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MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

Rasheed SA, Teo CR, Beillard EJ, Voorhoeve PM, Zhou W, Ghosh S, Casey PJ - Mol. Cancer (2015)

Bottom Line: Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity.Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity.Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, 169857, Singapore, Singapore. kabeer.rasheed@duke-nus.edu.sg.

ABSTRACT

Background: Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers.

Methods: To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays.

Results: Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels.

Conclusions: These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.

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GNA12 and GNA13 are highly expressed in aggressive cancer cells. (A) Immunoblot showing the GNA12 and GNA13 protein expression in different breast cancer cell lines. Tubulin is used as loading control. (B) Basal and FCS-induced invasion of MCF-10a and MDA-MB-231 cells. All values are expressed as fold change relative to basal invasion of MCF-10a cells.
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Fig1: GNA12 and GNA13 are highly expressed in aggressive cancer cells. (A) Immunoblot showing the GNA12 and GNA13 protein expression in different breast cancer cell lines. Tubulin is used as loading control. (B) Basal and FCS-induced invasion of MCF-10a and MDA-MB-231 cells. All values are expressed as fold change relative to basal invasion of MCF-10a cells.

Mentions: Previously others and we have shown that G12 proteins are highly expressed in prostate, breast and other cancer types [13,20,22,40]. Further, blockade of GNA12/13 signaling using the specific inhibitor p115RGS has been shown to inhibit breast and prostate cancer metastasis both in vitro and in vivo [20]. Since most of the previous studies focused on GNA12, we have recently begun to assess the specific role(s) of GNA13 in cancer cell invasion, and the control of its expression in cancers. These studies led to the above-noted findings that miR-182 and miR-200a control GNA13 expression in prostate cancer cells [28]. Similar to that observed previously in prostate cancer cell lines, examination of a panel of breast cancer cell lines revealed that GNA13 levels are greatest in more invasive cell lines such as MDA-MB-231 and MDA-MB-157 (Figure 1A). Both these cell lines were derived from metastatic breast cancers. However, more benign/less invasive breast epithelial cell lines such as MCF-10a and HMEC had much lower levels of GNA13 protein (Figure 1A). Further, the level of serum-stimulated cell invasiveness correlated with the GNA13 levels, as shown for the MCF-10a and MDA-MB-231 cell lines (Figure 1B). However, this correlation to invasiveness is only indicative of an important role of GNA13 in their invasive ability, as these cells may have multiple gene expression changes that contribute to this effect.Figure 1


MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

Rasheed SA, Teo CR, Beillard EJ, Voorhoeve PM, Zhou W, Ghosh S, Casey PJ - Mol. Cancer (2015)

GNA12 and GNA13 are highly expressed in aggressive cancer cells. (A) Immunoblot showing the GNA12 and GNA13 protein expression in different breast cancer cell lines. Tubulin is used as loading control. (B) Basal and FCS-induced invasion of MCF-10a and MDA-MB-231 cells. All values are expressed as fold change relative to basal invasion of MCF-10a cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379695&req=5

Fig1: GNA12 and GNA13 are highly expressed in aggressive cancer cells. (A) Immunoblot showing the GNA12 and GNA13 protein expression in different breast cancer cell lines. Tubulin is used as loading control. (B) Basal and FCS-induced invasion of MCF-10a and MDA-MB-231 cells. All values are expressed as fold change relative to basal invasion of MCF-10a cells.
Mentions: Previously others and we have shown that G12 proteins are highly expressed in prostate, breast and other cancer types [13,20,22,40]. Further, blockade of GNA12/13 signaling using the specific inhibitor p115RGS has been shown to inhibit breast and prostate cancer metastasis both in vitro and in vivo [20]. Since most of the previous studies focused on GNA12, we have recently begun to assess the specific role(s) of GNA13 in cancer cell invasion, and the control of its expression in cancers. These studies led to the above-noted findings that miR-182 and miR-200a control GNA13 expression in prostate cancer cells [28]. Similar to that observed previously in prostate cancer cell lines, examination of a panel of breast cancer cell lines revealed that GNA13 levels are greatest in more invasive cell lines such as MDA-MB-231 and MDA-MB-157 (Figure 1A). Both these cell lines were derived from metastatic breast cancers. However, more benign/less invasive breast epithelial cell lines such as MCF-10a and HMEC had much lower levels of GNA13 protein (Figure 1A). Further, the level of serum-stimulated cell invasiveness correlated with the GNA13 levels, as shown for the MCF-10a and MDA-MB-231 cell lines (Figure 1B). However, this correlation to invasiveness is only indicative of an important role of GNA13 in their invasive ability, as these cells may have multiple gene expression changes that contribute to this effect.Figure 1

Bottom Line: Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity.Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity.Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, 169857, Singapore, Singapore. kabeer.rasheed@duke-nus.edu.sg.

ABSTRACT

Background: Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers.

Methods: To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays.

Results: Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels.

Conclusions: These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.

Show MeSH
Related in: MedlinePlus