Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
Objective To investigate the effects and underlying mechanisms of human pituitary tumor-transforming gene 1 (hPTTG1) small interfering RNA (siRNA) on apoptosis of ovarian cancer cell line A2780. Methods hPTTG1 siRNA was transfected into A2780 with lipofectamine (the hPTTG1 siRNA group), and the normal group and the negative control group were set up. Detections were conducted 48 hours after transfection: the interfering efficiency of hPTTG1 mRNA was measured by real-time polymerase chain reaction, the expression of survivin gene and survivin protein was examined by semiquantitative reverse transcriptase-polymerase chain reaction and Western blot, cell apoptosis was detected by DNA fragmentation gel electrophoresis and propidium iodide staining kit, and the activity of caspase-3 was assayed by caspases colorimetric assay kit. Results The expression of hPTTG1 mRNA was expressly inhibited after hPTTG1 siRNA transfection. DNA ladder was observed in the hPTTG1 siRNA group. The apoptotic rate of hPTTG1 siRNA transfection in the hPTTG1 siRNA group was (17.53±2.17)%, higher than those in the normal group and the negative control group [(8.97±1.56)% and (9.64±1.31)%, respectively], with statistically significant differences between them (P<0.05). The expression levels of survivin mRNA and survivin protein were down-regulated. The activity of caspase-3 was raised. Conclusions siRNA targeting hPTTG1 could induce apoptosis of A2780 by inhibition of survivin expression and activation of caspase-3. It may be a potential target for gene therapy of ovarian cancer.