CircPVT1 up-regulation attenuates steroid-induced osteonecrosis of the femoral head through regulating miR-21-5p-mediated Smad7/TGFβ signalling pathway

Abstract

Steroid-induced osteonecrosis of the femoral head (SIONFH) has been a common disease following corticosteroid therapy. Presently, we aim to explore the functions of circular RNA (circ) PVT1 in SIONFH rats and the underlying mechanism. Glucocorticoid (GC) was used to treat SD rats and bone marrow-derived mesenchymal stem cells (BMSCs) to construct SIONFH model in vitro and in vivo, respectively. The pathological injury of the femoral head in the SIONFH rats was detected via haematoxylin-eosin (HE) staining and immunohistochemistry (IHC). The osteogenic differentiation, proliferation and apoptosis of BMSCs were detected. Western blot was used to detect Smad7, Bax, Bcl2 and Smad2/3. The potential targets of circPVT1 and miR-21-5p were validated through luciferase reporter gene assay and RNA pull-down assay, respectively. We found that CircPVT1 was decreased in the femoral head of SIONFH rats and GC-treated BMSCs, while miR-21-5p was markedly up-regulated. Overexpressed circPVT1 attenuated the apoptosis and cell viability inhibition of BMSCs induced by GC, while miR-21-5p up-regulation had the opposite effects. What's more, the in vivo experiments confirmed that up-regulating circPVT1 repressed osteonecrosis in SIONFH rats through repressing apoptosis. Mechanistically, circPVT1 functioned as a ceRNA of miR-21-5p, which targeted at the 3'untranslated region of Smad7. CircPVT1 enhancing Smad7 and mitigating GC activated TGFβ/Smad2/3 pathway through inhibiting miR-21-5p. In conclusion, CircPVT1 exerts protective effects against SIONFH via modulating miR-21-5p-mediated Smad7/TGFβ pathway.

Keywords: ceRNA; circPVT1; miR-21-5p; osteonecrosis; steroid.

FIGURE 1

Bone histomorphology examination of SIONFH rats. The SIONFH model was constructed in rats. A, HE staining was taken to check the pathological damage of femur. B, TRAP staining was adopted to detect osteoclast viability. C‐E, Measurement of bone cavity rate, cavity area and trabecular area. F‐J, Determination of bone volume fraction, trabecular bone quantity, trabecular bone thickness, trabecular bone separation and mode factor. *P < .05, **P < .01, ***P < .001 (vs.con group). N = 5

FIGURE 2

The effect of GC on the osteogenic differentiation ability of BMSCs and the expression level of CircPVT1/miR‐21‐5p. The BMSC was treated with 1600 mg of GC and tested their osteogenic differentiation ability. A‐D, RT‐PCR experiment was used to detect the levels of osteogenic differentiation‐specific markers ALP, RUNX2, CoL1a1 and OCN. E‐F, RT‐PCR experiment was applied to examine the expression of osteoclast‐related genes ACP5 and CTSK. G‐H, RT‐PCR was used to detect the expression of CircPVT1 and miR‐21‐5p in BMSCs. **P < .01, ***P < .001 (vs.con group). N = 3

FIGURE 3

Overexpression of CircPVT1 promoted differentiation, proliferation and inhibited apoptosis of BMSCs. After 1600 mg of GC, CircPVT1 overexpression plasmid was also given to treat BMSCs. A,B, The expression of CircPVT1 and miR‐21‐5p were detected by RT‐PCR. C, RT‐PCR was taken to assess the levels of osteogenic differentiation markers ALP, RUNX2, CoL1a1, OCN and the expression of osteoclast‐related genes ACP5 and CTSK. D,E, BrdU and flow cytometry were conducted to examine cell proliferation and apoptosis ability. F, Western blot detection of Smad7, Smad2/3, Bax and Bcl2 protein expression. *P < .05, **P < .01, ***P < .001 (vs.con group). ##P < .01, ###P < .001 (vs.GC group). N = 3

FIGURE 4

Overexpression of miR‐21‐5p inhibited the differentiation and proliferation of BMSCs and promoted apoptosis. MiR‐21‐5p mimics were transfected based on 1600 mg GC‐treated BMSCs. A,B, The expression of CircPVT1 and miR‐21‐5p were detected by RT‐PCR. C‐H, RT‐PCR was utilized to measure osteogenic differentiation markers ALP, RUNX2, CoL1a1, OCN and the expression of osteoclast‐related genes ACP5 CTSK. I‐J, BrdU and flow cytometry were taken to evaluate the effects of transfection of miR‐21‐5p mimics on the proliferation and apoptosis of BMSCs. K, Western blot detection of Smad7, Smad2/3, Bax, Bcl2 protein expression. *P < .05, **P < .01, ***P < .001 (vs.con group). ##P < .01, ###P < .001(vs.GC group). N = 3

FIGURE 5

MiR‐21‐5p is the target of CircPVT1 and Smad7. A, The relevant downstream molecular targets of CircPVT1 were analysed by Venn diagram. B, The expression of miRNAs after CircPVT1 overexpression was analysed by heat map. C, StarBase database (http://starbase.sysu.edu.cn/) was browsed to predict the binding site of miR‐21‐5p to CircPVT1 and Smad7. D, Dual‐luciferase activity experiment and RNA pull‐down assay (E) confirmed the binding relationship of miR‐21‐5p with CircPVT1, miR‐21‐5p and Smad7 in BMSCs cells. **P < .01, ***P < .001, nsP > 0.05 (vs.NC group). ###P < .001, nsP > 0.05 (vs.NC group). N = 3

FIGURE 6

Up‐regulation of CircPVT1 inhibited miR‐21‐5p‐induced osteonecrosis of BMSCs. BMSCs were transfected with miR‐21‐5p mimics and/or CircPVT1 overexpression plasmids. A‐F, RT‐PCR was employed to assess the levels of osteogenic differentiation markers ALP, RUNX2, CoL1a1, OCN and the expression of osteoclast‐related genes ACP5 and CTSK. G‐H, BrdU and flow cytometry were used to detect BMSC proliferation and apoptosis. I, Western blot detection of Smad7, Smad2/3, Bax, Bcl2 protein expression. **P < .01, ***P < .001 (vs.con group). ##P < .01, ###P < .001 (vs.miR‐21‐5p group). N = 3

FIGURE 7

The effect of overexpressed CircPVT1 on osteogenic differentiation of rat femoral head necrosis was verified in vivo. The CircPVT1 overexpression plasmid was injected into the ventricle of SIONFH rats. A, HE staining was taken to check the pathological damage of the femur. B, TRAP staining detected osteoclast viability. C‐E, Measurement of bone cavity rate, cavity area and trabecular area. F‐J, Determination of bone volume fraction, trabecular bone quantity, trabecular bone thickness, trabecular bone separation and mode factor. K‐P, RT‐PCR was used to detect osteogenic differentiation‐related genes ALP, RUNX2, CoL1a1, OCN and osteoclastogenesis‐related genes ACP5 and CTSK. NS P > .05, **P < .01, ***P < .001 (vs.vector group). N = 5

FIGURE 8

Effect of overexpressing CircPVT1 on SMAD7 and SMAD2/3 in vivo. The treatment method was the same as that in Figure 7. A,B, IHC was implemented to verify the positive expression of Smad7 and pro‐apoptotic protein Bax. C, Western blot was applied to monitor the expression of Smad7, Smad 2/3, Bcl2 and Bax. NS P > .05, ***P < .001 (vs.vector group). N = 5

FIGURE 9

The mechanistic diagram. CircPVT1 is down‐regulated in SIONFH. Overexpressing of circPVT1 down‐regulates TGFβ/Smad2/3 expression and promotes Smad7 activation by targeting miR‐21‐5p, thereby reducing SIONFN