Limits...
A novel role for microRNA-129-5p in inhibiting ovarian cancer cell proliferation and survival via direct suppression of transcriptional co-activators YAP and TAZ.

Tan G, Cao X, Dai Q, Zhang B, Huang J, Xiong S, Zhang Yy, Chen W, Yang J, Li H - Oncotarget (2015)

Bottom Line: Transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key oncogenes in mammalian cells.Activities of YAP and TAZ are largely restricted by the Hippo tumor suppressor pathway through phosphorylation-ubiquitination mechanisms.Furthermore, we reveal miR-129-5p inhibits ovarian cancer cell proliferation, survival and tumorigenicity, and that downregulation of miR-129-5p in ovarian cancer cells highly correlates with malignant progression and poor survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key oncogenes in mammalian cells. Activities of YAP and TAZ are largely restricted by the Hippo tumor suppressor pathway through phosphorylation-ubiquitination mechanisms. The involvement of microRNA in cancer progression has recently been reported, though whether they have a role in activating YAP and TAZ in human cancer cells remains unclear. Here, we report a microRNA, miR-129-5p, directly represses YAP and TAZ expression, leading to the inactivation of TEA domain (TEAD) transcription, and the downregulation of Hippo downstream genes, connective tissue growth factor (CTGF) and Cyclin A. Furthermore, we reveal miR-129-5p inhibits ovarian cancer cell proliferation, survival and tumorigenicity, and that downregulation of miR-129-5p in ovarian cancer cells highly correlates with malignant progression and poor survival. Hence, we demonstrate a novel mechanism for YAP and TAZ activation in cancers, indicating not only a potentially pivotal role for miR-129-5p in the progression of ovarian cancer, but also offering new therapeutic strategies to circumvent the disease.

No MeSH data available.


Related in: MedlinePlus

MiR-129-5p directly suppresses YAP and TAZ(A) Predicted miR-129-5p target sequence in 3′ UTRs of YAP and TAZ. Target sequences of YAP- and TAZ-3′ UTR were mutated. (B) Western blots of YAP and TAZ expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-YAP-3′UTR, pGL3-TAZ-3′UTR, pGL3-YAP-3′UTR-mut or pGL3-TAZ-3′UTR-mut reporter with miR-129-5p mimic, antagomiR-129-5p, mimic control or antagomiR control. (D) MiRNP IP assay showing the association between miR-129-5p with YAP and TAZ transcripts. GAPDH and 5s rRNA served as the negative controls. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496175&req=5

Figure 1: MiR-129-5p directly suppresses YAP and TAZ(A) Predicted miR-129-5p target sequence in 3′ UTRs of YAP and TAZ. Target sequences of YAP- and TAZ-3′ UTR were mutated. (B) Western blots of YAP and TAZ expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-YAP-3′UTR, pGL3-TAZ-3′UTR, pGL3-YAP-3′UTR-mut or pGL3-TAZ-3′UTR-mut reporter with miR-129-5p mimic, antagomiR-129-5p, mimic control or antagomiR control. (D) MiRNP IP assay showing the association between miR-129-5p with YAP and TAZ transcripts. GAPDH and 5s rRNA served as the negative controls. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.

Mentions: The transcriptional co-activators YAP and TAZ are major components of the Hippo signaling pathway and play a critical role in the development and progression of multiple cancer types, including ovarian cancer [21, 30]. To investigate the role microRNAs may play in regulating YAP and TAZ expression, we applied publicly available algorithms from TargetScan to reveal that YAP and TAZ might be potential targets of miR-129-5p (Figure 1A). Our western blot analysis revealed that expression levels of YAP and TAZ were significantly decreased in miR-129-5p-transfected cells, but increased in miR-129-5p-silenced cells (Figure 1B and Supplementary Figure 1A–1B), suggesting that miR-129-5p negatively regulates these two proteins. However, significant changes to YAP and TAZ expression were not observed when cells were transfected with miR-101, miR-124 and miR-18a, which are also predicted to target YAP and TAZ, and miR-129-5p has no effect on the LATS1 phosphorylation level (Supplementary Figure 1C), suggesting that YAP and TAZ are selectively regulated by miR-129-5p (data not shown). Furthermore, luciferase assays revealed miR-129-5p overexpression led to a decrease in reporter activity linked with the 3′ UTRs of YAP and TAZ transcripts, while in miR-129-5p silenced cells, an increase in reporter activity was observed (Figure 1C). However, neither overexpressing miR-129-5p nor silencing miR-129-5p exhibited effects on the reporter activities linked with mutant 3′ UTRs of YAP and TAZ. Importantly, micro-ribonucleoprotein (miRNP) immunoprecipitation (IP) assay showed that miR-129-5p overexpression enriched the transcripts of YAP and TAZ (and not GAPDH or 5s rRNA, that assembled into the miRNP complexes, indicating that miR-129-5p directly targets the mRNA 3′ UTR regions of these transcripts (Figure 1D). Taken together, our results demonstrate that YAP and TAZ are bona fide targets of miR-129-5p.


A novel role for microRNA-129-5p in inhibiting ovarian cancer cell proliferation and survival via direct suppression of transcriptional co-activators YAP and TAZ.

Tan G, Cao X, Dai Q, Zhang B, Huang J, Xiong S, Zhang Yy, Chen W, Yang J, Li H - Oncotarget (2015)

MiR-129-5p directly suppresses YAP and TAZ(A) Predicted miR-129-5p target sequence in 3′ UTRs of YAP and TAZ. Target sequences of YAP- and TAZ-3′ UTR were mutated. (B) Western blots of YAP and TAZ expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-YAP-3′UTR, pGL3-TAZ-3′UTR, pGL3-YAP-3′UTR-mut or pGL3-TAZ-3′UTR-mut reporter with miR-129-5p mimic, antagomiR-129-5p, mimic control or antagomiR control. (D) MiRNP IP assay showing the association between miR-129-5p with YAP and TAZ transcripts. GAPDH and 5s rRNA served as the negative controls. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496175&req=5

Figure 1: MiR-129-5p directly suppresses YAP and TAZ(A) Predicted miR-129-5p target sequence in 3′ UTRs of YAP and TAZ. Target sequences of YAP- and TAZ-3′ UTR were mutated. (B) Western blots of YAP and TAZ expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-YAP-3′UTR, pGL3-TAZ-3′UTR, pGL3-YAP-3′UTR-mut or pGL3-TAZ-3′UTR-mut reporter with miR-129-5p mimic, antagomiR-129-5p, mimic control or antagomiR control. (D) MiRNP IP assay showing the association between miR-129-5p with YAP and TAZ transcripts. GAPDH and 5s rRNA served as the negative controls. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
Mentions: The transcriptional co-activators YAP and TAZ are major components of the Hippo signaling pathway and play a critical role in the development and progression of multiple cancer types, including ovarian cancer [21, 30]. To investigate the role microRNAs may play in regulating YAP and TAZ expression, we applied publicly available algorithms from TargetScan to reveal that YAP and TAZ might be potential targets of miR-129-5p (Figure 1A). Our western blot analysis revealed that expression levels of YAP and TAZ were significantly decreased in miR-129-5p-transfected cells, but increased in miR-129-5p-silenced cells (Figure 1B and Supplementary Figure 1A–1B), suggesting that miR-129-5p negatively regulates these two proteins. However, significant changes to YAP and TAZ expression were not observed when cells were transfected with miR-101, miR-124 and miR-18a, which are also predicted to target YAP and TAZ, and miR-129-5p has no effect on the LATS1 phosphorylation level (Supplementary Figure 1C), suggesting that YAP and TAZ are selectively regulated by miR-129-5p (data not shown). Furthermore, luciferase assays revealed miR-129-5p overexpression led to a decrease in reporter activity linked with the 3′ UTRs of YAP and TAZ transcripts, while in miR-129-5p silenced cells, an increase in reporter activity was observed (Figure 1C). However, neither overexpressing miR-129-5p nor silencing miR-129-5p exhibited effects on the reporter activities linked with mutant 3′ UTRs of YAP and TAZ. Importantly, micro-ribonucleoprotein (miRNP) immunoprecipitation (IP) assay showed that miR-129-5p overexpression enriched the transcripts of YAP and TAZ (and not GAPDH or 5s rRNA, that assembled into the miRNP complexes, indicating that miR-129-5p directly targets the mRNA 3′ UTR regions of these transcripts (Figure 1D). Taken together, our results demonstrate that YAP and TAZ are bona fide targets of miR-129-5p.

Bottom Line: Transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key oncogenes in mammalian cells.Activities of YAP and TAZ are largely restricted by the Hippo tumor suppressor pathway through phosphorylation-ubiquitination mechanisms.Furthermore, we reveal miR-129-5p inhibits ovarian cancer cell proliferation, survival and tumorigenicity, and that downregulation of miR-129-5p in ovarian cancer cells highly correlates with malignant progression and poor survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key oncogenes in mammalian cells. Activities of YAP and TAZ are largely restricted by the Hippo tumor suppressor pathway through phosphorylation-ubiquitination mechanisms. The involvement of microRNA in cancer progression has recently been reported, though whether they have a role in activating YAP and TAZ in human cancer cells remains unclear. Here, we report a microRNA, miR-129-5p, directly represses YAP and TAZ expression, leading to the inactivation of TEA domain (TEAD) transcription, and the downregulation of Hippo downstream genes, connective tissue growth factor (CTGF) and Cyclin A. Furthermore, we reveal miR-129-5p inhibits ovarian cancer cell proliferation, survival and tumorigenicity, and that downregulation of miR-129-5p in ovarian cancer cells highly correlates with malignant progression and poor survival. Hence, we demonstrate a novel mechanism for YAP and TAZ activation in cancers, indicating not only a potentially pivotal role for miR-129-5p in the progression of ovarian cancer, but also offering new therapeutic strategies to circumvent the disease.

No MeSH data available.


Related in: MedlinePlus