SHIP2对喉癌Hep-2细胞生物学行为的影响及对放疗增敏的机制

Effect of SHIP2 on the biological behavior of laryngeal carcinoma Hep-2 cells and mechanism of radiosensitization

目的:探究抑制SHIP2对喉癌Hep-2细胞增殖、凋亡和侵袭、迁移的影响;探究X射线处理喉癌Hep-2细胞后细胞增殖、凋亡和周期的变化及SHIP2的表达情况;探究靶向抑制SHIP2基因联合放射治疗对喉癌Hep-2细胞增殖、凋亡和周期的影响及对放疗增敏的可能机制。方法:(1)应用小分子干扰RNA技术抑制喉癌Hep-2细胞SHIP2的表达,实验分为5组:空白对照组(Control)、阴性对照组(Negative)、干扰组1(siRNA1)、干扰组2(siRNA2)、干扰组3(siRNA3)],RT-qPCR、Western Blot检测转染24 h后干扰效果;(2)取对数生长期的喉癌Hep-2细胞,分别给予0,4 Gy X射线(SSD=100 cm)处理,CCK-8检测细胞增殖活力,AnnexinPI-FITC/PI双染检测细胞凋亡,PI单染检测细胞周期,RT-qPCR、Western Blot检测SHIP2 mRNA和蛋白的相对表达量;(3)取对数生长期的喉癌Hep-2细胞,实验分4组(空白对照组Control、单纯放射组4 Gy、干扰组siRNA-SHIP2、联合处理组siRNASHIP2+4 Gy)。Western Blot检测SHIP2、pSHIP2、pAKT、caspase-3、P21和P27蛋白的相对表达量。结果:(1)转染24 h后干扰组2 siRNA2下调作用最显著,较空白对照组SHIP2 mRNA下调达60. 1%(P<0. 05)、SHIP2蛋白下调达57. 8%(P<0. 05);与空白对照组对比,siRNA2抑制SHIP2表达后喉癌Hep-2细胞增殖活力、侵袭和迁移能力降低,凋亡增加(P<0. 05)。(2)与0 Gy组比较,放射组各组细胞增殖活力降低,凋亡和G2期阻滞增加(P<0. 05);4Gy射线处理后SHIP2 mRNA、SHIP2和pSHIP2蛋白的相对表达量较空白对照组降低(P<0. 05)。(3)与空白对照组、siRNA-SHIP2组和4 Gy组比较,联合处理组细胞增殖活力降低,凋亡和G2期阻滞增加(P<0. 05),SHIP2和pSHIP2、pAKT的相对表达量降低(P<0. 05),caspase-3、P21和P27蛋白的相对表达量升高(P<0. 05)。结论:靶向抑制SHIP2可与放射治疗协同抑制喉鳞癌Hep-2细胞的增殖活力,促进细胞凋亡和细胞周期G2期阻滞,从而发挥放射增敏的作用。其可能机制是靶向抑制SHIP2表达通过下调PI3K/Akt信号通路活性,进而解除对下游促凋亡因子caspase3和周期阻滞因子P21和P27的抑制作用,与放射线协同增强对喉癌Hep-2细胞的杀伤作用,从而发挥放疗增敏作用。

Objective: To investigate the effect of SHIP2 on the proliferation, apoptosis, invasion and migration of Hep-2 cells;to study the changes of proliferation, apoptosis, cell cycle and the expression of SHIP2 in Hep-2 cells after radiotherapy;and to investigate the effect of inhibition of SHIP2 combined with radiotherapy on the proliferation, apoptosis and cell cycle of laryngeal carcinoma Hep-2 cells,and the possible mechanism of radiotherapy sensitization.Methods: Firstly, the expression of SHIP2 gene in Hep-2 cells was inhibited by small interfering RNA technique. The study was divided into five groups: control group, Negative group, siRNA1, siRNA2 and siRNA3 groups. RT-qPCR and Western Blot were used to detect the effect of interference, CCK-8 was used to evaluate proliferation,Annexin PI-FITC/PI double staining was used to determine apoptosis,Transwell was used to assess invation, and Scratch test was used to examine migration. Secondly, lupine carcinoma Hep-2 cells were treated with 0, 2, 4 and 8 Gy X-ray(SSD=100 cm) for 48 h, proliferation, apoptosis, cell cycle were detected, and RT-qPCR and Western Blot were used to evaluate the relative expression of SHIP2 mRNA and protein. Finally, the experiment was divided into four groups: control group, radiotherapy group(4 Gy), siRNA-SHIP2 group, combined treatment group(siRNA-SHIP2+4 Gy).Proliferation was tested, and the expression of SHIP2, pSHIP2, pAKT, caspase-3, P21 and P27 protein was detected by Western Blot.Results: Firstly, the expression of SHIP2 mRNA and protein in Hep-2 cells was significantly down-regulated after transfection and the effect of siRNA2 down-regulation was the most significant, with deceases of SHIP2 mRNA to 60. 1%(P<0. 05), and protein to57. 8%(P<0. 05). So siRNA2 was chosen for posterior experimental study. Compared with the blank control group and the negative control group, siRNA2 group inhibited the proliferation, invasion and migration of Hep-2 cells and increased apoptosis(P<0. 05). Secondly, compared with the 0Gy group, the cell proliferation was inhibited, the apoptosis and G2 phase arrest were increased in the radiation group(P<0. 05). The relative expression of SHIP2 mRNA, SHIP2 and pSHIP2 protein after 4 Gy ray treatment was lower than that respectively of blank control group(P<0. 05). Finally,compared with those of the blank control group, siRNA-SHIP2 group and 4 Gy group, the cell proliferation decreased, the apoptosis and G2 arrest increased(P<0. 05), and the relative expression of SHIP2 and pSHIP2 and pAKT decreased(P<0. 05), Caspase-3, P21 and P27 proteins increased(P<0. 05) in the combined group.Conclusion: Targeted inhibition of SHIP2 combined with radiotherapy can inhibit cell proliferation and promote the apoptosis and G2 arrest of Hep-2 cells,thus increasing the radiosensitivity. The possible mechanism is that inhibiting the expression of SHIP2 in laryngeal carcinoma Hep-2 cells combined with radiation therapy can significantly down-regulate PI3K/Akt signaling pathway activity, then lift the downstream pro-apoptotic factor caspase3 and cycle arrest factors P21 and P27, promote cell apoptosis and cycle arrest, synergistically enhance the radiation sensitivity of laryngeal carcinoma Hep-2 cells.