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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

miR-125a-3p and miR-483-5p synergistically regulate the RhoA/ROCK1/ERK1/2 pathway.(A) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of total ERK1/2 (T-EKR1/2) and phosphorylated ERK1/2 (T-p-EKR1/2). (B) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. (C) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Total protein was extracted for immunoblotting of T-EKR1/2 and T-p-EKR1/2. (D) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. β-actin and PCNA served as loading controls. (E) hADSCs were transfected with miR-125a-3p agomir, pCEP4L-ERK1, miR-125a-3p agomir plus pCEP4L-ERK1, agomir-NC or pCEP4L-NC and induced for differentiation for 12days. Lipid droplet accumulation stained by Oil red O was observed under a microscope. (F) The hADSCs were induced differentiation for 12 days, the protein levels of PPARγ and C/EBPα were detected by western blot. β-Actin served as a loading control.
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f5: miR-125a-3p and miR-483-5p synergistically regulate the RhoA/ROCK1/ERK1/2 pathway.(A) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of total ERK1/2 (T-EKR1/2) and phosphorylated ERK1/2 (T-p-EKR1/2). (B) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. (C) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Total protein was extracted for immunoblotting of T-EKR1/2 and T-p-EKR1/2. (D) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. β-actin and PCNA served as loading controls. (E) hADSCs were transfected with miR-125a-3p agomir, pCEP4L-ERK1, miR-125a-3p agomir plus pCEP4L-ERK1, agomir-NC or pCEP4L-NC and induced for differentiation for 12days. Lipid droplet accumulation stained by Oil red O was observed under a microscope. (F) The hADSCs were induced differentiation for 12 days, the protein levels of PPARγ and C/EBPα were detected by western blot. β-Actin served as a loading control.

Mentions: To determine whether miR-125a-3p and miR-483-5p jointly regulate the activity of the RhoA/ROCK1/ERK1/2 pathway, we first detected the expression of T-ERK1/2, T-p-ERK1/2, n-T-ERK1/2, and n-p-ERK1/2 following transfection of hADSCs with the mimic or inhibitor of miR-125a-3p. Transfection with either the mimic or inhibitor of miR-125a-3p did not alter the T-ERK1/2 or T-p-ERK1/2 expression levels (Fig. 5A). However, the miR-125a-3p mimic apparently decreased the levels of n-T-ERK1/2 and n-p-ERK1/2; while the miR-125a-3p inhibitor increased the levels of n-T-ERK1/2 and n-p-ERK1/2 (Fig. 5B). Next, we cotransfected the miR-125a-3p inhibitor and miR-483-5p mimic to test ERK1/2 expression in hADSCs. In comparison to the control and miR-125a-3p inhibitor, cotransfection with the miR-125a-3p inhibitor and miR-483-5p mimic resulted in decreased expression of T-ERK1/2 and T-p-ERK1/2 to the levels of the miR-483-5p mimic group (Fig. 5C). Moreover, n-T-ERK1/2 and n-p-ERK1/2 were downregulated in the cotransfected group compared to that of the miR-125a-3p inhibitor group while upregulated compared to that of the miR-483-5p mimic group (Fig. 5D). Next, we transfected hADSCs with miR-125a-3p agomir or pCEP4L-ERK1, or cotransfected with the miR-125a-3p agomir and pCEP4L-ERK1 and induced differentiation for 12 days. miR-125a-3p agomir significantly promoted adipogenesis, and pCEP4L-ERK1 almost completely inhibited adipogenesis; however, cotransfection group partially restored adipogenesis (Fig. 5E). Similar protein expression of PPARγ and C/EBPα were observed in mature adipogenesis and cotransfection group increased PPARγ and C/EBPα compared to pCEP4L-ERK1 (Fig. 5F). These results suggested that miR-125a-3p and miR-483-5p might jointly regulate the activity of the RhoA/ROCK1/ERK1/2 signaling pathway.


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)

miR-125a-3p and miR-483-5p synergistically regulate the RhoA/ROCK1/ERK1/2 pathway.(A) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of total ERK1/2 (T-EKR1/2) and phosphorylated ERK1/2 (T-p-EKR1/2). (B) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. (C) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Total protein was extracted for immunoblotting of T-EKR1/2 and T-p-EKR1/2. (D) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. β-actin and PCNA served as loading controls. (E) hADSCs were transfected with miR-125a-3p agomir, pCEP4L-ERK1, miR-125a-3p agomir plus pCEP4L-ERK1, agomir-NC or pCEP4L-NC and induced for differentiation for 12days. Lipid droplet accumulation stained by Oil red O was observed under a microscope. (F) The hADSCs were induced differentiation for 12 days, the protein levels of PPARγ and C/EBPα were detected by western blot. β-Actin served as a loading control.
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f5: miR-125a-3p and miR-483-5p synergistically regulate the RhoA/ROCK1/ERK1/2 pathway.(A) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Total protein was extracted for immunoblotting of total ERK1/2 (T-EKR1/2) and phosphorylated ERK1/2 (T-p-EKR1/2). (B) hADSCs were transfected with the miR-125a-3p mimic, inhibitor, and controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. (C) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Total protein was extracted for immunoblotting of T-EKR1/2 and T-p-EKR1/2. (D) hADSCs were transfected with the miR-125a-3p inhibitor, miR-483-5p mimic, miR-125a-3p inhibitor plus miR-483-5p mimic, or controls for 72 h. Nuclear protein was extracted for immunoblotting of n-T-ERK1/2 and n-p-ERK1/2. β-actin and PCNA served as loading controls. (E) hADSCs were transfected with miR-125a-3p agomir, pCEP4L-ERK1, miR-125a-3p agomir plus pCEP4L-ERK1, agomir-NC or pCEP4L-NC and induced for differentiation for 12days. Lipid droplet accumulation stained by Oil red O was observed under a microscope. (F) The hADSCs were induced differentiation for 12 days, the protein levels of PPARγ and C/EBPα were detected by western blot. β-Actin served as a loading control.
Mentions: To determine whether miR-125a-3p and miR-483-5p jointly regulate the activity of the RhoA/ROCK1/ERK1/2 pathway, we first detected the expression of T-ERK1/2, T-p-ERK1/2, n-T-ERK1/2, and n-p-ERK1/2 following transfection of hADSCs with the mimic or inhibitor of miR-125a-3p. Transfection with either the mimic or inhibitor of miR-125a-3p did not alter the T-ERK1/2 or T-p-ERK1/2 expression levels (Fig. 5A). However, the miR-125a-3p mimic apparently decreased the levels of n-T-ERK1/2 and n-p-ERK1/2; while the miR-125a-3p inhibitor increased the levels of n-T-ERK1/2 and n-p-ERK1/2 (Fig. 5B). Next, we cotransfected the miR-125a-3p inhibitor and miR-483-5p mimic to test ERK1/2 expression in hADSCs. In comparison to the control and miR-125a-3p inhibitor, cotransfection with the miR-125a-3p inhibitor and miR-483-5p mimic resulted in decreased expression of T-ERK1/2 and T-p-ERK1/2 to the levels of the miR-483-5p mimic group (Fig. 5C). Moreover, n-T-ERK1/2 and n-p-ERK1/2 were downregulated in the cotransfected group compared to that of the miR-125a-3p inhibitor group while upregulated compared to that of the miR-483-5p mimic group (Fig. 5D). Next, we transfected hADSCs with miR-125a-3p agomir or pCEP4L-ERK1, or cotransfected with the miR-125a-3p agomir and pCEP4L-ERK1 and induced differentiation for 12 days. miR-125a-3p agomir significantly promoted adipogenesis, and pCEP4L-ERK1 almost completely inhibited adipogenesis; however, cotransfection group partially restored adipogenesis (Fig. 5E). Similar protein expression of PPARγ and C/EBPα were observed in mature adipogenesis and cotransfection group increased PPARγ and C/EBPα compared to pCEP4L-ERK1 (Fig. 5F). These results suggested that miR-125a-3p and miR-483-5p might jointly regulate the activity of the RhoA/ROCK1/ERK1/2 signaling pathway.

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