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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

Analysis of miR-34 family members using breast cancer TCGA data. Pair-wise scatter-plots of the expression of miR-34a, −34b and -34c in 658 miRNA HiSeq breast tumor samples from TCGA (A-C). Box plots demonstrate log2 expression levels of miR-34a (D), miR-34b (E) and miR-34c (F) in basal-like tumors, non-basal-like tumors and non-malignant breast tissue. Indicated p-values were calculated with a t-test comparing the group with the basal-like tumor samples. Kaplan-Meier analysis curves were constructed using the 310 TCGA tumors that had miRNA HiSeq data and follow-up data (G-I). The expression data were divided based on median expression and new tumor event was used as end point.
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Fig1: Analysis of miR-34 family members using breast cancer TCGA data. Pair-wise scatter-plots of the expression of miR-34a, −34b and -34c in 658 miRNA HiSeq breast tumor samples from TCGA (A-C). Box plots demonstrate log2 expression levels of miR-34a (D), miR-34b (E) and miR-34c (F) in basal-like tumors, non-basal-like tumors and non-malignant breast tissue. Indicated p-values were calculated with a t-test comparing the group with the basal-like tumor samples. Kaplan-Meier analysis curves were constructed using the 310 TCGA tumors that had miRNA HiSeq data and follow-up data (G-I). The expression data were divided based on median expression and new tumor event was used as end point.

Mentions: To assess putative roles of miR-34 family members in breast cancer, miRNA HiSeq expression data from 658 tumors and 86 normal breast tissue samples from the TCGA (The Cancer Genome Atlas) database were used. There was a clear correlation between miR-34b and miR-34c levels, whereas neither of them displayed a strong correlation with miR-34a (Figure 1A-C). This likely reflects the fact that miR-34b and 34c are located at the same locus on chromosome 11q23 whereas miR-34a is located at 1p36. The TCGA tumors were clustered using mRNA data, which separated the tumors in two major groups which correspond to basal-like and non-basal-like tumors. For miR-34c, lower levels were seen in basal-like tumors compared both to non-basal-like tumors and to normal breast tissue, whereas no substantial difference was observed for miR-34a or miR-34b (Figure 1D-F).The miR-34 expression data from the 310 tumors with follow-up information were split into two groups based on the median of the expression and survival curves with new tumor event as end point were generated (Figure 1G-I). No obvious difference between tumors expressing high or low levels of miR-34a could be seen. However, for both miR-34b and miR-34c, there was a difference in new tumor events between the low- and high-expressing groups, with more new tumor events in the group with miR-34 expression below median.Figure 1


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)

Analysis of miR-34 family members using breast cancer TCGA data. Pair-wise scatter-plots of the expression of miR-34a, −34b and -34c in 658 miRNA HiSeq breast tumor samples from TCGA (A-C). Box plots demonstrate log2 expression levels of miR-34a (D), miR-34b (E) and miR-34c (F) in basal-like tumors, non-basal-like tumors and non-malignant breast tissue. Indicated p-values were calculated with a t-test comparing the group with the basal-like tumor samples. Kaplan-Meier analysis curves were constructed using the 310 TCGA tumors that had miRNA HiSeq data and follow-up data (G-I). The expression data were divided based on median expression and new tumor event was used as end point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Analysis of miR-34 family members using breast cancer TCGA data. Pair-wise scatter-plots of the expression of miR-34a, −34b and -34c in 658 miRNA HiSeq breast tumor samples from TCGA (A-C). Box plots demonstrate log2 expression levels of miR-34a (D), miR-34b (E) and miR-34c (F) in basal-like tumors, non-basal-like tumors and non-malignant breast tissue. Indicated p-values were calculated with a t-test comparing the group with the basal-like tumor samples. Kaplan-Meier analysis curves were constructed using the 310 TCGA tumors that had miRNA HiSeq data and follow-up data (G-I). The expression data were divided based on median expression and new tumor event was used as end point.
Mentions: To assess putative roles of miR-34 family members in breast cancer, miRNA HiSeq expression data from 658 tumors and 86 normal breast tissue samples from the TCGA (The Cancer Genome Atlas) database were used. There was a clear correlation between miR-34b and miR-34c levels, whereas neither of them displayed a strong correlation with miR-34a (Figure 1A-C). This likely reflects the fact that miR-34b and 34c are located at the same locus on chromosome 11q23 whereas miR-34a is located at 1p36. The TCGA tumors were clustered using mRNA data, which separated the tumors in two major groups which correspond to basal-like and non-basal-like tumors. For miR-34c, lower levels were seen in basal-like tumors compared both to non-basal-like tumors and to normal breast tissue, whereas no substantial difference was observed for miR-34a or miR-34b (Figure 1D-F).The miR-34 expression data from the 310 tumors with follow-up information were split into two groups based on the median of the expression and survival curves with new tumor event as end point were generated (Figure 1G-I). No obvious difference between tumors expressing high or low levels of miR-34a could be seen. However, for both miR-34b and miR-34c, there was a difference in new tumor events between the low- and high-expressing groups, with more new tumor events in the group with miR-34 expression below median.Figure 1

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