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miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis.

Chen K, He H, Xie Y, Zhao L, Zhao S, Wan X, Yang W, Mo Z - Sci Rep (2015)

Bottom Line: Most importantly, we found that miR-125a-3p and miR-483-5p promoted de novo adipose tissue formation in nude mice.These results demonstrated that miR-125a-3p and miR-483-5p coordinately promoted adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway.Our findings may provide novel strategies for the management and treatment of MSL or obesity.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Third Xiangya Hospital of Central South University, Changsha, Hunan, 410013, China.

ABSTRACT
Multiple symmetric lipomatosis (MSL) is a rare disease characterized by symmetric and abnormal distribution of subcutaneous adipose tissue (SAT); however, the etiology is largely unknown. We report here that miR-125a-3p and miR-483-5p are upregulated in the SAT of MSL patients, promoting adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. TaqMan microRNA (miR) array analysis revealed that 18 miRs were upregulated in the SAT of MSL patients. Transfection of human adipose-derived mesenchymal stem cells (hADSCs) with the individual agomirs of these 18 miRs showed that miR-125a-3p and miR-483-5p significantly promoted adipogenesis. A dual-luciferase assay showed that RhoA and ERK1 were the targets of miR-125a-3p and miR-483-5p, respectively. Moreover, transfection of hADSCs with mimics of miR-125a-3p and miR-483-5p resulted in a pronounced decrease of ERK1/2 phosphorylation in the nucleus; conversely, transfection of hADSCs with inhibitors of miR-125a-3p and miR-483-5p led to a significant increase of ERK1/2 phosphorylation in the nucleus. Most importantly, we found that miR-125a-3p and miR-483-5p promoted de novo adipose tissue formation in nude mice. These results demonstrated that miR-125a-3p and miR-483-5p coordinately promoted adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. Our findings may provide novel strategies for the management and treatment of MSL or obesity.

No MeSH data available.


Related in: MedlinePlus

RhoA and ERK1 are the respective target genes of miR-125a-3p and miR-483-5p.(A) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-RhoA-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-RhoA-3′-UTR) was cotransfected with the miR-125a-3p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (B) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-ERK1-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-ERK1-3′-UTR) was cotransfected with the miR-483-5p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (C) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of RhoA and ROCK1. (D) hADSCs were transfected with the miR-125a-3p agomir, antagomir, and controls for 72 h, and induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA and ROCK1. (E) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole cell protein was extracted for immunoblotting to detect total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2). (F) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole nuclear protein was extracted to detect nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2).*p < 0.05, compared to 293T cells transfected with miR control mimics. All measurements were preformed by three independent experiments.
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f3: RhoA and ERK1 are the respective target genes of miR-125a-3p and miR-483-5p.(A) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-RhoA-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-RhoA-3′-UTR) was cotransfected with the miR-125a-3p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (B) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-ERK1-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-ERK1-3′-UTR) was cotransfected with the miR-483-5p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (C) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of RhoA and ROCK1. (D) hADSCs were transfected with the miR-125a-3p agomir, antagomir, and controls for 72 h, and induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA and ROCK1. (E) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole cell protein was extracted for immunoblotting to detect total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2). (F) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole nuclear protein was extracted to detect nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2).*p < 0.05, compared to 293T cells transfected with miR control mimics. All measurements were preformed by three independent experiments.

Mentions: To further support previous observations1827, we designed different mutant site sequences in the 3′-UTR of RhoA and ERK1 and verified the targeting of RhoA and ERK1 by miR-125a-3p and miR-483-5p, respectively. We found that the miR-125a-3p mimic significantly downregulated the luciferase activity of WT-PmiR-RhoA-3′-UTR compared to the control mimic. In sharp contrast, the miR-125a-3p mimic had no effect on the luciferase activity of MUT-PmiR-RhoA-3′-UTR (Fig. 3A). Similar results were observed in miR-483-5p and ERK1 (Fig. 3B). Next, we transfected hADSCs with the miR-125a-3p or miR-483-5p mimic or inhibitor for 72 h and determined the protein levels of RhoA or ERK1/2 by immunoblotting. The results showed that the miR-125a-3p mimic significantly downregulated RhoA and its downstream factor ROCK1 and ROCK2; conversely, the miR-125a-3p inhibitor significantly upregulated RhoA, ROCK1 and ROCK2 (Fig. 3C). Similar results were observed in mature adipocytes after hADSCs were transfected with the miR-125a-3p agomir or antagomir for 72 h and induced for 12 days (Fig. 3D). Total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2) from whole cell protein lysate were downregulated by the miR-483-5p mimic, while they were upregulated by the miR-483-5p inhibitor in hADSCs following transfection for 72 h (Fig. 3E). Similar results were found for nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2) in whole nuclear protein lysate (Fig. 3F). We did not investigate the protein expression T-p-ERK1/2, n-T-ERK1/2 and n-p-ERK1/2 due to their very low expression in mature adipoctye. These results clearly demonstrated that RhoA and ERK1 are the direct target genes of miR-125a-3p and miR-483-5p, respectively.


miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis.

Chen K, He H, Xie Y, Zhao L, Zhao S, Wan X, Yang W, Mo Z - Sci Rep (2015)

RhoA and ERK1 are the respective target genes of miR-125a-3p and miR-483-5p.(A) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-RhoA-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-RhoA-3′-UTR) was cotransfected with the miR-125a-3p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (B) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-ERK1-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-ERK1-3′-UTR) was cotransfected with the miR-483-5p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (C) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of RhoA and ROCK1. (D) hADSCs were transfected with the miR-125a-3p agomir, antagomir, and controls for 72 h, and induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA and ROCK1. (E) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole cell protein was extracted for immunoblotting to detect total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2). (F) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole nuclear protein was extracted to detect nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2).*p < 0.05, compared to 293T cells transfected with miR control mimics. All measurements were preformed by three independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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f3: RhoA and ERK1 are the respective target genes of miR-125a-3p and miR-483-5p.(A) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-RhoA-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-RhoA-3′-UTR) was cotransfected with the miR-125a-3p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (B) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-ERK1-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-ERK1-3′-UTR) was cotransfected with the miR-483-5p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (C) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of RhoA and ROCK1. (D) hADSCs were transfected with the miR-125a-3p agomir, antagomir, and controls for 72 h, and induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA and ROCK1. (E) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole cell protein was extracted for immunoblotting to detect total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2). (F) hADSCs were transfected with the miR-483-5p mimic, inhibitor, and controls for 72 h. Whole nuclear protein was extracted to detect nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2).*p < 0.05, compared to 293T cells transfected with miR control mimics. All measurements were preformed by three independent experiments.
Mentions: To further support previous observations1827, we designed different mutant site sequences in the 3′-UTR of RhoA and ERK1 and verified the targeting of RhoA and ERK1 by miR-125a-3p and miR-483-5p, respectively. We found that the miR-125a-3p mimic significantly downregulated the luciferase activity of WT-PmiR-RhoA-3′-UTR compared to the control mimic. In sharp contrast, the miR-125a-3p mimic had no effect on the luciferase activity of MUT-PmiR-RhoA-3′-UTR (Fig. 3A). Similar results were observed in miR-483-5p and ERK1 (Fig. 3B). Next, we transfected hADSCs with the miR-125a-3p or miR-483-5p mimic or inhibitor for 72 h and determined the protein levels of RhoA or ERK1/2 by immunoblotting. The results showed that the miR-125a-3p mimic significantly downregulated RhoA and its downstream factor ROCK1 and ROCK2; conversely, the miR-125a-3p inhibitor significantly upregulated RhoA, ROCK1 and ROCK2 (Fig. 3C). Similar results were observed in mature adipocytes after hADSCs were transfected with the miR-125a-3p agomir or antagomir for 72 h and induced for 12 days (Fig. 3D). Total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2) from whole cell protein lysate were downregulated by the miR-483-5p mimic, while they were upregulated by the miR-483-5p inhibitor in hADSCs following transfection for 72 h (Fig. 3E). Similar results were found for nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2) in whole nuclear protein lysate (Fig. 3F). We did not investigate the protein expression T-p-ERK1/2, n-T-ERK1/2 and n-p-ERK1/2 due to their very low expression in mature adipoctye. These results clearly demonstrated that RhoA and ERK1 are the direct target genes of miR-125a-3p and miR-483-5p, respectively.

Bottom Line: Most importantly, we found that miR-125a-3p and miR-483-5p promoted de novo adipose tissue formation in nude mice.These results demonstrated that miR-125a-3p and miR-483-5p coordinately promoted adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway.Our findings may provide novel strategies for the management and treatment of MSL or obesity.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Third Xiangya Hospital of Central South University, Changsha, Hunan, 410013, China.

ABSTRACT
Multiple symmetric lipomatosis (MSL) is a rare disease characterized by symmetric and abnormal distribution of subcutaneous adipose tissue (SAT); however, the etiology is largely unknown. We report here that miR-125a-3p and miR-483-5p are upregulated in the SAT of MSL patients, promoting adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. TaqMan microRNA (miR) array analysis revealed that 18 miRs were upregulated in the SAT of MSL patients. Transfection of human adipose-derived mesenchymal stem cells (hADSCs) with the individual agomirs of these 18 miRs showed that miR-125a-3p and miR-483-5p significantly promoted adipogenesis. A dual-luciferase assay showed that RhoA and ERK1 were the targets of miR-125a-3p and miR-483-5p, respectively. Moreover, transfection of hADSCs with mimics of miR-125a-3p and miR-483-5p resulted in a pronounced decrease of ERK1/2 phosphorylation in the nucleus; conversely, transfection of hADSCs with inhibitors of miR-125a-3p and miR-483-5p led to a significant increase of ERK1/2 phosphorylation in the nucleus. Most importantly, we found that miR-125a-3p and miR-483-5p promoted de novo adipose tissue formation in nude mice. These results demonstrated that miR-125a-3p and miR-483-5p coordinately promoted adipogenesis through suppressing the RhoA/ROCK1/ERK1/2 pathway. Our findings may provide novel strategies for the management and treatment of MSL or obesity.

No MeSH data available.


Related in: MedlinePlus