Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions

Abstract

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

Keywords: H19 gene; LncRNA; breast cancer; cancer stem cell; miR-675; tumoral progression.

Figure 1

H19 and miR-675 both promote cancer cell invasion in vivo. (A) Invasive capacities of MDA-MB-231 stably overexpressing H19 and the control, stained with lipophilic tracers, in transgenic zebrafish. Fluorescent pictures were captured using automated image acquisition software. (B) Invasive capacities of MDA-MB-231 stably overexpressing miR-675 and the control, stained with lipophilic tracers, in transgenic zebrafish. Fluorescent pictures were captured using automated image acquisition software. (C) Quantification of invasive cells per zebrafish. (D) mCherry protein fluorescence in SUM159PT transfected or not with pH19-mCherry plasmid. Fluorescence intensity is categorized in mCherry^neg and mCherry^high cellular subpopulations. Relative H19 expression in those subpopulations is figured. (E) Invasive capacities of mCherry^neg and mCherry^high cellular subpopulations, stained with lipophilic tracers, in transgenic zebrafish. Fluorescent pictures were captured using automated image acquisition software. (F) Quantification of invasive cells per zebrafish. For each experiment, forty embryos were used. *p < 0.05 and **p < 0.01.

Figure 2

H19 and miR-675 both promote breast cancer cell migrations. (A) Control (mock) or H19-stably overexpressing cells (H19) were cultured in Transwell for 24 h. Migrated cells were then incubated with 1-mM Hoescht 33258 and counted. (B) Migratory capacities of the control (siCtrl) and H19-knockdown cells (siH19) determined by Transwell assay. (C) MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675) were cultured in Transwell for 24 h. Migrated cells were then incubated with 1-mM Hoescht 33258 and counted. (D) Migratory capacities of miR-675-transfected cells (miR) or miR-675-specific inhibitor transfected cells (anti-miR) determined by Transwell assay. Results are presented as a percentage of the control. (E) Relative migratory capacities of SUM159PT-pH19-mCherry^neg versus SUM159PT-pH19-mCherry^high determined by Transwell assay. (F) Migratory capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the control. (G) Migratory capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the mCherry^neg condition. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 2

H19 and miR-675 both promote breast cancer cell migrations. (A) Control (mock) or H19-stably overexpressing cells (H19) were cultured in Transwell for 24 h. Migrated cells were then incubated with 1-mM Hoescht 33258 and counted. (B) Migratory capacities of the control (siCtrl) and H19-knockdown cells (siH19) determined by Transwell assay. (C) MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675) were cultured in Transwell for 24 h. Migrated cells were then incubated with 1-mM Hoescht 33258 and counted. (D) Migratory capacities of miR-675-transfected cells (miR) or miR-675-specific inhibitor transfected cells (anti-miR) determined by Transwell assay. Results are presented as a percentage of the control. (E) Relative migratory capacities of SUM159PT-pH19-mCherry^neg versus SUM159PT-pH19-mCherry^high determined by Transwell assay. (F) Migratory capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the control. (G) Migratory capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the mCherry^neg condition. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 3

H19 and miR-675 participate in the breast cancer cells invasion. (A) Control (mock) or H19-stably overexpressing cells (H19) were cultured in Transwell for 24 h. Invasive cells were then incubated with 1-mM Hoescht 33258 and counted. (B) Invasive capacities of the control (siCtrl) and H19-knockdowned cells (siH19) determined by Transwell assay. (C) MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675) were cultured in Transwell for 24 h. Invasive cells were then incubated with 1-mM Hoescht 33258 and counted. (D) Invasive capacities of miR-675-transfected cells (miR) or miR-675-specific inhibitor transfected cells (anti-miR) determined by Transwell assay. Results are presented as the percentage of the control. (E) Relative invasive capacities of SUM159PT-pH19-mCherry^neg versus SUM159PT-pH19-mCherry^high determined by Transwell assay. (F) Invasive capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as the percentage of the control. (G) Invasive capacities of miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the mCherry^neg condition. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 4

The effects of H19 and miR-675 on the expressions of the epithelial-to-mesenchymal transition (EMT) markers. (A) EMT protein expressions in the MCF-7 control (mock) or H19-stably overexpressing cells (H19), determined by Western blot analysis. (B) EMT protein expressions in SUM159PT-pH19-mCherry^neg (mCherry^neg) or SUM159PT-pH19-mCherry^high (mCherry^high), determined by Western blot analysis. (C) EMT protein expressions in the MDA-MB-231 control (mock) or H19-stably overexpressing cells (H19), determined by Western blot analysis. (D) EMT protein expressions in the MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675), determined by Western blot analysis. For each panel, actin was used as the equi-loading control. The relative signal intensities were quantified by ImageJ and shown above the protein bands for the representative experiment figure. The quantification of the triplicate is figured in the graph beside. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant. Uncropped blots are shown in Figures S1–S22.

Figure 4

The effects of H19 and miR-675 on the expressions of the epithelial-to-mesenchymal transition (EMT) markers. (A) EMT protein expressions in the MCF-7 control (mock) or H19-stably overexpressing cells (H19), determined by Western blot analysis. (B) EMT protein expressions in SUM159PT-pH19-mCherry^neg (mCherry^neg) or SUM159PT-pH19-mCherry^high (mCherry^high), determined by Western blot analysis. (C) EMT protein expressions in the MDA-MB-231 control (mock) or H19-stably overexpressing cells (H19), determined by Western blot analysis. (D) EMT protein expressions in the MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675), determined by Western blot analysis. For each panel, actin was used as the equi-loading control. The relative signal intensities were quantified by ImageJ and shown above the protein bands for the representative experiment figure. The quantification of the triplicate is figured in the graph beside. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant. Uncropped blots are shown in Figures S1–S22.

Figure 5

Effects of H19 and miR-675 in colony formations. (A) The clonogenic capacities of the control (mock) or H19-overexpressing cells (H19). (B) The relative clonogenic capacities of SUM159PT-pH19-mCherry^neg versus SUM159PT-pH19-mCherry^high. (C) The clonogenic capacities of the control (siCtrl) and H19-knockdown cells (siH19). (D) The clonogenic capacities of the MDA-MB-231 control (mock) or miR-675-overexpressing cells (miR-675). (E) The clonogenic capacities of the miR-675-transfected cells (miR) or miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the control. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 6

The expression of H19, miR-675 and different stem cell markers. (A) The expression of the H19 gene in mammary tumors expressing or not stem cell signatures. The table represents the H19 gene expression dependent on the gene signature of the tumors. Two gene signatures were used, one obtained in cell expressing ALDHA1 and the other in CD44₊/CD24^-. (B) The relative expression of H19, Sox2, Oct3/4, Notch1, Nanog, Abcg2, Aldh1a1 and Aldh1a3 genes and miR-675 in MCF-7, MDA-MB-231 and SUM159PT cells. The expression levels were related to the expression levels in hTERT cells indexed to 1. For panel A, a Fisher’s exact test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 7

H19 and miR-675 enhance the sphere formation of breast cancer cells. (A) Sphere-forming capacities of the control (mock) or H19-stably overexpressing cells (H19). (B) The relative sphere-forming capacities of SUM159PT-pH19-mCherry^neg versus SUM159PT-pH19-mCherry^high. (C) The sphere-forming capacities of the control (siCtrl) and H19-knockdown cells (siH19). (D) The sphere-forming capacities of the MDA-MB-231 control (mock) or miR-675-stably overexpressing cells (miR-675). (E) The sphere-forming capacities of miR-675-transfected cells (miR) or miR-675-specific inhibitor transfected cells (anti-miR). Results are presented as a percentage of the control. (F) The sphere-forming capacities of MCF-7 H19-stably overexpressing cells transfected with miR-675 (miR) or the miR-675-specific inhibitor (anti-miR). Results are presented as a percentage of the control. Representative pictures for each condition are shown. (G) The sphere-forming capacities of MDA-MB-231 H19-stably overexpressing cells transfected with miR-675 (miR) or the miR-675-specific inhibitor (anti-miR). Results are presented as a percentage of the control. Representative pictures of each condition are shown. (H) ALDEFLUOR-positive subpopulations defined by the ALDEFLUOR assay in H19-knockdown cells (siH19), miR-675-transfected cells (miR) or miR-675-knockdown cells (anti-miR). Results are presented as a percentage of the ALDEFLUOR-positive subpopulation in the native cells. (I) The expression levels of H19 and miR-675 in the cell lines cultured in sphere-forming conditions versus the same cell lines cultured in 2D conditions. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 8

The relative contribution of long non-coding (lnc)RNA H19 and its miR-675 in breast cancer progression.