Limits...
Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells.

Achari C, Winslow S, Ceder Y, Larsson C - BMC Cancer (2014)

Bottom Line: Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase.We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines.In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Translational Cancer Research, Lund University, Medicon Village, Building 404:C3, 223 81 Lund, Sweden. Christer.Larsson@med.lu.se.

ABSTRACT

Background: MicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells.

Methods: Data from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot.

Results: TCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c.

Conclusions: Together, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

Show MeSH

Related in: MedlinePlus

Effect of miR-34c on cell cycle distribution of breast cancer cells. Following transfection of MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cells with miR-34c mimic or negative control for 96 h, nuclei were stained with propidium iodide solution and analyzed for DNA content by flow cytometry. Data (mean ± SEM, n = 5) represent percentage cells in different phases of the cell cycle with miR-34c related to scramble treatment. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4125691&req=5

Fig3: Effect of miR-34c on cell cycle distribution of breast cancer cells. Following transfection of MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cells with miR-34c mimic or negative control for 96 h, nuclei were stained with propidium iodide solution and analyzed for DNA content by flow cytometry. Data (mean ± SEM, n = 5) represent percentage cells in different phases of the cell cycle with miR-34c related to scramble treatment. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.

Mentions: The suppressed [3H]-thymidine incorporation suggests that miR-34c may influence the cell cycle. The cell cycle distribution was thus analyzed by FACS following nuclear staining with propidium iodide (Additional file 2). Ectopic expression of miR-34c induced an accumulation of cells in the G2/M phase compared to control in MDA-MB-231 (Figure 3A) and MDA-MB-468 (Figure 3B) cells. A similar tendency was observed for BT-549 cells (Figure 3C). In all three cell lines a significant increase in sub-G1 phase was detected along with a reduction of cells in G1 phase (Figure 3A-C), but the G1-arrest that has been reported for other cell types [18, 31] was not detected in the breast cancer cells.Figure 3


Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells.

Achari C, Winslow S, Ceder Y, Larsson C - BMC Cancer (2014)

Effect of miR-34c on cell cycle distribution of breast cancer cells. Following transfection of MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cells with miR-34c mimic or negative control for 96 h, nuclei were stained with propidium iodide solution and analyzed for DNA content by flow cytometry. Data (mean ± SEM, n = 5) represent percentage cells in different phases of the cell cycle with miR-34c related to scramble treatment. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125691&req=5

Fig3: Effect of miR-34c on cell cycle distribution of breast cancer cells. Following transfection of MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cells with miR-34c mimic or negative control for 96 h, nuclei were stained with propidium iodide solution and analyzed for DNA content by flow cytometry. Data (mean ± SEM, n = 5) represent percentage cells in different phases of the cell cycle with miR-34c related to scramble treatment. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.
Mentions: The suppressed [3H]-thymidine incorporation suggests that miR-34c may influence the cell cycle. The cell cycle distribution was thus analyzed by FACS following nuclear staining with propidium iodide (Additional file 2). Ectopic expression of miR-34c induced an accumulation of cells in the G2/M phase compared to control in MDA-MB-231 (Figure 3A) and MDA-MB-468 (Figure 3B) cells. A similar tendency was observed for BT-549 cells (Figure 3C). In all three cell lines a significant increase in sub-G1 phase was detected along with a reduction of cells in G1 phase (Figure 3A-C), but the G1-arrest that has been reported for other cell types [18, 31] was not detected in the breast cancer cells.Figure 3

Bottom Line: Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase.We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines.In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Translational Cancer Research, Lund University, Medicon Village, Building 404:C3, 223 81 Lund, Sweden. Christer.Larsson@med.lu.se.

ABSTRACT

Background: MicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells.

Methods: Data from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot.

Results: TCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c.

Conclusions: Together, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

Show MeSH
Related in: MedlinePlus