Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins

Abstract

Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway.

Keywords: Akt; PI3K; PTEN; mTOR; miR301.

Figure 1. Effect of miR301 on PI3K-Akt pathway

A. In order to study changes in miR levels upon Akt upregulation cells were transiently transfected with Akt. Western blot confirms overexpression of Akt as compared to empty plasmid (control) in MCF7 and in MDAMB468 cells. B. Quantitative RT-PCR shows significant downregulation of miR301 expression mediated by transient transfection with Akt construct, as compared to cells transfected with empty vector, in MCF7 and MDAMB468 cells C. Quantitative RT-PCR shows significant downregulation miR301-expressions by Akt overexpression, even more downregulation by miR301 inhibitor and upregulation by miR301-mimic, as compared to Ctrl (negative control) miR in breast cancer cells. D. Co-transfection of Akt construct with miR301-modulators and Akt alone shows downregulation of miR301 expression in comparison with the control (empty plasmid and Ctrl miR). Since the effect is observed regardless of use of miR301-inhibitor or -mimics, it is likely due to Akt-overexpression, and confirms our results from (A). Akt transfection with miR301-inhibitor showed strong downregulation of miR301 expression compared to control (* p < 0.05).

Figure 2. Akt mediated increase in cell survival and migration upon down-regulation of miR301

A. MTT-assay was performed to measure the cell survival upon downregulation of the miR301 with Akt. MCF7 and MDAMB468 breast cancer cells transiently transfected with miR301 inhibitor in the presence of Akt shows a significant increase in cell survival compared to control. B. Scratch assay was preformed to assess cell migration upon inhibition of miR301 in Akt-overexpressing cells. 72h post transfection, the cell migration was highely pronounced in cells co-expressing miR301 inhibitor and Akt, as compared to control, in MCF7 cells (* p < 0.05). C. Quantitative assessment of scratch shows significantly higher wound closure at 72h post transfection, upon miR301 inhibition in the presence of Akt compared to control in MCF7 cells (* p < 0.05).

Figure 3. Role of miR301 in the presence of Akt on cell cycle progression

A. Cell cycle analysis shows significant increase in the G2/M phase upon inhibition of miR301 expression in the presence of Akt compared to control and miR301-mimic+Akt in MCF7 breast cancer cells. Lower panel represents quantification of data (A). B. Western blot represents detection of p21^Waf1/Cip1 and p27^kip1 cell cycle inhibitory proteins. The levels of total and phophorylated p21^Waf1/Cip1 and p27^kip1 protein were increased upon inhibition of miR301 in the presence of Akt compared to control and miR301-mimic+Akt in MCF7 cells. C. Western blot analysis of Cyclin D1 and Cyclin B1 cell cycle regulatory proteins. Levels of Cyclin D1 and Cyclin B1 were increased upon the expression of miR301 inhibitor in the presence of overexpressed Akt, as compared to control, in MCF7 cells (* p < 0.05).

Figure 4. miR301 inhibition in the presence of Akt reduced cell death

A. Cell death was assessed using Po-Pro (apoptotic) and 7-AAD (necrotic) markers. Transient transfection of miR301 inhibitor and Akt resulted in reduced cell death compared to control MCF7 cells. B. Quantitative representation of data from “A” shows significantly decreased in cell death as compared to control (* p < 0.05).

Figure 5. miR301 targeted gene expression in breast cancer cells

A. Western blot analysis of miR301 targets PI3K, PTEN and FoxF2 indicates that total protein levels were significantly increased upon miR301 inhibition in Akt-overexpressing MCF7-cells, as compared to control. Phosphorylated PI3K levels were increased as compared to control and phosphorylated PTEN protein expression was not significantly changed in cells expressing miR301-modulators and Akt. B. Confocal-microscopy images show enhanced nuclear localization of PTEN (red) in MCF7-cells transiently-transfected with miR301 inhibitor and Akt. Transfection with miR301 mimic and Akt caused PTEN's cytoplasmic localization, in MCF7 cells. For better visualization of nuclei, cells were counterstained with DAPI (blue). C. Western blot analysis shows increased levels of phosphorylated Akt (pAkt), pmTOR and P70S6 in MCF7 cells transiently transfected with miR301 inhibitor and Akt.

Figure 6. Schematic outline of the miR-301's functions in breast cancer cells

In the proposed model overexpression of Akt causes down-regulation of miR301. This effect can be also reached and strengthened by applying miR301 specific inhibitor. Down-regulation of miR301 leads to the increase of expression of PI3K, FOXF2, mTOR, p70s6 and nuclear localized PTEN. At the same time several cell cycle related proteins (p21^Waf1/Cip1, p27^kip1, Cyclin D1 and Cyclin B1) are also overexpressed, which results in higher proliferation rate and accumulation of cells in G2 phase. This all cause increase of cell migration and prevents the induction of apoptosis.