Stim1在骨肉瘤组织和细胞中的表达水平及与顺铂耐药性的关系

目的:探讨Stim1在骨肉瘤组织和细胞中的表达水平及与顺铂耐药性的关系。方法:采用免疫组织化学染色检测了50例骨肉瘤患者的肿瘤组织中Stim1的表达。通过用高浓度的顺铂处理人骨肉瘤细胞系U-2 OS来建立耐药性骨肉瘤细胞（U-2 OS/DDP细胞）。通过RT-PCR和蛋白质印迹检测细胞中Stim1、ATF4、CHOP和GRP78的表达。通过转染人Stim1-siRNA或过表达Stim1的pCDNA3-HA质粒来下调或上调骨肉瘤细胞中Stim1的表达，并检测了细胞的钙离子内流情况。通过CCK-8法检测细胞增殖，通过Annexin V-FITC/PI双重染色法检测细胞凋亡。结果:Stim1的阳性表达与骨肉瘤患者的TNM分期和远处转移有关，Stim1阳性患者中，74.19%（23例）为TNMⅢ期，87.10%（27例）发生远处转移。而Stim1阴性患者中，26.31%（5例）为TNMⅢ期，26.31%（5例）发生远处转移。顺铂耐药的骨肉瘤U-2 OS/DDP细胞中的Stim1 mRNA和蛋白表达水平显著高于非耐药U-2 OS细胞（P＜0.05）。下调Stim1表达明显减少了U-2 OS/DDP细胞中的钙离子内流（P＜0.05）；下调Stim1表达抑制了细胞的增殖并促进了细胞凋亡（P＜0.05）。此外，下调Stim1进一步提高了内质网应激分子标志物ATF4、CHOP和GRP78的表达（P＜0.05）。相反，上调Stim1的表达则发挥了相反的作用。结论:Stim1在骨肉瘤患者中高表达并与不良预后有关。Stim1的高表达可增加骨肉瘤细胞的顺铂耐药性。下调Stim1通过抑制钙离子内流并促进内质网应激诱导的细胞凋亡来降低骨肉瘤细胞的顺铂耐药性。

Stim1 expression in osteosarcoma tissues and cells and its relationship with cisplatin resistance

Objective:To investigate the expression level of Stim1 in osteosarcoma tissues and cells and its relationship with cisplatin resistance.Methods:The expression of Stim1 in tumor tissues of 50 patients with osteosarcoma was detected by immunohistochemical staining.Drug-resistant osteosarcoma cells(U-2 OS/DDP cells)were established by treating the human osteosarcoma cell line U-2 OS with high concentrations of cisplatin.The expression of Stim1,ATF4,CHOP and GRP78 in the cells was detected by RT-PCR and Western blot.Transfection of human Stim1-siRNA or pCDNA3-HA plasmid over-expressing Stim1 down-regulated or up-regulated the expression of Stim1 in osteosarcoma cells,and the calcium influx in the cells was detected.Cell proliferation was detected by the CCK-8 method,and apoptosis was detected by the Annexin V-FITC/PI dual staining method.Results:The positive expression of Stim1 was related to TNM staging and distant metastasis in patients with osteosarcoma.Among Stim1 positive patients,74.19%(23 cases)were TNM stage Ⅲ,and 87.10%(27 cases)had distant metastases.Among Stim1-negative patients,26.31%(5 cases)were TNM stage Ⅲ,and 26.31%(5 cases)had distant metastases.The expression levels of Stim1 mRNA and protein in cisplatin-resistant osteosarcoma U-2 OS/DDP cells were significantly higher than those in non-resistant U-2 OS cells(P<0.05).Down-regulating Stim1 significantly reduced calcium ion influx in U-2 OS/DDP cells(P<0.05).Down-regulating Stim1 inhibited cell proliferation and promoted apoptosis(P<0.05).In addition,down-regulating Stim1 further increased the expression of endoplasmic reticulum stress molecular markers ATF4,CHOP and GRP78(P<0.05).In contrast,up-regulating the expression of Stim1 played the opposite role.Conclusion:Stim1 is highly expressed in patients with osteosarcoma and is associated with poor prognosis.High expression of Stim1 can increase cisplatin resistance in osteosarcoma cells.Down-regulating Stim1 reduces cisplatin resistance of osteosarcoma cells by inhibiting calcium influx and promoting endoplasmic reticulum stress-induced apoptosis.