SP1 regulates BMSC osteogenic differentiation through the miR-133a-3p/MAPK3 axis : SP1 regulates osteogenic differentiation of BMSCs

Abstract

Background: The progression of osteoporosis (OP) can dramatically increase the risk of fractures, which seriously disturb the life of elderly individuals. Specific protein 1 (SP1) is involved in OP progression. However, the mechanism by which SP1 regulates OP progression remains unclear.

Objective: This study investigated the mechanism underlying the function of SP1 in OP.

Methods: SAMP6 mice were used to establish an in vivo model of age-dependent OP, and BALB/c mice were used as controls. BMSCs were extracted from two subtypes of mice. Hematoxylin and eosin staining were performed to mark the intramedullary trabecular bone structure to evaluate histological changes. ChIP assay was used to assess the targeted regulation between SP1 and miR-133a-3p. The binding sites between MAPK3 and miR-133a-3p were verified using a dual-luciferase reporter assay. The mRNA levels of miR-133a-3p and MAPK3 were detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The protein expression of SP1, MAPK3, Colla1, OCN, and Runx2 was examined using Western blotting. Alkaline phosphatase (ALP) kit and Alizarin Red S staining were used to investigate ALP activity and mineralized nodules, respectively.

Results: The levels of SP1 and miR-133a-3p were upregulated, whereas the expression of MAPK3 was downregulated in BMSCs from SAMP6 mice, and miR-133a-3p inhibitor accelerated osteogenic differentiation in BMSCs. SP1 directly targeted miR-133a-3p, and MAPK3 was the downstream mRNA of miR-133a-3p. Mechanically, SP1 accelerated osteogenic differentiation in BMSCs via transcriptional mediation of the miR-133a-3p/MAPK3 axis.

Conclusion: SP1 regulates osteogenic differentiation by mediating the miR-133a-3p/MAPK3 axis, which would shed new light on strategies for treating senile OP.

Keywords: BMSCs; MAPK3; MiR-133a-3p; Osteoporosis; SP1.

Fig. 1

SP1 and miR-133a-3p were upregulated, MAPK3 was downregulated, and osteogenic differentiation was inhibited in SAMP6 mouse-derived BMSCs. SAMP6 mice were considered as an in vivo model of age-dependent OP, and BALB/c mice were considered as controls. A, The histological changes in OP were investigated using H&E staining (The scale bar was 100 μm). B, the levels of SP1 and MAPK3 were detected using IHC staining (the scale bar was 100 μm). C, the level of miR-133a-3p in mouse femur bone tissues and BMSCs was investigated using RT-qPCR. D, The contents of PINP in the serum of mice were assessed using commercial kits. E, The expression of SP1 and MAPK3 was examined using Western blotting. F, ALP activity was detected using the Alkaline Phosphatase Assay Kit, and Alizarin Red S staining was performed to examine the mineralized nodules after culture with osteogenic OM for 21 days (the scale bar was 100 μm). G, The contents of PINP in the serum of mice were assessed using commercial kits. The protein levels of Colla1, OCN, and Runx2 were assessed using Western blotting. N = 8, **p < 0.01

Fig. 2

miR-133a-3p inhibitor accelerated the osteogenic differentiation of BMSCs. BMSCs were derived from the bone tissues of SAMP6 mice and then cultured with OM for 21 days. BMSCs were transfected with an miR-133a-3p/NC inhibitor. BMSCs as blank controls were cultured in CM. A, The level of miR-133a-3p in BMSCs was detected using RT-qPCR. B, Alkaline Phosphatase Kit was used to detect ALP activity, and Alizarin Red S staining was used to examine the mineralized nodules (the scale bar was 100 μm). C, The contents of PINP in BMSCs derived from mice were investigated using commercial kits. D, The protein levels of Colla1, OCN, and Runx2 in BMSCs were detected using Western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

SP1 accelerated the osteogenic differentiation of BMSCs by transcriptionally modulating miR-133a-3p. A, The binding site of SP1 and MIR133A1 promoter region were analyzed using JASPAR. B, The binding relationship between SP1 and the MIR133A1 promoter region was verified using the ChIP assay. C, The binding between SP1 and the MIR133A1 promoter region was verified using dual-luciferase reporter assay. Subsequently, BMSCs were transfected with oe-SP1, oe-NC, oe-SP1 + NC inhibitor, or oe-SP1 + miR-133a-3p inhibitor. D, The level of miR-133a-3p in BMSCs was examined using RT-qPCR. E, ALP activity was detected using the Alkaline Phosphatase Assay Kit, and Alizarin Red S staining was performed to analyze mineralized nodules (the scale bar was 100 μm). F, The contents of PINP in BMSCs were examined using commercial kits. G, The protein expression of SP1, Colla1, OCN, and Runx2 was examined using Western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

MiR-133a-3p promoted the osteogenic differentiation of BMSCs by targeting MAPK3. A, The binding site of miR-133a-3p and MAPK3 were predicted using Starbase. B, The binding between miR-133a-3p and MAPK3 was analyzed using dual-luciferase reporter assay. Subsequently, BMSCs were transfected with NC/miR-133a-3p mimics, miR-133a-3p mimics + oe-NC, or miR-133a-3p mimics + oe-MAPK3. C, The level of miR-133a-3p was examined using RT-qPCR. D, The expression of MAPK3 was examined using RT-qPCR. E, ALP activity was examined using Alkaline Phosphatase Kit, and the mineralized nodules in BMSCs were detected by Alizarin Red S staining (The scale bar was 100 μm). F, The content of PINP in BMSCs was assessed by commercial kits. G, The protein expression of MAPK3, Colla1, OCN, and Runx2 was assessed using Western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Knockdown of SP1 significantly alleviated the symptom of OPin vivo. (A) The histological changes in mice were observed using H&E staining. (B) The levels of MAPK3 and SP1 in mice were detected using IHC staining. (C) The protein levels of SP1 and MAPK3 in the tissues of mice were detected using Western blotting. (D) The level of miR-133a-3p in the tissues of mice was evaluated using RT-qPCR. (E) The protein levels of Colla1, OCN, and Runx2 in mice were examined using Western blotting. (F) The content of PINP in the serum of mice was measured using commercial kits. N = 6, *p < 0.05, **p < 0.01