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Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells.

Weiner-Gorzel K, Dempsey E, Milewska M, McGoldrick A, Toh V, Walsh A, Lindsay S, Gubbins L, Cannon A, Sharpe D, O'Sullivan J, Murphy M, Madden SF, Kell M, McCann A, Furlong F - Cancer Med (2015)

Bottom Line: Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene.Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect.Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment.

View Article: PubMed Central - PubMed

Affiliation: UCD School of Medicine and Medical Science (SMMS), UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.

No MeSH data available.


Related in: MedlinePlus

Stable expression of miR-433 in A2780 cells attenuates cellular apoptosis and results in a distinct morphological and proliferative change. (A) Quantitative real-time PCR (qRT-PCR) values for miR-433 in the miR-433-stable A2780 cells compared to miR-433-stable cells showing a significant (P < 0.05) fold increase in miR-433 levels. (B) Western blot analysis showing downregulation of two miR-433 targets, MAD2 and HDAC6 in the A2780 miR-433-stable expressing cell line. (C) Histogram representation of the response of the stable miR-433 versus control-miR cell lines post 24 h paclitaxel (10, 25, 50 nmol/L) treatment. Student's t-test was used for the comparison of means and demonstrates an increased resistance of the miR-433-stable cell line compared to controls at 25 and 50 nmol/L paclitaxel (P < 0.05). (D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage in the miR-433-stable and miR-control-stable cell line treated with 50 nmol/L paclitaxel for 24 h demonstrating a decreased PARP cleavage in the miR-433-stable expressing A2780 cells. (E) Increased miR-433 expression influences the cellular morphology of A2780 cells appreciated by green fluorescent protein (GFP) expression with a resultant flattened and enlarged cellular morphology. Magnification 20×. (F) Colony forming assay of miR-control and miR-433 stable tranfected A2780 cells stained with crystal violet (magnification 4×) demonstrating a significantly lower colony formation ability in the miR-433 stable transfectants. Error bars represent SEM. *P < 0.05, ***P < 0.001.
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fig01: Stable expression of miR-433 in A2780 cells attenuates cellular apoptosis and results in a distinct morphological and proliferative change. (A) Quantitative real-time PCR (qRT-PCR) values for miR-433 in the miR-433-stable A2780 cells compared to miR-433-stable cells showing a significant (P < 0.05) fold increase in miR-433 levels. (B) Western blot analysis showing downregulation of two miR-433 targets, MAD2 and HDAC6 in the A2780 miR-433-stable expressing cell line. (C) Histogram representation of the response of the stable miR-433 versus control-miR cell lines post 24 h paclitaxel (10, 25, 50 nmol/L) treatment. Student's t-test was used for the comparison of means and demonstrates an increased resistance of the miR-433-stable cell line compared to controls at 25 and 50 nmol/L paclitaxel (P < 0.05). (D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage in the miR-433-stable and miR-control-stable cell line treated with 50 nmol/L paclitaxel for 24 h demonstrating a decreased PARP cleavage in the miR-433-stable expressing A2780 cells. (E) Increased miR-433 expression influences the cellular morphology of A2780 cells appreciated by green fluorescent protein (GFP) expression with a resultant flattened and enlarged cellular morphology. Magnification 20×. (F) Colony forming assay of miR-control and miR-433 stable tranfected A2780 cells stained with crystal violet (magnification 4×) demonstrating a significantly lower colony formation ability in the miR-433 stable transfectants. Error bars represent SEM. *P < 0.05, ***P < 0.001.

Mentions: miR-433 expression was confirmed by qRT-PCR analysis and was significantly upregulated in the stably transfected cells compared to control (P < 0.001) (Fig.1A). To confirm that stable overexpressed miR-433 was functionally active, we examined the protein expression of two known miR-433 target genes, MAD2 5 and HDAC6 15. The levels of both proteins were downregulated in the miR-433 overexpressing cells when compared to controls (Fig.1B).


Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells.

Weiner-Gorzel K, Dempsey E, Milewska M, McGoldrick A, Toh V, Walsh A, Lindsay S, Gubbins L, Cannon A, Sharpe D, O'Sullivan J, Murphy M, Madden SF, Kell M, McCann A, Furlong F - Cancer Med (2015)

Stable expression of miR-433 in A2780 cells attenuates cellular apoptosis and results in a distinct morphological and proliferative change. (A) Quantitative real-time PCR (qRT-PCR) values for miR-433 in the miR-433-stable A2780 cells compared to miR-433-stable cells showing a significant (P < 0.05) fold increase in miR-433 levels. (B) Western blot analysis showing downregulation of two miR-433 targets, MAD2 and HDAC6 in the A2780 miR-433-stable expressing cell line. (C) Histogram representation of the response of the stable miR-433 versus control-miR cell lines post 24 h paclitaxel (10, 25, 50 nmol/L) treatment. Student's t-test was used for the comparison of means and demonstrates an increased resistance of the miR-433-stable cell line compared to controls at 25 and 50 nmol/L paclitaxel (P < 0.05). (D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage in the miR-433-stable and miR-control-stable cell line treated with 50 nmol/L paclitaxel for 24 h demonstrating a decreased PARP cleavage in the miR-433-stable expressing A2780 cells. (E) Increased miR-433 expression influences the cellular morphology of A2780 cells appreciated by green fluorescent protein (GFP) expression with a resultant flattened and enlarged cellular morphology. Magnification 20×. (F) Colony forming assay of miR-control and miR-433 stable tranfected A2780 cells stained with crystal violet (magnification 4×) demonstrating a significantly lower colony formation ability in the miR-433 stable transfectants. Error bars represent SEM. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Stable expression of miR-433 in A2780 cells attenuates cellular apoptosis and results in a distinct morphological and proliferative change. (A) Quantitative real-time PCR (qRT-PCR) values for miR-433 in the miR-433-stable A2780 cells compared to miR-433-stable cells showing a significant (P < 0.05) fold increase in miR-433 levels. (B) Western blot analysis showing downregulation of two miR-433 targets, MAD2 and HDAC6 in the A2780 miR-433-stable expressing cell line. (C) Histogram representation of the response of the stable miR-433 versus control-miR cell lines post 24 h paclitaxel (10, 25, 50 nmol/L) treatment. Student's t-test was used for the comparison of means and demonstrates an increased resistance of the miR-433-stable cell line compared to controls at 25 and 50 nmol/L paclitaxel (P < 0.05). (D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage in the miR-433-stable and miR-control-stable cell line treated with 50 nmol/L paclitaxel for 24 h demonstrating a decreased PARP cleavage in the miR-433-stable expressing A2780 cells. (E) Increased miR-433 expression influences the cellular morphology of A2780 cells appreciated by green fluorescent protein (GFP) expression with a resultant flattened and enlarged cellular morphology. Magnification 20×. (F) Colony forming assay of miR-control and miR-433 stable tranfected A2780 cells stained with crystal violet (magnification 4×) demonstrating a significantly lower colony formation ability in the miR-433 stable transfectants. Error bars represent SEM. *P < 0.05, ***P < 0.001.
Mentions: miR-433 expression was confirmed by qRT-PCR analysis and was significantly upregulated in the stably transfected cells compared to control (P < 0.001) (Fig.1A). To confirm that stable overexpressed miR-433 was functionally active, we examined the protein expression of two known miR-433 target genes, MAD2 5 and HDAC6 15. The levels of both proteins were downregulated in the miR-433 overexpressing cells when compared to controls (Fig.1B).

Bottom Line: Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene.Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect.Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment.

View Article: PubMed Central - PubMed

Affiliation: UCD School of Medicine and Medical Science (SMMS), UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.

No MeSH data available.


Related in: MedlinePlus