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. 2014 Jul 1;23(13):1452-63.
doi: 10.1089/scd.2013.0600. Epub 2014 May 22.

let-7 enhances osteogenesis and bone formation while repressing adipogenesis of human stromal/mesenchymal stem cells by regulating HMGA2

Jianfeng Wei  1 , Hongling Li, Shihua Wang, Tangping Li, Junfen Fan, Xiaolei Liang, Jing Li, Qin Han, Li Zhu, Linyuan Fan, Robert Chunhua Zhao
Affiliations

let-7 enhances osteogenesis and bone formation while repressing adipogenesis of human stromal/mesenchymal stem cells by regulating HMGA2

Jianfeng Wei et al. Stem Cells Dev. .

Abstract

Bone and fat cells share a common progenitor, stromal/mesenchymal stem cells (MSCs), that can differentiate into osteoblasts or adipocytes. Osteogenesis and adipogenesis of MSCs maintain homeostasis under physiological conditions. The disruption of this homeostasis leads to bone-related metabolic diseases. For instance, reduction in bone formation, which is usually accompanied by an increase in bone marrow adipogenesis, occurs with aging, immobility, or osteoporosis. Thus, it is crucial to gain an understanding of how osteogenic and adipogenic lineages of MSCs are regulated. Here, we present evidence that let-7 is a positive regulator of bone development. Using gain- and loss-of-function approaches, we demonstrate that let-7 markedly promotes osteogenesis and suppresses adipogenesis of MSCs in vitro. Moreover, let-7 could promote ectopic bone formation of MSCs in vivo. Subsequent studies further demonstrated that let-7's effects are mediated through the repression of high-mobility group AT-hook 2 (HMGA2) expression. RNAi depletion of HMGA2 could also enhance osteogenesis and repress adipogenesis. Overall, we found a novel role of let-7/HMGA2 axis in regulating the balance of osteogenesis and adipogenesis of MSCs. Thus, let-7 can be used as a novel therapeutic target for disorders that are associated with bone loss and adipocyte accumulation.

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Figures

<b>FIG. 1.</b>
FIG. 1.
let-7 expression profiles during bone formation and osteogenesis differentiation. (A, B) The expression of let-7c and let-7d, respectively, in different developmental stages of the mouse femur was detected using TaqMan qRT-PCR. U6 was used as an internal normalization control. The data are presented as the mean±SD from three independent trials. (C, D) TaqMan qRT-PCR analysis of the dynamic expression pattern of let-7c was performed in human adipose-derived mesenchymal stem cells (hADSCs) at days 0, 2, 4, and 6 of osteogenic differentiation. U6 was used as an internal normalization control. The data are presented as the mean±SD from three independent trials.
<b>FIG. 2.</b>
FIG. 2.
Overexpression of let-7 promotes osteogenic differentiation and suppresses adipogenesis of hADSCs; the expression of let-7 was up-regulated by expressing the let-7c precursor with a lentiviral vector (Lenti-let-7c), and the same lentiviral vector expressing a scrambled sequence (Lenti-scr) was used as a parallel control. (A) Alkaline phosphatase (ALP) staining indicated the effect on osteogenic differentiation of Lenti-let-7c-infected hADSCs compared with Lenti-scr-infected cells at day 4. n=5. Scale bars: 100 μm. (B) ALP-positive cells were counted under a high power field and normalized by the total cell numbers in infected cells. The data are presented as the mean±SD from five independent images of each group. (C) ALP activity was analyzed in hADSCs infected with Lenti-let-7c and Lenti-scr during osteogenic differentiation at day 4. n=3. (D) Alizarin red staining indicated the mineralization in hADSCs infected with Lenti-let-7c and Lenti-scr after induction to the osteogenic lineage at day 14. n=3. Scale bars: 100 μm. (E, F) Immunofluorescence analysis of the expression of ALP and Runt-related transcription factor 2 (Runx2) with Hoechst 33342 counterstaining in hADSCs infected with Lenti-let-7c and Lenti-scr after induction to the osteogenic lineage at day 4. Scale bars: 100 μm. (G) The protein expression of ALP, osteopontin (OPN), and Runx2 was measured by western blot analysis in hADSCs infected with Lenti-let-7c and Lenti-scr after inducing osteogenic differentiation at day 4. n=3. (H) The mRNA expression levels of Runx2, ALP, OPN, and osteocalcin (OC) were analyzed by qRT-PCR in hADSCs infected with Lenti-let-7c and Lenti-scr after induction to the osteogenic lineage at day 4. The data are presented as the mean±SD from three independent trials. (I) Oil red O staining indicated the effect of let-7 overexpression on adipogenic differentiation of hADSCs at day 12. n=4. Scale bars: 100 μm. (J) The dye in cells was extracted with isopropanol, and the OD value was measured at a wavelength of 510 nm using a microplate reader for quantitation. The data are presented as the mean±SD from five independent trials. Color images available online at www.liebertpub.com/scd
<b>FIG. 3.</b>
FIG. 3.
Down-regulation of endogenous let-7 suppresses osteogenic differentiation and promotes adipogenic differentiation of hADSCs; a specific inhibitor of let-7 (let-7I) was transfected into hADSCs to inhibit its endogenous expression. (A) ALP staining indicated the effect of let-7 down-regulation on osteogenic differentiation of hADSCs at day 6. n=3. Scale bars: 100 μm. (B) ALP-positive cells were counted under a high power field and normalized by the total cell number of hADSCs transfected with let-7 inhibitor and negative control miRNA inhibitor (miR-NCI) after inducing osteogenic differentiation at day 6. The data are presented as the mean±SD from five independent images of each group. (C) ALP activity was calculated in let-7c-down-regulated and negative control hADSCs after induction to the osteogenic lineage at day 6. n=3. (D) Alizarin red staining for mineralization indicated the effect of osteogenic differentiation of let-7-down-regulated and negative control hADSCs at day 14. n=3. Scale bars: 100 μm. (E) Western blot analysis evaluated the expression of ALP, OPN, and Runx2 in let-7-down-regulated and negative control hADSCs undergoing osteogenic differentiation at day 6. n=3. (F) Oil red O staining indicated the effect of let-7 down-regulation on adipogenesis of hADSCs at day 12. n=3. Scale bars: 100 μm. (G) The dye of oil red O-positive cells was extracted by isopropanol, and the OD value was quantified at 510 nm wavelength. The data are presented as the mean±SD from four independent trials. miR-NCI was used as a control. let-7I, let-7 inhibitor. Color images available online at www.liebertpub.com/scd
<b>FIG. 4.</b>
FIG. 4.
let-7 promotes the ectopic bone formation of hADSCs in vivo. The expression of let-7 was up-regulated by expressing the let-7c precursor with a lentiviral vector (Lenti-let-7c), and the same lentiviral vector expressing a scrambled sequence (Lenti-scr) was used as a parallel control. (A, B) The hADSCs infected with Lenti-let-7c and Lenti-scr were loaded on PLGA scaffolds and implanted into 6-week-old male athymic mice (BALB/c nu/nu strain) for 60 days. Black arrows indicate xenografts (12 mice/group). (C) Hematoxylin and eosin staining of xenografts. Scale bars: 50 μm. (D) Masson's trichrome staining analyzed the osteoid formation in xenografts. Scale bars: 50 μm. (E) Immunohistochemical staining showed the expression levels of Runx2, bone sialoprotein (BSP), type I collagen alpha 1 (Col1α1), ALP, and OPN in xenografts. Scale bars: 50 μm. (F) Western blot analysis indicated the expression of ALP, OPN, and Runx2 in xenografts. n=3. (G) The ultrastructure of Lenti-let-7c-infected xenografts was observed by transmission electron microscopy. Scale bars: 1 μm. Color images available online at www.liebertpub.com/scd
<b>FIG. 5.</b>
FIG. 5.
Prediction and confirmation of the direct target of let-7 in regulating osteogenic differentiation of hADSCs. (A) Bioinformatics analysis showed that target sequences of let-7 on the 3′-untranslated region (3′-UTR) of its candidate target genes. (B) Schematic representation of the luciferase reporter vectors construct containing that had either a wild-type 3′-UTR or a mutant 3′-UTR of high-mobility group AT-hook 2 (HMGA2) coding sequence in the let-7 binding site. (C) let-7 specifically represses its targets in the luciferase assay in 293T cells. The data are presented as the mean±SD from three independent experiments. (D) Western blot analysis was used to evaluate the expression of HMGA2 when endogenous HMGA2 was inhibited in hADSCs using let-7. miR-NC was used as a negative control. n=3.
<b>FIG. 6.</b>
FIG. 6.
Down-regulation of HMGA2 promotes osteogenic differentiation and inhibits adipogenic differentiation of hADSCs. (A) ALP staining indicated osteogenic differentiation of hADSCs at day 6 when HMGA2 was down-regulated by RNA interference. Scrambled siRNA was used as a negative control. n=3. Scale bars: 100 μm. (B) The number of ALP-positive cells was determined by counting under a high power field and normalized by total cell number. The data are presented as the mean±SD from five independent images of each group. (C) ALP activity was calculated during osteogenic differentiation of hADSCs at day 6 when HMGA2 was down-regulated by RNA interference. n=3. (D) Alizarin red staining for mineralization indicated the effect of HMGA2 down-regulation on osteogenic differentiation of hADSCs at day 14 by RNA interference. Scrambled siRNA was used as a negative control. n=3. Scale bars: 100 μm. (E) qRT-PCR revealed the mRNA expression levels of Runx2, OSX, ALP, and OC during the osteogenic differentiation of HMGA2-inhibited hADSCs at day 6. Scrambled siRNA was used as a negative control. n=3. (F) Western blot analysis detected the expression of ALP, OPN, and Runx2 during the osteogenic differentiation of HMGA2-inhibited hADSCs at day 6. Scrambled siRNA was used as a negative control. n=3. (G) Down-regulation of HMGA2 affected the adipogenesis of hADSCs as indicated by oil red O staining at day 12. Scrambled siRNA was used as a negative control. n=5. Scale bars: 100 μm. Color images available online at www.liebertpub.com/scd
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