沉默钙离子通道相关基因CACNA1A对人骨肉瘤细胞增殖、侵袭、迁移能力的影响

Effect of silencing calcium channel-related gene CACNA1A on proliferation,invasion and migration ability of human osteosarcoma cells

目的:探讨沉默钙离子(Ca^(2+))通道相关基因CACNA1A对骨肉瘤(OS)细胞增殖、侵袭、迁移能力的影响。方法:采用实时荧光定量PCR(RT-qPCR)法检测CACNA1A在临床OS患者瘤组织及其瘤旁组织中的表达。比较成骨细胞(hFOB1.19细胞)与OS细胞系(MNNG和SJSA-1细胞)中CACNA1A的表达。将敲除CACNA1A的小干扰RNA(siCACNA1A)及其对照(siControl)分别转入MNNG、SJSA-1细胞中,观察沉默CACNA1A表达后OS细胞内的Ca^(2+)分布情况;采用细胞计数试剂盒(CCK-8)实验、划痕实验和Transwell实验检测细胞的增殖、侵袭和迁移能力。建立裸鼠皮下移植瘤模型,比较siCACNA1A组和siControl组成瘤体积和瘤重,免疫组织化学染色(IHC)检测成瘤组织中CACNA1A蛋白表达水平。结果:与瘤旁组织比较,OS患者瘤组织CACNA1A表达水平升高(P<0.05);与h FOB1.19细胞比较,MNNG和SJSA-1细胞中CACNA1A表达水平升高(P<0.05);与siControl组相比,siCACNA1A组OS细胞内的Ca^(2+)分布减少,细胞增殖、侵袭和迁移能力受到显著抑制(P<0.05)。动物实验显示,与siControl组比较,siCACNA1A组裸鼠肿瘤体积和重量显著减小(P<0.05)。IHC结果显示,与siControl组相比,siCACNA1A组皮下肿瘤的CACNA1A相对免疫染色强度降低(P<0.001)。结论:沉默Ca^(2+)通道相关基因CACNA1A可抑制OS细胞的增殖和转移以及裸鼠体内肿瘤的生长。

Objective:To investigate the effect of silencing calcium(Ca^(2+))channel-related gene CACNA1A on proliferation,invasion and migration of osteosarcoma(OS)cells.Methods:Reverse transcription-quantitative PCR(RT-qPCR)was used to detect the expression of CACNA1A in tumor tissues and adjacent tissues of clinical patients with OS.The expression of CACNA1A in osteoblasts(hFOB1.19 cells)and OS cell lines(MNNG and SJSA-1 cells)was compared.The small interfering RNA(siCACNA1A)knocking out CACNA1A and its control(si-Control)were transferred into MNNG and SJSA-1 cells respectively to observe the Ca^(2+)distribution in the OS cells after silencing CACNA1A expression.Cell counting kit-8(CCK-8)assay,scratch assay and Transwell assay were used to detect the cell proliferation,invasion and migration.A subcutaneous transplanted tumor model was established in nude mice,the volume and weight of tumorigenesis in the siCACNA1A group and the siControl group were compared,and the expression level of CACNA1A protein in tumorigenic tissues was detected by immunohistochemical staining(IHC).Results:Compared with adjacent tissues,the expression level of CACNA1A in tumor tissues of OS patients was increased(P<0.05).Compared with hFOB1.19 cells,the expression level of CACNA1A in MNNG and SJSA-1 cells was increased(P<0.05).Compared with the siControl group,the Ca^(2+)distribution in the OS cells in the siCACNA1A group was reduced,and the cell proliferation and migration ability was significantly inhibited.The results of animal experiments showed that the tumor volume and weight in the si-CACNA1A group were significantly reduced compared with the siControl group(P<0.05).IHC results showed that the relative immunostaining intensity of CACNA1A in subcutaneous tumors in the siCACNA1A group was lower than that in the siControl group(P<0.001).Conclusion:The silencing Ca^(2+)channel-related gene CACNA1A can inhibit the proliferation and metastasis of OS cells and the growth of tumors in nude mice.