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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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Related in: MedlinePlus

Relationship of miR-31 and GNA13 in breast cancer cells. (A) Immunoblot analysis of GNA13 protein expression in breast and prostate cancer cell lines. Tubulin is used as loading control. (B) Relative basal expression of the microRNAs predicted to bind to the GNA13-3′-UTR in breast and prostate cancer cells. Relative microRNA expression is plotted in the y-axis. All values were calibrated to basal miR-31 expression in MDA-MB-231 cells.
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Fig3: Relationship of miR-31 and GNA13 in breast cancer cells. (A) Immunoblot analysis of GNA13 protein expression in breast and prostate cancer cell lines. Tubulin is used as loading control. (B) Relative basal expression of the microRNAs predicted to bind to the GNA13-3′-UTR in breast and prostate cancer cells. Relative microRNA expression is plotted in the y-axis. All values were calibrated to basal miR-31 expression in MDA-MB-231 cells.

Mentions: To obtain evidence for which, if any, of the potential regulatory miRNAs might be impacting GNA13 expression in breast cancer cells, we performed a comparison of GNA13 protein levels (by immunoblot analysis) and of the respective miRNAs (by real time PCR) (Figure 3A, B), respectively. Surprisingly, both miR-182 and miR-200a/141, which showed an inverse correlation to GNA13 protein expression in prostate cancer cells as reported earlier [28], were highly suppressed in breast cancer cells and hence had no correlation to GNA13 protein expression (Figure 3A, B). Interestingly, however, miR-31 showed a clear inverse correlation to GNA13 protein expression in the breast cancer cells (Figure 3A, B). Levels of this miRNA were much higher in cells containing low GNA13 protein levels such as HMECs and MCF-10a (Figure 3A, B). MDA-MB-231 and MDA-MB-157 cells expressed high GNA13 protein and had no or little detectable miR-31 expression (Figure 3A, B). These data implicate miR-31 in control of GNA13 expression in breast cancer cells.Figure 3


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)

Relationship of miR-31 and GNA13 in breast cancer cells. (A) Immunoblot analysis of GNA13 protein expression in breast and prostate cancer cell lines. Tubulin is used as loading control. (B) Relative basal expression of the microRNAs predicted to bind to the GNA13-3′-UTR in breast and prostate cancer cells. Relative microRNA expression is plotted in the y-axis. All values were calibrated to basal miR-31 expression in MDA-MB-231 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Relationship of miR-31 and GNA13 in breast cancer cells. (A) Immunoblot analysis of GNA13 protein expression in breast and prostate cancer cell lines. Tubulin is used as loading control. (B) Relative basal expression of the microRNAs predicted to bind to the GNA13-3′-UTR in breast and prostate cancer cells. Relative microRNA expression is plotted in the y-axis. All values were calibrated to basal miR-31 expression in MDA-MB-231 cells.
Mentions: To obtain evidence for which, if any, of the potential regulatory miRNAs might be impacting GNA13 expression in breast cancer cells, we performed a comparison of GNA13 protein levels (by immunoblot analysis) and of the respective miRNAs (by real time PCR) (Figure 3A, B), respectively. Surprisingly, both miR-182 and miR-200a/141, which showed an inverse correlation to GNA13 protein expression in prostate cancer cells as reported earlier [28], were highly suppressed in breast cancer cells and hence had no correlation to GNA13 protein expression (Figure 3A, B). Interestingly, however, miR-31 showed a clear inverse correlation to GNA13 protein expression in the breast cancer cells (Figure 3A, B). Levels of this miRNA were much higher in cells containing low GNA13 protein levels such as HMECs and MCF-10a (Figure 3A, B). MDA-MB-231 and MDA-MB-157 cells expressed high GNA13 protein and had no or little detectable miR-31 expression (Figure 3A, B). These data implicate miR-31 in control of GNA13 expression in breast cancer cells.Figure 3

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