胸腺素β10在人肺腺癌细胞株A549中抑制细胞凋亡、促进细胞增殖机制研究

Mechanism of Thymosin Beta 10 Inhibiting the Apoptosis and Prompting Proliferation in A549 Cells

背景与目的胸腺素β10(thymisinβ10,Tβ10)是胸腺素家族的成员之一,它的分子量在5 k Da左右,是哺乳动物体内含量最丰富的β胸腺素之一,作为一种肌动蛋白结合蛋白,它可能通过调控肌动蛋白的结构改变细胞的生长、死亡、粘附和迁移。Tβ10在肿瘤的增殖、凋亡、血管形成方面也发挥重要的作用。然而Tβ10在不同类型的肿瘤中所发挥的作用有很大差异且Tβ10对肺癌细胞增殖和凋亡的影响尚未见文献报道。本研究选择肺腺癌细胞系A549作为研究对象,通过加入Tβ10或用小干扰RNA干扰Tβ10的方法 ,检测肺癌细胞凋亡、增殖及细胞周期的变化,探讨Tβ10对肺癌细胞这几种生物学行为的影响及其可能的机制。方法流式双染检测加入Tβ10或干扰Tβ10后细胞凋亡的变化,PI染色后检测细胞周期的变化,CCK-8法检测细胞增殖能力的变化,Real-time PCR及蛋白免疫印迹检测增殖、凋亡相关基因的变化。结果加入Tβ10能抑制A549细胞的凋亡,促进细胞的增殖,增加S期和G2期/M期细胞的比率,减少Caspase-3、P53表达的同时增加Cyclin A、Cyclin E表达;干扰Tβ10能促进A549细胞的凋亡,抑制细胞的增殖,增加G0期/G1期细胞的比率,增加Caspase-3、P53表达的同时减少Cyclin A、Cyclin E表达。结论在肺癌细胞系中Tβ10能够通过抑制P53的表达抑制细胞凋亡,能够通过上调Cyclin A、Cyclin E的表达水平,促进细胞周期进程,促进细胞的增殖。Tβ10可能成为肺癌诊断的分子标记物及治疗靶标。

Background and objective Thymosin beta 10 （Tβ10） is one ofβ-thymosin family members, has a highly conserved polar 5 kDa peptides. hTis peptide is now regarded to be a small actin-binding protein and thereby induce depolymerization of the intracellular F-actin networks. Alteration of Tβ10 expression may alter the balance of cell growth, cell death, cell attachment and cell migration. Tβ10 also affects cell metastasis as well as proliferation, apoptosis and vasculariza-tion of cancer cells. But function of Tβ10 appear to be rather different between cancer cells, and the molecular mechanisms ofβ-thymosins to regulate cell apoptosis and proliferation in NSCLC （non-small cell lung cancer） cell lines are unclear. In this study, we used lung adenocarcinoma cell line A549, added Tβ10 or down-regulated the expression of Tβ10. We observed the change of apoptosis, proliferation and cell cyclin ability in A549 and the mechanisms underline them were also identiifed. Methods Atfer A549 was treated with 100 ng/mL recombinant human Tβ10 or siTβ10, apoptosis rate of A549 and cell cycle distribution were detected by lfow cytometry （FCM）. CCK-8 assay was employed to determine the proliferation of A549. hTe mRNA level of P53, Caspase-3, Cyclin A and Cyclin E were determined by real-time PCR. hTe protein level of P53, Caspase-3, Cyclin A and Cyclin E were detected by Western blot. Results Add Tβ10 can inhibit the apoptosis and prompt the prolifera-tion of A549. It can also increase the cell rates of S-phrase and G2/M-phrase, decrease the expression of P53 and Caspase-3,but increase the expression of Cyclin A and Cyclin E. Interferance of Tβ10 can prompt the apoptosis and inhibit the prolifera-tion of A549. It can also increase the cell rates of G0/G1-phrase, increase the expression of P53 and Caspase-3, but decrease the expression of Cyclin A and Cyclin E. Conclusion In lung cancer cell line, Tβ10 can inhibit the apoptosis by increase P53, drive cells into the S and G2/M-phase, prompt cell proliferation by increase the expression of Cyclin A and Cyclin E. Tβ10 may become a potential biomarker and therapy target for non-small cell lung cancer.