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A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis.

Shen S, Guo X, Yan H, Lu Y, Ji X, Li L, Liang T, Zhou D, Feng XH, Zhao JC, Yu J, Gong XG, Zhang L, Zhao B - Cell Res. (2015)

Bottom Line: Organ size determination is one of the most intriguing unsolved mysteries in biology.Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals.Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute and Innovation Center for Cell Signaling Network Hangzhou, Zhejiang 310058, China.

ABSTRACT
Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

miR-130a activates YAP through repression of VGLL4. (A) miR-130a activates YAP transcriptional activity. CTGF-luciferase reporter was co-transfected with other plasmids into HEK293T cells as indicated for luciferase assay. Experiments were performed in duplicates. (B) miR-130a inhibition suppresses YAP transcriptional activity in a VGLL4-dependent manner. HEK293T cells stably expressing CTGF-luciferase reporter were transfected as indicated for luciferase assay. Experiments were performed in duplicates. * indicates a P-value of < 0.05 as calculated by student's t-test. (C) miR-130a promotes YAP target gene expression, which is blocked by VGLL4. Control or VGLL4-overexpressing HepG2 cells were transfected with NC or miR-130a mimic for gene expression analysis. Experiments were performed in triplicates. (D) miR-130a promotes Hippo pathway target gene expression in a YAP-and-TAZ-dependent manner. HepG2 cells were transfected as indicated for gene expression assay. Experiments were performed in triplicates. (E) miR-130a inhibition suppresses YAP target gene expression in a VGLL4-dependent manner. HMLE cells were transfected and gene expression was determined. Experiments were performed in triplicates. (F) miR-130a regulates YAP binding to target gene promoter. ChIP was performed using control IgG or antibodies against TEAD1 and YAP on HepG2 stable cell lysates. Relative enrichment of CTGF promoter was determined by quantitative PCR in triplicates. (G) miR-130a regulates YAP-TEAD1 interaction. HepG2 cells stably expressing miR-130a precursor or sponge and respective empty vectors were lysed and immunoprecipitated with anti-YAP or anti-TEAD1 antibodies. Lysates and immunoprecipitates were analyzed by western blots.
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fig4: miR-130a activates YAP through repression of VGLL4. (A) miR-130a activates YAP transcriptional activity. CTGF-luciferase reporter was co-transfected with other plasmids into HEK293T cells as indicated for luciferase assay. Experiments were performed in duplicates. (B) miR-130a inhibition suppresses YAP transcriptional activity in a VGLL4-dependent manner. HEK293T cells stably expressing CTGF-luciferase reporter were transfected as indicated for luciferase assay. Experiments were performed in duplicates. * indicates a P-value of < 0.05 as calculated by student's t-test. (C) miR-130a promotes YAP target gene expression, which is blocked by VGLL4. Control or VGLL4-overexpressing HepG2 cells were transfected with NC or miR-130a mimic for gene expression analysis. Experiments were performed in triplicates. (D) miR-130a promotes Hippo pathway target gene expression in a YAP-and-TAZ-dependent manner. HepG2 cells were transfected as indicated for gene expression assay. Experiments were performed in triplicates. (E) miR-130a inhibition suppresses YAP target gene expression in a VGLL4-dependent manner. HMLE cells were transfected and gene expression was determined. Experiments were performed in triplicates. (F) miR-130a regulates YAP binding to target gene promoter. ChIP was performed using control IgG or antibodies against TEAD1 and YAP on HepG2 stable cell lysates. Relative enrichment of CTGF promoter was determined by quantitative PCR in triplicates. (G) miR-130a regulates YAP-TEAD1 interaction. HepG2 cells stably expressing miR-130a precursor or sponge and respective empty vectors were lysed and immunoprecipitated with anti-YAP or anti-TEAD1 antibodies. Lysates and immunoprecipitates were analyzed by western blots.

Mentions: VGLL4 is a repressor of YAP activity through competition for TEAD binding. Therefore, by repressing VGLL4, the YAP target gene miR-130a could potentially act as an activator of YAP. Indeed, expression of miR-130a activated a CTGF reporter, a well-defined readout of YAP transcriptional activity (Figure 4A and Supplementary information, Figure S4A). However, co-expression of miR-130a-insensitive VGLL4 from constructs lacking the 3′UTR normalized the reporter activity dose-dependently (Figure 4A). More importantly, inhibition of miR-130a attenuated CTGF reporter activity in a VGLL4-dependent manner (Figure 4B and Supplementary information, Figure S4A), suggesting that YAP is regulated by endogenous miR-130a through VGLL4. Furthermore, expression of YAP target genes CTGF and Cyr61 was enhanced or repressed by activation or inhibition of miR-130a, respectively (Supplementary information, Figure S4B, S4C, S4D). Consistently, VGLL4 overexpression or YAP and TAZ knockdown (Supplementary information, Figure S4E, S4F) blocked YAP target gene induction by miR-130a mimic (Figure 4C, 4D), while knockdown of VGLL4 (Supplementary information, Figure S4G) rescued YAP target gene expression in the presence of miR-130a inhibitor (Figure 4E). Thus, miR-130a promotes Hippo pathway transcriptional output by repressing VGLL4 protein level. Such a mechanism implies that YAP binding to target gene promoters would be regulated by miR-130a. Indeed, we observed an enhanced or suppressed binding of YAP to CTGF promoter by pre-miR-130a and the miR-130a sponge, respectively (Figure 4F). Notably, the binding of TEAD1 to CTGF promoter was not affected. The mechanism would also predict that miR-130a promotes the binding between YAP and TEADs. By immunoprecipitating endogenous YAP or TEAD1 from miR-130a precursor or sponge expressing cells we indeed observed that pre-miR130a inhibited VGLL4-TEAD1 interaction and promoted YAP-TEAD1 interaction while miR-130a sponge promoted VGLL4-TEAD1 interaction and repressed YAP-TEAD1 interaction (Figure 4G). Taken together, miR-130a is not only a Hippo pathway target but also an activator of YAP.


A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis.

Shen S, Guo X, Yan H, Lu Y, Ji X, Li L, Liang T, Zhou D, Feng XH, Zhao JC, Yu J, Gong XG, Zhang L, Zhao B - Cell Res. (2015)

miR-130a activates YAP through repression of VGLL4. (A) miR-130a activates YAP transcriptional activity. CTGF-luciferase reporter was co-transfected with other plasmids into HEK293T cells as indicated for luciferase assay. Experiments were performed in duplicates. (B) miR-130a inhibition suppresses YAP transcriptional activity in a VGLL4-dependent manner. HEK293T cells stably expressing CTGF-luciferase reporter were transfected as indicated for luciferase assay. Experiments were performed in duplicates. * indicates a P-value of < 0.05 as calculated by student's t-test. (C) miR-130a promotes YAP target gene expression, which is blocked by VGLL4. Control or VGLL4-overexpressing HepG2 cells were transfected with NC or miR-130a mimic for gene expression analysis. Experiments were performed in triplicates. (D) miR-130a promotes Hippo pathway target gene expression in a YAP-and-TAZ-dependent manner. HepG2 cells were transfected as indicated for gene expression assay. Experiments were performed in triplicates. (E) miR-130a inhibition suppresses YAP target gene expression in a VGLL4-dependent manner. HMLE cells were transfected and gene expression was determined. Experiments were performed in triplicates. (F) miR-130a regulates YAP binding to target gene promoter. ChIP was performed using control IgG or antibodies against TEAD1 and YAP on HepG2 stable cell lysates. Relative enrichment of CTGF promoter was determined by quantitative PCR in triplicates. (G) miR-130a regulates YAP-TEAD1 interaction. HepG2 cells stably expressing miR-130a precursor or sponge and respective empty vectors were lysed and immunoprecipitated with anti-YAP or anti-TEAD1 antibodies. Lysates and immunoprecipitates were analyzed by western blots.
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fig4: miR-130a activates YAP through repression of VGLL4. (A) miR-130a activates YAP transcriptional activity. CTGF-luciferase reporter was co-transfected with other plasmids into HEK293T cells as indicated for luciferase assay. Experiments were performed in duplicates. (B) miR-130a inhibition suppresses YAP transcriptional activity in a VGLL4-dependent manner. HEK293T cells stably expressing CTGF-luciferase reporter were transfected as indicated for luciferase assay. Experiments were performed in duplicates. * indicates a P-value of < 0.05 as calculated by student's t-test. (C) miR-130a promotes YAP target gene expression, which is blocked by VGLL4. Control or VGLL4-overexpressing HepG2 cells were transfected with NC or miR-130a mimic for gene expression analysis. Experiments were performed in triplicates. (D) miR-130a promotes Hippo pathway target gene expression in a YAP-and-TAZ-dependent manner. HepG2 cells were transfected as indicated for gene expression assay. Experiments were performed in triplicates. (E) miR-130a inhibition suppresses YAP target gene expression in a VGLL4-dependent manner. HMLE cells were transfected and gene expression was determined. Experiments were performed in triplicates. (F) miR-130a regulates YAP binding to target gene promoter. ChIP was performed using control IgG or antibodies against TEAD1 and YAP on HepG2 stable cell lysates. Relative enrichment of CTGF promoter was determined by quantitative PCR in triplicates. (G) miR-130a regulates YAP-TEAD1 interaction. HepG2 cells stably expressing miR-130a precursor or sponge and respective empty vectors were lysed and immunoprecipitated with anti-YAP or anti-TEAD1 antibodies. Lysates and immunoprecipitates were analyzed by western blots.
Mentions: VGLL4 is a repressor of YAP activity through competition for TEAD binding. Therefore, by repressing VGLL4, the YAP target gene miR-130a could potentially act as an activator of YAP. Indeed, expression of miR-130a activated a CTGF reporter, a well-defined readout of YAP transcriptional activity (Figure 4A and Supplementary information, Figure S4A). However, co-expression of miR-130a-insensitive VGLL4 from constructs lacking the 3′UTR normalized the reporter activity dose-dependently (Figure 4A). More importantly, inhibition of miR-130a attenuated CTGF reporter activity in a VGLL4-dependent manner (Figure 4B and Supplementary information, Figure S4A), suggesting that YAP is regulated by endogenous miR-130a through VGLL4. Furthermore, expression of YAP target genes CTGF and Cyr61 was enhanced or repressed by activation or inhibition of miR-130a, respectively (Supplementary information, Figure S4B, S4C, S4D). Consistently, VGLL4 overexpression or YAP and TAZ knockdown (Supplementary information, Figure S4E, S4F) blocked YAP target gene induction by miR-130a mimic (Figure 4C, 4D), while knockdown of VGLL4 (Supplementary information, Figure S4G) rescued YAP target gene expression in the presence of miR-130a inhibitor (Figure 4E). Thus, miR-130a promotes Hippo pathway transcriptional output by repressing VGLL4 protein level. Such a mechanism implies that YAP binding to target gene promoters would be regulated by miR-130a. Indeed, we observed an enhanced or suppressed binding of YAP to CTGF promoter by pre-miR-130a and the miR-130a sponge, respectively (Figure 4F). Notably, the binding of TEAD1 to CTGF promoter was not affected. The mechanism would also predict that miR-130a promotes the binding between YAP and TEADs. By immunoprecipitating endogenous YAP or TEAD1 from miR-130a precursor or sponge expressing cells we indeed observed that pre-miR130a inhibited VGLL4-TEAD1 interaction and promoted YAP-TEAD1 interaction while miR-130a sponge promoted VGLL4-TEAD1 interaction and repressed YAP-TEAD1 interaction (Figure 4G). Taken together, miR-130a is not only a Hippo pathway target but also an activator of YAP.

Bottom Line: Organ size determination is one of the most intriguing unsolved mysteries in biology.Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals.Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute and Innovation Center for Cell Signaling Network Hangzhou, Zhejiang 310058, China.

ABSTRACT
Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus