MicroRNA-497 elevation or LRG1 knockdown promotes osteoblast proliferation and collagen synthesis in osteoporosis via TGF-β1/Smads signalling pathway

Abstract

MicroRNAs (miRNAs) have been corroborated to engage in the process of cellular activities in osteoporosis. However, few researches have been conducted to expose the integrated role of miR-497, leucine-rich alpha-2-glycoprotein-1 (LRG1) and transforming growth factor beta 1 (TGF-β1)/Smads signalling pathway in osteoporosis. Thereafter, the study is set out to delve into miR-497/LRG1/TGF-β1/Smads signalling pathway axis in osteoporosis. Osteoporosis bone tissues and normal bone tissues were collected. Rat osteoporosis models were constructed via ovariectomy. Model rats were injected with restored miR-497 or depleted LRG1 to explore their roles in osteoporosis. Rat osteoblasts were extracted from osteoporosis rats and transfected with restored miR-497 or depleted LRG1 for further verification. MiR-497 and LRG1 expression in femoral head tissues and osteoblasts of osteoporosis rats were detected. TGF-β1/Smads signalling pathway-related factors were detected. MiR-497 was poorly expressed while LRG1 was highly expressed and TGF-β1/Smads signalling pathway activation was inhibited in osteoporosis. MiR-497 up-regulation or LRG1 down-regulation activated TGF-β1/Smads signalling pathway, promoted collagen type 1 synthesis and suppressed oxidative stress in femoral head tissues in osteoporosis. MiR-497 restoration or LRG1 knockdown activated TGF-β1/Smads signalling pathway, promoted viability and suppressed apoptosis of osteoblasts in osteoporosis. Our study suggests that miR-497 up-regulation or LRG1 down-regulation promotes osteoblast viability and collagen synthesis via activating TGF-β1/Smads signalling pathway, which may provide a novel reference for osteoporosis treatment.

Keywords: collagen synthesis; leucine-rich alpha-2-glycoprotein-1; microRNA-497; osteoblast; osteoporosis; transforming growth factor β1/Smads signalling pathway; viability.

FIGURE 1

MiR‐497 expression reduces and LRG1 expression increases in femoral head tissues of patients with osteoporosis. A, MiR‐497 expression in femoral head tissues in control and osteoporosis; B, LRG1 mRNA expression in femoral head tissues in control and osteoporosis; C, LRG1 protein bands in femoral head tissues in control and osteoporosis; D, LRG1 protein expression in femoral head tissues in control and osteoporosis; E, Pearson's analysis of the correlation between miR‐497 and LRG1 in femoral head tissues in osteoporosis patients; F, schematic diagram of the miR‐497 binding site in the LRG1 based on RNA22 software; G, luciferase activity verification; in figure A‐E, control group (n = 20), osteoporosis group (n = 20); in Figure G, N = 3. The data were expressed as the mean ± standard deviation. Comparisons between two groups were analysed by unpaired t test; In Figure E, Pearson correlation analysis was performed

FIGURE 2

Up‐regulation of miR‐497 or down‐regulation of LRG1 activates TGF‐β1/Smads signalling pathway in rats with osteoporosis. A, MiR‐497 expression in femoral head tissues of rats; B, LRG1 mRNA expression in femoral head tissues of rats; C, LRG1 protein bands in femoral head tissues of rats; D, LRG1 protein expression in femoral head tissues of rats; E, TGF‐β1 protein bands in femoral head tissues of rats; F, TGF‐β1 protein expression in femoral head tissues of rats; G, Smad3, Smad4 and Smad7 protein bands in femoral head tissues of rats; H, Smad3, Smad4 and Smad7 protein expression in femoral head tissues of rats; I, p‐Smad2/3 protein bands in femoral head tissues of rats; J, p‐Smad2/3 protein expression in femoral head tissues of rats; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; n = 8. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 3

Up‐regulation of miR‐497 or down‐regulation of LRG1 increases blood calcium and blood phosphorus levels, reduces urinary calcium, urinary phosphorus, Hyp and ALP levels and lowers OC, BALP, PINP and PICP contents in rats with osteoporosis. A, Urinary calcium and phosphorus levels of rats; B, urinary Hyp of rats; C, blood calcium and phosphorus levels of rats; D, blood ALP content of rats; E, OC and BALP contents in blood of rats; F, PINP and PICP contents in blood of rats; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; n = 8. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 4

Up‐regulation of miR‐497 or down‐regulation of LRG1 attenuates pathological femoral tissue damage, raises Col‐1 expression and inhibits oxidative stress in femoral head tissues of rats with osteoporosis. A, HE staining of femoral head tissues of rats; B, Col‐1 mRNA expression in femoral head tissues of rats; C, Col‐1 protein bands in femoral head tissues of rats; D, Col‐1 protein expression in femoral head tissues of rats; E, SOD activities in rat femoral head tissues; F, MDA contents in rat femoral head tissues; G, GSH‐Px activities in rat femoral head tissues; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; n = 8. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 5

MiR‐497 is down‐regulated while LRG1 is up‐regulated in osteoblasts of rats with osteoporosis, and miR‐497 elevation or LRG1 depletion activates TGF‐β1/Smads signalling pathway. A, Osteoblast morphology at different time‐points; B, ALP staining of osteoblasts; C, MiR‐497 expression in rat osteoblasts; D, LRG1 mRNA expression in rat osteoblasts; E, LRG1 protein bands in rat osteoblasts; F, LRG1 protein expression in rat osteoblasts; G, TGF‐β1 protein band in rat osteoblasts; H, TGF‐β1 protein expression in rat osteoblasts; I, Smad3, Smad4 and Smad7 protein bands in rat osteoblasts; J, Smad3, Smad4 and Smad7 protein expression in rat osteoblasts; K, p‐Smad2/3 protein bands in rat osteoblasts; L, p‐Smad2/3 protein expression in rat osteoblasts; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; N = 3. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 6

Up‐regulation of miR‐497 or down‐regulation of LRG1 inhibits osteoblast apoptosis and promotes Col‐1 synthesis of rats with osteoporosis. A, Protein bands of Bax and Bcl‐2 in rat osteoblasts; B, Bax and Bcl‐2 protein expression in rat osteoblasts; C, protein bands of Cyclin D1 and CDK4 in rat osteoblasts; D, cyclin D1 and CDK4 protein expression in rat osteoblasts; E, Col‐1 mRNA expression in rat osteoblasts; F,Col‐1 protein bands in rat osteoblasts; G, Col‐1 protein expression in rat osteoblasts; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; N = 3. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 7

Up‐regulation of miR‐497 or down‐regulation of LRG1 promotes viability and suppresses apoptosis in osteoblasts of rats with osteoporosis. A, Osteoblast viability detected by MTT assay; B, cell cycle distribution of osteoblasts; C, proportion of osteoblast cell cycle; D, osteoblast apoptosis; E, apoptosis rate of osteoblasts; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; N = 3. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

FIGURE 8

The mechanistic diagrams indicate that in osteoporosis, miR‐497 could promote osteoblast proliferation and collagen synthesis, and inhibit osteoblast apoptosis through inhibiting LRG1 and activating TGF‐β1/Smads signalling pathway. The miR‐497 mimics inhibited the expression of the LRG1 gene and activated TGF‐β1/Smads signalling pathway, including elevated expression of TGF‐β1, Smad3, Smad4 and p‐Smad2/3, and decreased expression of Smad7, thereby promoting osteoblast proliferation and collagen synthesis, and suppressing osteoblast apoptosis