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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 represses VGLL4. (A) Conservation of miR-130a binding sites in VGLL4 3′UTR in vertebrates. VGLL4 3′UTR from different species are aligned together with miR-130a. The seed region and its matching nucleotides are in red. Base paring is indicated by a line. Mutation of VGLL4 3′UTR sensor is also shown. (B) miR-130a regulates VGLL4 sensor activity. HEK293T cells were transfected with WT or mutant VGLL4 3′UTR sensor together with miR-130a mimic or inhibitor. Sensor activities were determined by luciferase assay. Experiments were performed in duplicates. * and *** indicate a P-value of < 0.05 and < 0.001, respectively, as calculated by student's t-test. (C, D) miR-130a represses VGLL4 protein level. Cells were transfected with NC or miR-130a mimic (C) or infected with empty vector or pre-miR-130a (D). Cell lysates were subjected to immunoblotting. (E, F) miR-130a inhibition increases VGLL4 protein level. Cells were transfected with NC or miR-130a inhibitor (E) or infected with empty vector or the miR-130a sponge (F) and analyzed for VGLL4 protein level. (G) miR-130a promotes cell proliferation by repressing VGLL4. Cells were transduced with pre-miR-130a and VGLL4 (left panel, HMLE cells) or transfected with miR-130a inhibitor and VGLL4 siRNA (right panel, HepG2 cells) as indicated. Cell viability was determined by CellTiter-Blue assay.
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fig3: miR-130a represses VGLL4. (A) Conservation of miR-130a binding sites in VGLL4 3′UTR in vertebrates. VGLL4 3′UTR from different species are aligned together with miR-130a. The seed region and its matching nucleotides are in red. Base paring is indicated by a line. Mutation of VGLL4 3′UTR sensor is also shown. (B) miR-130a regulates VGLL4 sensor activity. HEK293T cells were transfected with WT or mutant VGLL4 3′UTR sensor together with miR-130a mimic or inhibitor. Sensor activities were determined by luciferase assay. Experiments were performed in duplicates. * and *** indicate a P-value of < 0.05 and < 0.001, respectively, as calculated by student's t-test. (C, D) miR-130a represses VGLL4 protein level. Cells were transfected with NC or miR-130a mimic (C) or infected with empty vector or pre-miR-130a (D). Cell lysates were subjected to immunoblotting. (E, F) miR-130a inhibition increases VGLL4 protein level. Cells were transfected with NC or miR-130a inhibitor (E) or infected with empty vector or the miR-130a sponge (F) and analyzed for VGLL4 protein level. (G) miR-130a promotes cell proliferation by repressing VGLL4. Cells were transduced with pre-miR-130a and VGLL4 (left panel, HMLE cells) or transfected with miR-130a inhibitor and VGLL4 siRNA (right panel, HepG2 cells) as indicated. Cell viability was determined by CellTiter-Blue assay.

Mentions: To dissect how miR-130a functions downstream of YAP, we used the Targetscan algorithm to predict potential miR-130a targets (Supplementary information, Table S1). Interestingly, one of the hits was VGLL4, a known YAP inhibitor. In line with miR-130a as a vertebrate-specific microRNA, a miR-130a seed-binding site in VGLL4 3′UTR is conserved in vertebrates, suggesting its functional importance (Figure 3A). To determine the functionality of this predicted site, we constructed a VGLL4 3′UTR sensor. Despite substantial repression and activation of the wild-type (WT) sensor by miR-130a mimic and inhibitor, respectively, the seed-matching region mutant sensor remained unresponsive (Figure 3B). Therefore, miR-130a could specifically bind to VGLL4 3′UTR. Furthermore, transfection of miR-130a mimic or expression of pre-miR-130a substantially repressed endogenous VGLL4 protein level (Figure 3C, 3D) as indicated by a validated antibody (Supplementary information, Figure S3A). More importantly, inhibition of endogenous miR-130a by an inhibitor antisense oligomer or microRNA sponge clearly increased VGLL4 protein level in multiple cell lines from different tissue origins (Figure 3E, 3F). However, miR-130a did not significantly alter VGLL4 mRNA level (Supplementary information, Figure S3B), suggesting a mechanism of translation repression. In support of the functional importance of VGLL4 as a miR-130a target, miR-130a-induced cell proliferation was blocked by VGLL4 overexpression (Figure 3G). Furthermore, knockdown of VGLL4 rescued cell proliferation defects caused by miR-130a inhibition (Figure 3G). Taken together, VGLL4 is a miR-130a target critical for growth regulation.


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 represses VGLL4. (A) Conservation of miR-130a binding sites in VGLL4 3′UTR in vertebrates. VGLL4 3′UTR from different species are aligned together with miR-130a. The seed region and its matching nucleotides are in red. Base paring is indicated by a line. Mutation of VGLL4 3′UTR sensor is also shown. (B) miR-130a regulates VGLL4 sensor activity. HEK293T cells were transfected with WT or mutant VGLL4 3′UTR sensor together with miR-130a mimic or inhibitor. Sensor activities were determined by luciferase assay. Experiments were performed in duplicates. * and *** indicate a P-value of < 0.05 and < 0.001, respectively, as calculated by student's t-test. (C, D) miR-130a represses VGLL4 protein level. Cells were transfected with NC or miR-130a mimic (C) or infected with empty vector or pre-miR-130a (D). Cell lysates were subjected to immunoblotting. (E, F) miR-130a inhibition increases VGLL4 protein level. Cells were transfected with NC or miR-130a inhibitor (E) or infected with empty vector or the miR-130a sponge (F) and analyzed for VGLL4 protein level. (G) miR-130a promotes cell proliferation by repressing VGLL4. Cells were transduced with pre-miR-130a and VGLL4 (left panel, HMLE cells) or transfected with miR-130a inhibitor and VGLL4 siRNA (right panel, HepG2 cells) as indicated. Cell viability was determined by CellTiter-Blue assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: miR-130a represses VGLL4. (A) Conservation of miR-130a binding sites in VGLL4 3′UTR in vertebrates. VGLL4 3′UTR from different species are aligned together with miR-130a. The seed region and its matching nucleotides are in red. Base paring is indicated by a line. Mutation of VGLL4 3′UTR sensor is also shown. (B) miR-130a regulates VGLL4 sensor activity. HEK293T cells were transfected with WT or mutant VGLL4 3′UTR sensor together with miR-130a mimic or inhibitor. Sensor activities were determined by luciferase assay. Experiments were performed in duplicates. * and *** indicate a P-value of < 0.05 and < 0.001, respectively, as calculated by student's t-test. (C, D) miR-130a represses VGLL4 protein level. Cells were transfected with NC or miR-130a mimic (C) or infected with empty vector or pre-miR-130a (D). Cell lysates were subjected to immunoblotting. (E, F) miR-130a inhibition increases VGLL4 protein level. Cells were transfected with NC or miR-130a inhibitor (E) or infected with empty vector or the miR-130a sponge (F) and analyzed for VGLL4 protein level. (G) miR-130a promotes cell proliferation by repressing VGLL4. Cells were transduced with pre-miR-130a and VGLL4 (left panel, HMLE cells) or transfected with miR-130a inhibitor and VGLL4 siRNA (right panel, HepG2 cells) as indicated. Cell viability was determined by CellTiter-Blue assay.
Mentions: To dissect how miR-130a functions downstream of YAP, we used the Targetscan algorithm to predict potential miR-130a targets (Supplementary information, Table S1). Interestingly, one of the hits was VGLL4, a known YAP inhibitor. In line with miR-130a as a vertebrate-specific microRNA, a miR-130a seed-binding site in VGLL4 3′UTR is conserved in vertebrates, suggesting its functional importance (Figure 3A). To determine the functionality of this predicted site, we constructed a VGLL4 3′UTR sensor. Despite substantial repression and activation of the wild-type (WT) sensor by miR-130a mimic and inhibitor, respectively, the seed-matching region mutant sensor remained unresponsive (Figure 3B). Therefore, miR-130a could specifically bind to VGLL4 3′UTR. Furthermore, transfection of miR-130a mimic or expression of pre-miR-130a substantially repressed endogenous VGLL4 protein level (Figure 3C, 3D) as indicated by a validated antibody (Supplementary information, Figure S3A). More importantly, inhibition of endogenous miR-130a by an inhibitor antisense oligomer or microRNA sponge clearly increased VGLL4 protein level in multiple cell lines from different tissue origins (Figure 3E, 3F). However, miR-130a did not significantly alter VGLL4 mRNA level (Supplementary information, Figure S3B), suggesting a mechanism of translation repression. In support of the functional importance of VGLL4 as a miR-130a target, miR-130a-induced cell proliferation was blocked by VGLL4 overexpression (Figure 3G). Furthermore, knockdown of VGLL4 rescued cell proliferation defects caused by miR-130a inhibition (Figure 3G). Taken together, VGLL4 is a miR-130a target critical for growth regulation.

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