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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 regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis.hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, (A) De novo adipose tissue formation was observed (n = 5); (B) The weights of the de novo adipose tissues were measured (n = 5); (C) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); (D) The de novo adipose tissue was stained to observe the histology (n = 5); (E) Adipoctye size were analyzed by ImageJ software (F) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and (G) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p < 0.05, **p < 0.01 compared to control or LV-NC group. Δp < 0.01 compared to miR-125a-3p or miR-483-3p group.
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f6: miR-125a-3p and miR-483-5p regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis.hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, (A) De novo adipose tissue formation was observed (n = 5); (B) The weights of the de novo adipose tissues were measured (n = 5); (C) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); (D) The de novo adipose tissue was stained to observe the histology (n = 5); (E) Adipoctye size were analyzed by ImageJ software (F) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and (G) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p < 0.05, **p < 0.01 compared to control or LV-NC group. Δp < 0.01 compared to miR-125a-3p or miR-483-3p group.

Mentions: To investigate the roles of miR-125a-3p and miR-483-5p in regulating adipogenesis in vivo, we transfected hADSCs with lentivirus containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), LV-miR-125a + 483 or negative control miR (LV-NC). Almost 100% transfection efficiency was verified by fluorescence microscopy (data not shown). Each group of cells were transplanted the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, we observed the de novo adipose tissue formation. No de novo adipose tissue formation was observed in the PBS group. Interestingly, capsular de novo adipose tissues were found in the LV-miR-125a, LV-miR-483, and cotransfection group; however, no capsular formation was seen in the Con or LV-NC groups. The de novo adipose tissue formation in LV-miR-125a, LV-miR-483 and cotransfection group were much larger than that of the Con or LV-NC groups, especially in cotransfection group (Fig. 6A,B). In the de novo adipose tissues of LV-miR-125a or LV-miR-483 group, we found that miR-125a-3p and miR-483-5p expression was significantly higher than miR-125a-5p and miR-483-3p expression (Fig. 6C). In addition, adipose tissue staining showed cell size of miR-125a or miR-483 was bigger than Con or LV-NC group, but smaller than cotransfection group (Fig. 6D,E). Lastly, we assessed the activity of the RhoA/ROCK1/ERK1/2 pathway. In the LV-miR-125a group, there was no apparent alteration of the T-ERK1/2 and T-p-ERK1/2 levels, but the RhoA and ROCK1 levels were decreased. In contrast, T-ERK1/2 and T-p-ERK1/2 were both decreased in the LV-miR-483 and cotransfection group compared to that of the control, LV-NC, or LV-miR-125a groups, while LV-miR-483 did not affect RhoA and ROCK1 levels (Fig. 6F). n-T-ERK1/2 and n-p-ERK1/2 were downregulated in the LV-miR-125a and LV-miR-483 groups, and even greater downregulation was observed in the cotransfection group compared to the LV-miR-125a and LV-miR-483 groups (Fig. 6G). Our results suggest that miR-125a-3p and miR-483-5p may jointly promote adipogenesis in vivo through regulating the RhoA/ROCK1/ERK1/2 pathway, at least in part.


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 regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis.hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, (A) De novo adipose tissue formation was observed (n = 5); (B) The weights of the de novo adipose tissues were measured (n = 5); (C) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); (D) The de novo adipose tissue was stained to observe the histology (n = 5); (E) Adipoctye size were analyzed by ImageJ software (F) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and (G) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p < 0.05, **p < 0.01 compared to control or LV-NC group. Δp < 0.01 compared to miR-125a-3p or miR-483-3p group.
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f6: miR-125a-3p and miR-483-5p regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis.hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, (A) De novo adipose tissue formation was observed (n = 5); (B) The weights of the de novo adipose tissues were measured (n = 5); (C) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); (D) The de novo adipose tissue was stained to observe the histology (n = 5); (E) Adipoctye size were analyzed by ImageJ software (F) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and (G) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p < 0.05, **p < 0.01 compared to control or LV-NC group. Δp < 0.01 compared to miR-125a-3p or miR-483-3p group.
Mentions: To investigate the roles of miR-125a-3p and miR-483-5p in regulating adipogenesis in vivo, we transfected hADSCs with lentivirus containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), LV-miR-125a + 483 or negative control miR (LV-NC). Almost 100% transfection efficiency was verified by fluorescence microscopy (data not shown). Each group of cells were transplanted the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, we observed the de novo adipose tissue formation. No de novo adipose tissue formation was observed in the PBS group. Interestingly, capsular de novo adipose tissues were found in the LV-miR-125a, LV-miR-483, and cotransfection group; however, no capsular formation was seen in the Con or LV-NC groups. The de novo adipose tissue formation in LV-miR-125a, LV-miR-483 and cotransfection group were much larger than that of the Con or LV-NC groups, especially in cotransfection group (Fig. 6A,B). In the de novo adipose tissues of LV-miR-125a or LV-miR-483 group, we found that miR-125a-3p and miR-483-5p expression was significantly higher than miR-125a-5p and miR-483-3p expression (Fig. 6C). In addition, adipose tissue staining showed cell size of miR-125a or miR-483 was bigger than Con or LV-NC group, but smaller than cotransfection group (Fig. 6D,E). Lastly, we assessed the activity of the RhoA/ROCK1/ERK1/2 pathway. In the LV-miR-125a group, there was no apparent alteration of the T-ERK1/2 and T-p-ERK1/2 levels, but the RhoA and ROCK1 levels were decreased. In contrast, T-ERK1/2 and T-p-ERK1/2 were both decreased in the LV-miR-483 and cotransfection group compared to that of the control, LV-NC, or LV-miR-125a groups, while LV-miR-483 did not affect RhoA and ROCK1 levels (Fig. 6F). n-T-ERK1/2 and n-p-ERK1/2 were downregulated in the LV-miR-125a and LV-miR-483 groups, and even greater downregulation was observed in the cotransfection group compared to the LV-miR-125a and LV-miR-483 groups (Fig. 6G). Our results suggest that miR-125a-3p and miR-483-5p may jointly promote adipogenesis in vivo through regulating the RhoA/ROCK1/ERK1/2 pathway, at least in part.

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