCF-7 cell proliferation and these are reversed by TP63 target protector. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) Impact of TP63 target protector and pre-miR-196a* on TP63 mRNA. MCF-7 cells were transfected with 100 nM TP63 miScript target protector or negative control protector (Ctrl) with or without 10 nM pre-miR-196a*. After 48 h, cells were treated with 0.1% ethanol (veh) or 10 nM E2 for 24 h and RNA was then isolated for gene expression analysis. ** p-value < 0.01. (B) Impact of TP63 target protector and pre-miR-196a* on MCF-7 cell proliferation. Cells were transfected with 100 nM TP63 miScript target protector or negative control protector (Ctrl) with or without 10 nM pre-miR-196a*. Cells were then grown for 4 days and proliferation was assessed using WST-1 reagent. ** p-value < 0.01. (C) Effect of siTP63 treatment on TP63 mRNA level in cells treated with 0.1% ethanol (veh) or 10 nM E2 for 24 h. (D) Impact of TP63 knockdown on MCF-7 cell proliferation. Cells were transfected with 20 nM siGENOME Ctrl (siCtrl) or siTP63 and then were treated with 0.1% ethanol or 10 nM E2 for 4 days and cell proliferation was monitored. ** p-value < 0.01.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
5.
Figure 8

Figure 8. Model schematically depicting the effect of estradiol (E2), ERα, and ERK2 on hsa-miR-196a2* and TP63 regulation and their impact on breast cancer cell phenotypic properties. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

ERα and ERK2 up-regulate hsa-miR-196a2* upon cell treatment with E2, resulting in the down-regulation of TP63, with consequent decrease in cell proliferation. Although E2-ERα, in collaboration with ERK2, is overall pro-proliferative in ERα-positive breast cancer cells, this model, based upon our findings, reveals that E2-ERα-ERK2 does regulate a pathway that has a net antiproliferative and tumor-suppressive effect. Potential modulation of this hsa-miR-196a2*-TP63 circuit might be capable of reducing the growth stimulatory actions of estrogens in ERα-positive breast cancer. Our findings also suggest that enhancement of this axis in ERα-negative breast cancers by alteration in miR-196a2* expression might also suppress cancer cell invasiveness and the aggressiveness of these breast cancers in a manner independent of ERα.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
6.
Figure 7

Figure 7. Regulation of cell proliferation, colony formation, and invasion by hsa-miR-196a2* and effect of TP63 target protector in three ER-negative breast cancer cell lines (MDA-MB-453, MDA-MB-468 and SKBR3). From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) Impact of pre-miR-196a* overexpression and TP63 target protector on cell proliferation. Cells were transfected with negative control pre-miR (Ctrl), 10 nM pre-miR-196a* alone or 10 nM pre-miR-196a* with 100 nM TP63 miScript target protector, and then were grown for 4 days. Cell proliferation was assessed using WST-1 reagent. **** p-value < 0.0001. (B) Cells were transfected with negative control pre-miR (Ctrl), 10 nM pre-miR-196a* alone or 10 nM pre-miR-196a* with 100 nM TP63 miScript target protector as indicated, and were then analyzed in in vitro invasion assays. *, p-value <0.1; **, p-value < 0.01; ***, p-value < 0.001. (C) Cells were transfected with negative control pre-miR (Ctrl), 10 nM pre-miR-196a* alone or 10 nM pre-miR-196a* with 100 nM TP63 miScript target protector and then analyzed for soft agar colony formation. Images are at 4x magnification. Colony numbers per field were determined. Colony size was measured using ImageJ software. *, p-value <0.1; **, p-value < 0.01; ***, p-value < 0.001.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
7.
Figure 3

Figure 3. Relative levels of hsa-miR-196a2* and hsa-miR-196a2 in MCF-7 cells; time course of the E2 stimulated increase in hsa-miR-196a2* and the requirement for ERα and ERK2; prediction of miR-196a2* target genes; and impact of ERα and ERK2 knockdown on target gene expression. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) Relative levels of hsa-miR-196a2* and hsa-miR-196a2 in control MCF-7 cells. (B) Time course monitoring changes in the level of hsa-miR-196a2* and hsa-miR-196a2 after treatment with 10 nM E2 for the times indicated. (C) Impact of ERα or ERK2 knockdown on the response of hsa-miR-196a2* to E2 treatment. MCF-7 cells were transfected with siCtrl, siERα or siERK2 for 48 h and then treated with 0.1% ethanol (veh) or 10 nM E2 for the indicated times. Total RNA was harvested and the expression level of hsa-miR-196a2* was determined by quantitative RT-PCR. (D) List of E2 down-regulated genes that are potential targets of hsa-miR-196a2*. (E) Effect of ERα or ERK2 knockdown on expression of three potential target mRNAs of hsa-miR-196a2*, TP63, SPRY1, and TFAP2. MCF-7 cells were transfected with siCtrl, siERα or siERK2 for 48 h and then treated with 0.1% ethanol (veh) or 10 nM E2 for 24 h. (F) Effect of siERα or siERK2 treatment on the level of TP63 (ΔNp63α) protein, and on ERα and ERK2 and pERK1/2 protein levels monitored by western blot analysis. Cells were treated as described in panel E. β-actin was used as a loading control.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.
8.
Figure 6

Figure 6. TP63, hsa-miR-196a, and ERα expression in human breast tumors, and TP63-mediated regulation of cell growth and invasion by hsa-miR-196a2* overexpression and effect of TP63 target protector in MDA-MB-231 cells. From: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties.

(A) Expression of the miRNAs, hsa-miR-196a1 and a2 (representing the same miR sequence but from a gene on a different chromosome) and hsa-miR-196a2* (complementary to the a2) and TP63 mRNA expression in human breast tumors. Analyses were performed by us on datasets of miRNA and mRNA sequencing in 132 human breast tumors from [47] as described in Methods. Tumor data for ERα, PGR, HER2, and potential hsa-miR-196a2* target genes (TFAP2A, SPRY1, TP63, NEUROD1, MGAT4A, and IGF1) are also shown in this Tree View Java hierarchical clustering analysis. (B) Relative levels of TP63 ΔNp63α protein in MCF-7 cells, MDA-MB-231 cells and MDA-MB-231cells stably containing ERα. (C) Relative levels of hsa-miR-196a2* in MCF-7 cells, MDA-MB-231 cells, and MDA-MB-231 ERα-positive cells. (D) Impact of pre-miR-196a* overexpression and TP63 protector on MDA-MB-231 cell proliferation. Cells were transfected with 100 nM TP63 miScript target protector or negative control protector (Ctrl) with or without 10 nM pre-miR-196a*, or cells were transfected with pre-miR-196a* alone, and then were grown for 4 days. Cell proliferation was assessed using WST-1 reagent. ** p-value < 0.01. (E) MDA-MB-231 cells were transfected with pre-miR-196a* (or negative control, Ctrl), or with target protector oligonucleotides or siTP63 alone or together as indicated, and were then analyzed in in vitro invasion assays. ** p-value < 0.01. Western blots shown at right indicate effects of these treatments on ΔNp63α protein level. Total ERK2 was monitored as the internal reference loading protein. (F) MDA-MB-231 cells were transfected with negative control pre-miR (Ctrl), pre-miR-196a* or pre-miR-196a* and TP63 target protector and then analyzed for soft agar colony formation. Images are at 4x magnification. Colony numbers per field were determined. Colony size was measured using ImageJ software. * p-value < 0.1; **, p-value < 0.01.

Kyuri Kim, et al. Horm Cancer. ;4(2):78-91.

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