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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.

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Related in: MedlinePlus

Evaluation of miR-34c targets. Following transfection of MDA-MB-231, MDA-MB-468 and BT-549 (breast cancer cells with miR-34c mimic or negative control for 96 h, cells were analyzed for expression of PRKCA(B), CCND1(C), CDK4(D), CDK6(E) or CDC23(G) mRNA with real-time quantitative PCR or for protein expression with Western blot (AandF). Data represent mean ± SEM from 2–3 independent experiments and the blots shown are representative of three independent experiments. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.
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Fig5: Evaluation of miR-34c targets. Following transfection of MDA-MB-231, MDA-MB-468 and BT-549 (breast cancer cells with miR-34c mimic or negative control for 96 h, cells were analyzed for expression of PRKCA(B), CCND1(C), CDK4(D), CDK6(E) or CDC23(G) mRNA with real-time quantitative PCR or for protein expression with Western blot (AandF). Data represent mean ± SEM from 2–3 independent experiments and the blots shown are representative of three independent experiments. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.

Mentions: As mentioned in the introduction, PRKCA is a predicted miR-34c target. Since PKCα is important for optimal breast cancer cell proliferation [28–30] we analyzed the effects of miR-34c on PKCα expression. We could not detect any effects on the protein levels (Figure 5A) despite the observation that PRKCA mRNA levels in MDA-MB-231 and MDA-MB-468 were affected by miR-34c (Figure 5B). This suggests that PKCα downregulation is not a mediator of the effects seen by miR-34c in breast cancer cells.Figure 5


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)

Evaluation of miR-34c targets. Following transfection of MDA-MB-231, MDA-MB-468 and BT-549 (breast cancer cells with miR-34c mimic or negative control for 96 h, cells were analyzed for expression of PRKCA(B), CCND1(C), CDK4(D), CDK6(E) or CDC23(G) mRNA with real-time quantitative PCR or for protein expression with Western blot (AandF). Data represent mean ± SEM from 2–3 independent experiments and the blots shown are representative of three independent experiments. 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

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Show All Figures
getmorefigures.php?uid=PMC4125691&req=5

Fig5: Evaluation of miR-34c targets. Following transfection of MDA-MB-231, MDA-MB-468 and BT-549 (breast cancer cells with miR-34c mimic or negative control for 96 h, cells were analyzed for expression of PRKCA(B), CCND1(C), CDK4(D), CDK6(E) or CDC23(G) mRNA with real-time quantitative PCR or for protein expression with Western blot (AandF). Data represent mean ± SEM from 2–3 independent experiments and the blots shown are representative of three independent experiments. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test) compared to control cells.
Mentions: As mentioned in the introduction, PRKCA is a predicted miR-34c target. Since PKCα is important for optimal breast cancer cell proliferation [28–30] we analyzed the effects of miR-34c on PKCα expression. We could not detect any effects on the protein levels (Figure 5A) despite the observation that PRKCA mRNA levels in MDA-MB-231 and MDA-MB-468 were affected by miR-34c (Figure 5B). This suggests that PKCα downregulation is not a mediator of the effects seen by miR-34c in breast cancer cells.Figure 5

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