Figure 5.
CircVMA21 alleviated IVDD in vivo. (A) A flow diagram of the experiments in vivo. A total of 32 rats were randomly divided into four groups: non-puncture group (control), non-injection with puncture group (non-injection), circVMA21 injection with puncture group (circVMA21), and circVMA21 mutant injection with puncture group (circVMA21-mut). (B) Radiographs of the indicated groups were obtained 9 weeks after needle puncture. Co6/7, Co8/9 and Co10/11 were punctured with Co7/8 and Co9/10 left intact. (C) Changes in disc height index (DHI) of the indicated groups after needle puncture. The DHI was measured at weeks 0, 1, 5 and 9 time point. A significant decrease of the %DHI was observed in all puncture groups at 1 week after surgery (P<0.01). At each time point after puncture, a significant decrease of %DHI was noted in all puncture groups compared with the control group (P<0.01). No significant difference was found in the %DHI between all puncture groups. (D) MRIs of the indicated groups were obtained 9 weeks after needle puncture. Co6/7, Co8/9 and Co10/11 were punctured with Co7/8 and Co9/10 left intact. (E) The MRI grade in the indicated groups at 9 weeks after needle puncture. The degree of disc degeneration by MRI grade was significantly lower in the circVMA21 group than in the non-injection group. *P<0.05, **P<0.01. (F) In vivo RNA fluorescence in situ hybridisation found circVMA21 located in the NP region. Blue fluorescence (4,6-diamidino-2-phenylindole, DAPI) indicating cell nucleus; green fluorescence (Alexa 488) indicating circVMA21. Scale bar=100 µm. (G) qRT-PCR showed that the decreased levels of circVMA21 in the punctured IVDs were rescued by the injection of circVMA21. **P<0.01. (H) qRT-PCR showed that the increased levels of miR-200c in the punctured IVDs were depressed by the injection of circVMA21. *P<0.05, **P<0.01. (I) Western blot analysed the expression of XIAP, apoptotic effector caspases (caspase-3, caspase-7 and caspase-9), catabolic enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5) and extracellular matrix (ECM) compositions (collagen II, aggrecan) in the rat NP tissues. The injection of circVMA21 alleviated the degenerative changes of the NP such as enhanced apoptotic and catabolic response, and reduced the expression of ECM compositions in the rat model of IVDD. (J) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining of the IVDs in the indicated groups at 9 weeks after needle puncture. Blue fluorescence (DAPI) indicating total cells; green fluorescence (fluorescein isothiocyanate) indicating TUNEL positive cells. Scale bar=100 µm. (K) A significant decrease in the apoptosis rate was noted in the circVMA21 group compared with the non-injection group. *P<0.05, **P<0.01. (L) H&E (left) and safranin-O/fast green (right) staining of the IVDs in the indicated groups at 9 weeks after needle puncture. Scale bar=100 µm. (M) A significant decrease in the grade of IVDD was noted in the circVMA21 group compared with the non-injection group. *P<0.05, **P<0.01. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IVD, intervertebral disc; IVDD, intervertebral disc degeneration; MMP, matrix metalloproteinases; NP, nucleus pulposus; qRT-PCR, quantitative real-time reverse transcription-PCR; XIAP, X linked inhibitor-of-apoptosis protein.