论文部分内容阅读
目的探讨特异性沉默人舌鳞癌细胞系TCA8113中PTP4A1基因的表达,并研究沉默该基因表达后对TCA8113细胞体内外生物学行为的影响。方法采用siRNA的慢病毒载体siRNA-PTP4A1作为阳性干扰组(KD),空病毒载体作为阴性对照组(NC),未作任何处理的TCA8113细胞作为空白对照组(CON)。Real-time PCR和Western blot检测PTP4A1基因的干扰效果,MTT法检测3组细胞的增殖能力,流式细胞技术分析3组细胞凋亡和细胞周期情况,平板克隆形成实验检测体外克隆形成能力,Western blot分别检测3组细胞抑制凋亡蛋白Bcl-2、促凋亡蛋白Bax的表达情况,3组细胞分别裸鼠成瘤后观察移植瘤的生长情况。结果 siRNA-PTPA1转染TCA8113细胞成功,Real-time PCR和Western blot检测PTP4A1基因的抑制率达到55%和60%以上(P<0.05);MTT结果显示KD组细胞增殖能力小于NC、CON组;细胞凋亡率也明显增加,KD组早期凋亡为(17.48±0.70)%,而NC、CON组的早期细胞凋亡率为(7.34±0.70)%和(4.86±0.25)%(P<0.01);KD组细胞周期阻滞在G1/G0和S期(P<0.05);Western blot结果显示Bcl-2蛋白表达降低(P<0.01),Bax蛋白表达增高(P<0.01);KD组体外克隆形成数小于NC、CON组(P<0.01);KD组裸鼠移植瘤的生长速度缓慢,质量和体积均小于NC、CON组(P<0.01)。结论 siRNA PTP4A1在体内外均能有效抑制舌鳞癌TCA8113细胞的恶性生物学行为,PTP4A1基因可能与舌鳞癌的发生、发展相关。
Objective To investigate the expression of PTP4A1 gene in human tongue squamous cell carcinoma cell line TCA8113 and to study the effect of silencing the gene on the biological behavior of TCA8113 cells in vitro and in vivo. Methods Lentiviral siRNA siRNA-PTP4A1 was used as positive interference group (KD) and empty vector as negative control (NC). TCA8113 cells without any treatment were used as blank control group (CON). Real-time PCR and Western blot were used to detect the interference effect of PTP4A1 gene. The proliferation of three groups of cells was detected by MTT assay. The apoptosis and cell cycle were analyzed by flow cytometry. The clonogenic assay was used to detect the clonogenic capacity in vitro. blot were used to detect the expression of Bcl-2 and Bax in the three groups of cells. The growth of the xenografts in the three groups of nude mice were observed. Results The siRNA-PTPA1 transfected TCA8113 cells were successfully transfected, and the inhibitory rates of PTP4A1 gene were 55% and 60% (P <0.05) by Real-time PCR and Western blot respectively. The MTT results showed that the cell proliferation ability of KD group was less than that of NC and CON groups (17.48 ± 0.70)% in KD group, and (7.34 ± 0.70)% and (4.86 ± 0.25)% in NC and CON group, respectively (P <0.01) ). The cell cycle arrest in G1, G2 and G0 phases and S phase in KD group (P <0.05). The Western blot results showed that the expression of Bcl-2 protein was decreased (P <0.01) and the protein expression of Bax was increased The number of colony formation was smaller than that in NC and CON groups (P <0.01). The growth of nude mice in KD group was slower than that in NC and CON groups (P <0.01). Conclusion siRNA PTP4A1 can effectively inhibit the malignant biological behavior of tongue squamous cell carcinoma TCA8113 cells both in vitro and in vivo, and PTP4A1 gene may be related to the occurrence and development of tongue squamous cell carcinoma.