LncRNA HCG11 Inhibits Adipocyte Differentiation in Human Adipose-Derived Mesenchymal Stem Cells by Sponging miR-204-5p to Upregulate SIRT1

Abstract

Long noncoding RNAs (lncRNAs) have been discovered to play a key role in adipogenesis, while the role of lncRNA human leukocyte antigen complex group 11 (HCG11) in adipocyte differentiation has not been studied clearly. We used human adipose-derived mesenchymal stem cells (hAdMSCs) to establish a model of cell differentiation in vitro and found that expression of lncRNA HCG11 was decreased during adipogenesis through real-time quantitative polymerase chain reaction analysis. Then, hAdMSCs were transfected with pcDNA-HCG11 or HCG11-shRNA (sh-HCG11); the adipogenic marker proteins were detected by Western blot, and the activity of lipogenesis enzymes was detected by spectrophotometry. The expression of CCAAT-enhancer-binding protein α, fatty acid-binding protein, peroxisome proliferator-activated receptor gamma 2 and the levels of acetyl coenzyme A carboxylase and fatty acid synthase FAS were significantly downregulated in hAdMSCs at different stages transfected with pcDNA-HCG11, while knockdown of lncRNA HCG11 promoted adipocyte differentiation. Bioinformatic analysis indicated that miR-204-5p was a potential target gene of HCG11, which was confirmed by luciferase reporter gene analysis and RNA pull-down analysis. In addition, miR-204-5p directly targeting the 3'-untranslated region of SIRT1 was also predicted by StarBase and verified by luciferase reporter gene analysis. Enforced expression of miR-204-5p negatively regulated the SIRT1 protein level. Furthermore, SIRT1 overexpression significantly inhibited adipogenic marker protein, levels of lipogenesis enzymes, and the proliferation of hAdMSCs. When pcDNA-HCG11 and miR-204-5p mimic were co-transfected into hAdMSCs, we found that the miR-204-5p mimic reversed the suppressor effect of pcDNA-HCG11. Taken together, we found that HCG11 negatively regulated cell proliferation and adipogenesis by the miR-204-5p/SIRT1 axis. Our findings might provide a new target for the study of adipogenesis in hAdMSCs and obesity.

Keywords: HCG11; SIRT1; adipogenesis; miR-204-5p; obesity.

Figure 1.

Identification of a model that induced hAdMSCs to adipocytes differentiation. (A) Expression of adipogenic marker proteins in different stages (D0, D3, D6, D9, and D12) was detected by Western blot, and GAPDH was used as a loading control in each sample. Expression of (B) C/EBPα, (C) PPARγ2, (D) AdipoQ, (E) FABP4, and (F) LPL were quantified using Image J software. The levels of lipogenesis enzymes, such as (G) ACC and (H) FAS were detected by spectrophotometry. (I) Expression of HCG11 in different stages (D0, D3, D6, D9, and D12) was detected by RT-qPCR. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. **P < 0.01 and ***P < 0.001 versus D0. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; RT-qPCR: real-time quantitative polymerase chain reaction.

Figure 2.

Differentiation of adipocytes overexpressing lncRNA HCG11. (A) Expression of HCG11 in different stages (D0, D6, and D12) of hAdMSCs transfected with pcDNA-HCG11 was detected by RT-qPCR. (B) Expression of adipogenic marker protein was detected by Western blot, and GAPDH was used as a loading control in each sample. The expression of (C) C/EBPα, (D) PPARγ2, (E) AdipoQ, (F) FABP4, and (G) LPL in different stages (D0, D6, and D12) of hAdMSCs transfected with pcDNA-HCG11 and control was quantified using Image J software. The levels of lipogenesis enzymes such as (H) ACC and (I) FAS were detected by spectrophotometry. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control; ^# P < 0.05 and ^## P < 0.01 versus vector. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; RT-qPCR: real-time quantitative polymerase chain reaction.

Figure 3.

Differentiation of adipocytes interfering with sh-HCG11. (A) Expression of HCG11 in different stages (D0, D6, and D12) hAdMSCs transfected with sh-HCG11 was detected by RT-qPCR. (B) Expression of adipogenic marker protein such as (C) C/EBPα, (D) PPARγ2, (E) AdipoQ, (F) FABP4, and (G) LPL was detected by Western blot and quantified using Image J software. The levels of lipogenesis enzymes such as (H) ACC and (I) FAS were detected by spectrophotometry. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control; ^# P < 0.05 and ^## P < 0.01 versus vector. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; RT-qPCR: real-time quantitative polymerase chain reaction.

Figure 4.

HCG11 directly targeted to miR-204-5p. (A) Online database StarBase showed the binding sites of HCG11 and miR-204-5p. (B) The luciferase reporter gene assay was performed in HEK-293T cells to validate the binding of HCG11 and miR-204-5p. Firefly and Renilla luciferase activities were determined. (C) HEK-293T cells were transfected with biotinylated miR-204-5p (Bio-204-5p-wt) or its mutant form (Bio-204-5p-mut), and then a biotin-based pull-down assay was performed to detect HCG11 expression and normalized to a biotinylated mimic control (Bio-NC). (D) Expression of miR-204-5p in hAdMSCs transfected with pcDNA-HCG11 or sh-HCG11 was detected by RT-qPCR. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. **P < 0.01 and ***P < 0.001 versus control; ^# P < 0.05 versus scramble. hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; HEK-293T: human embryonic kidney 293T; RT-qPCR: real-time quantitative polymerase chain reaction.

Figure 5.

Overexpression of miR-204-5p promoted cell proliferation and adipocytes differentiation in hAdMSCs. The hAdMSCs were transfected with NC mimic (20 nM), miR-204-5p mimic (20 nM), NC inhibitor (20 nM), and miR-204-5p inhibitor (20 nM) for 48 h. (A) Expression of miR-204-5p was detected by RT-qPCR. (B) The expression of adipogenic marker protein and inflammatory factor was detected by Western blot. Expression of (C) C/EBPα, (D) PPARγ2, (E) AdipoQ, (F) FABP4, (G) LPL, (H) IL-6, and (I) TNF-α was quantified using Image J software. The levels of lipogenesis enzymes such as ACC (J) and FAS (K) were detected by spectrophotometry. (L) Cell proliferation ability was tested by CCK-8. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; CCK: cell counting kit; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; hAdMSCs: human adipose-derived mesenchymal stem cells; IL-6: interleukin-6; LPL: lipoprotein lipase; NC: normal control; PPARγ: peroxisome proliferator-activated receptor gamma; TNF-α: tumor necrosis factor-alpha.

Figure 6.

MiR-204-5p directly targeted SIRT1. (A) Online database StarBase showed the sequence alignment between miR-204-5p and SIRT1. (B) The luciferase reporter gene assay was performed in HEK-293T cells to validate the binding of miR-204-5p and SIRT1. Firefly and Renilla luciferase activities were determined. (C) Expression of SIRT1 in hAdMSCs transfected with miR-204-5p mimic, inhibitor, and their control was detected by Western blotting. (D) The expression of adipogenic marker protein and inflammatory factor was detected by Western blot. Expression of (E) C/EBPα, (F) PPARγ2, (G) AdipoQ, (H) FABP4, (I) LPL, (J) IL-6, and (K) TNF-α in hAdMSCs transfected with miR-204-5p mimic, Res, and pcDNA-SIRT1. The levels of lipogenesis enzymes such as ACC (L) and FAS (M) were detected by spectrophotometry. (N) Cell proliferation ability was tested by CCK-8. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; CCK: cell counting kit; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; hAdMSCs: human adipose-derived mesenchymal stem cells; IL-6: interleukin-6; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; TNF-α: tumor necrosis factor alpha.

Figure 7.

Overexpression of miR-204-5p reversed the effect of HCG11 on hAdMSCs. (A) The expression of adipogenic marker protein and inflammatory factor was detected by Western blot. Expression of (B) C/EBPα, (C) PPARγ2, (D) AdipoQ, (E) FABP4, (F) LPL, (G) IL-6, and (H) TNF-α in hAdMSCs transfected with miR-204-5p mimic, pcDNA-HCG11, and their control was quantified using Image J software. The levels of lipogenesis enzymes such as (I) ACC and (J) FAS were detected by spectrophotometry. (K) Cell proliferation ability was tested by CCK-8. Statistical significance was determined using an independent sample t-test. Values were expressed as mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 versus control. ACC: acetyl coenzyme A carboxylase; AdipoQ: adiponectin; C/EBPα: CCAAT-enhancer-binding protein α; CCK: cell counting kit; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; hAdMSCs: human adipose-derived mesenchymal stem cells; HCG11: human leukocyte antigen complex group 11; IL-6: interleukin-6; LPL: lipoprotein lipase; PPARγ: peroxisome proliferator-activated receptor gamma; TNF-α: tumor necrosis factor alpha.