Fig 1: miR-185-5p inhibition rescues the inhibition of matrix mineralization in osteoblasts by H19 knockdown. a The mRNA and protein expression of OCN, ALP and Collagen I in H19 knocked-down MC3T3-E1 cells transfected with miR-185-5p inhibitor and NC by qPCR and western blot. GAPDH was used for normalization. b ELISA analysis of the amount of Collagen I and BALP protein in the supernatant of in H19 knocked-down MC3T3-E1 cells transfected with miR-185-5p inhibitor and NC. c Representative images of ALP staining of transfected MC3T3-E1 cells. Scale bars, 800 μm. d Staining of calcium deposition by Alizarin Red in MC3T3-E1 cells transfected with si-H19, miR-185-5p inhibitor and NC. Scale bars, 800 μm. The asterisks show difference significant as ** p < 0.01, * p < 0.05
Fig 2: Schematic diagram of proposed mechanism. lncRNA H19 promoted matrix mineralization of osteoblasts through miR-185-5p/IGF1 axis. In mineralization condition, H19 is up-regulated and sponge miR-185-5p. The decreased in miR-185-5p availability, increases the level and translation of IGF1 mRNA, which in turn up-regulates the expression of OCN, ALP and Col1agen I involved in bone matrix mineralization. By contrast, when miR-185-5p is up-regulated, because H19 is decreased, the level of IGF1 is diminished and subsequent inhibition of mineralization, with arrows indicating the process
Fig 3: H19 knockdown inhibits matrix mineralization of osteoblasts. a, b The mRNA expression of OCN, ALP and Collagen I in control and H19 knockdown group of MC3T3-E1 cells were analysed by qPCR and western blot. GAPDH was used for normalization. c ELISA analysis of the amount of Collagen I and BALP protein in the supernatant of MC3T3-E1 cells after treatment with NC and H19 siRNA. d Representative images of ALP staining of MC3T3-E1 cells on day 7 after treatment with NC and H19 siRNA. Scale bars, 800 μm. e Staining of calcium deposition by Alizarin Red in MC3T3-E1 cells on day 14 treated with si-NC and si-H19. Scale bars, 800 μm. The asterisks show difference significant as ** p < 0.01, * p < 0.05
Fig 4: miR-185-5p overexpression inhibits matrix mineralization of osteoblasts. a The expression of IGF1 in MC3T3-E1 cells transfected with miR-185-5p mimics and miR-NC by western blot. GAPDH was used for normalization. b The mRNA expression of OCN, ALP and Collagen I in in MC3T3-E1 cells transfected with miR-185-5p and NC by qPCR and western blot. GAPDH was used for normalization. c ELISA analysis of the amount of Collagen I and BALP protein in the supernatant of MC3T3-E1 cells. d Representative images of ALP staining of MC3T3-E1 cells after transfection. Scale bars, 800 μm. e Staining of calcium deposition by Alizarin Red in MC3T3-E1 cells after treatment with miR-NC and miR-185-5p mimics. Scale bars, 800 μm. The asterisks show difference significant as ** p < 0.01, * p < 0.05
Fig 5: The expression of lncRNA H19 and miR-185-5p in mineralized osteoblasts. a The results of Alizarin Red staining, mineralization nodules and ralative areas at 7, 14 and 21 day after induced mineralization of MC3T3-E1 cells. Scale bars, 800 μm. b, c The mRNA expression of OCN, ALP and Collagen I in control and mineralized MC3T3-E1 cells were assessed by qPCR and western blot on day 7. GAPDH was used for normalization. d The expression of H19, IGF1 and miR-185-5p in control and mineralized MC3T3-E1 cells were detected by qPCR. GAPDH or U6 was used for normalization, respectively. The asterisks show difference significant as ** p < 0.01, * p < 0.05
Supplier Page from Wuhan Fine Biotech Co., Ltd. for Mouse BALP(Bone Alkaline Phosphatase) ELISA Kit