AUF1负向调控p21对骨肉瘤细胞增殖和凋亡的影响

The effect of AUF1 negatively regulating p21 on the proliferation and apoptosis of osteosarcoma cells

目的 探讨AU结合因子1(AUF1)调控p21对骨肉瘤细胞增殖和凋亡的影响及机制。方法 采用qRT-PCR和Western blot分别检测AUF1在骨肉瘤组织mRNA和蛋白表达水平。在骨肉瘤U2OS和HOS细胞中分别转染sh-NC和sh-AUF1后,CCK8检测细胞增殖情况;qRT-PCR检测增殖相关分子PCNA的表达水平;流式细胞术检测细胞凋亡情况;Western blot检测凋亡相关分子Cleaved-caspase-3的表达水平。RNA结合蛋白免疫沉淀实验和RNA半衰期实验检测AUF1调控p21的机制。结果 AUF1 mRNA和蛋白的表达水平在骨肉瘤组织中明显高于癌旁正常组织(P<0.05),AUF1 mRNA的表达水平在骨肉瘤伴转移的患者中明显高于骨肉瘤不伴转移的患者(P<0.05)。在U2OS和HOS细胞中分别转染sh-NC和sh-AUF1,AUF1 mRNA和蛋白的表达水平在转染sh-AUF1组较sh-NC组明显下降(P<0.05)。低表达AUF1能够明显抑制U2OS和HOS细胞的增殖和增殖相关分子PCNA的表达(P<0.05),促进细胞凋亡和凋亡相关分子Cleaved-caspase-3的表达(P<0.05)。低表达AUF1能够明显促进U2OS和HOS细胞中p21 mRNA和蛋白的表达水平(P<0.05)。AUF1蛋白能够与p21 mRNA结合,同时低表达AUF1能够明显抑制p21 mRNA的降解速度(P<005)。结论 AUF1在骨肉瘤组织和细胞中明显高表达,低表达AUF1能够抑制p21 mRNA降解,增加p21蛋白的表达,进而抑制骨肉瘤细胞增殖和促进细胞凋亡。

Objective To investigate the effect and mechanism of AUF1 regulating p21 on the proliferation and apoptosis of osteosarcoma cells.Methods qRT-PCR and Western blot were used to detect the expression level of AUF1 in osteosarcoma tissues and cells.After transfection of sh-NC and sh-AUF1 into U2OS and HOS cells,CCK8 was used to detect cell proliferation.qRT-PCR was used to detect the expression level of proliferation-related molecule PCNA.The flow cytometry was used to detect cell apoptosis.Western blot was used to detect the expression level of apoptosis-related molecule Cleaved-caspase-3.RIP and RNA half-life experiments were used to measure the mechanism of AUF1 regulating p21.Results The expression level of AUF1 mRNA and protein in osteosarcoma tissues and cells was significantly higher than that in normal tissues adjacent to cancer(P<0.05),and the expression level of AUF1 mRNA in osteosarcoma patients with metastasis was significantly higher than in patients without metastasis(P<0.05).U2OS and HOS cells were transfected with sh-NC and sh-AUF1,respectively.The expression levels of AUF1 mRNA and protein in the sh-AUF1 group were significantly lower than those in the sh-NC group(P<0.05).Low expression of AUF1 could significantly inhibit the proliferation of U2OS and HOS cells and the expression of proliferation-related molecule PCNA(P<0.05),and promote cell apoptosis and the expression of apoptosis-related molecule Cleaved-caspase-3(P<0.05).Low expression of AUF1 significantly promoted the expression of p21 mRNA and protein in U2OS and HOS cells(P<0.05).AUF1 protein bound to p21 mRNA(P<0.05),while low expression of AUF1 significantly inhibited the degradation rate of p21 mRNA(P<0.05).Conclusion AUF1 was highly expressed in osteosarcoma tissues and cells.The low expression of AUF1 could inhibit the degradation of p21 mRNA and increase the protein expression of p21,thereby inhibiting the proliferation of osteosarcoma cells and promoting cell apoptosis.