Sox2对人非小细胞肺癌A549/DDP干样特征影响研究

Effects of Sox2 on cisplatin-resistant cells A549/DDP stem-like properties in lung adenocarcinoma

目的Sry相关的HMG盒基因2 [Sry-related （high mobility group, HMG） Box Gene 2, Sox2 ]蛋白在多个肿瘤的发生发展过程中具有重要作用。本研究旨在探讨Sox2对人非小细胞肺癌（non-small cell lung cancer,NSCLC）获得性耐药株A549／DDP干样特征的影响。方法通过药物诱导培养A获得性耐药株549／DDP，进而使用CCK-8、微球体形成实验、集落形成实验、蛋白质印迹法和流式细胞术等方法了解Sox2对A549／DDP细胞干样特征的影响。利用Graph—Pad Prism5进行数据统计。结果成功建立的获得性耐药株A549／DDP具有较强的克隆形成能力和微球体形成能力等干样特征。细胞免疫荧光和蛋白质印迹法结果显示Sox2高表达于部分A549／DDP细胞核中。siRNA—Sox2组的克隆形成数为（16．8±1．5）个，而sIRNA对照组克隆数为（35．3±1．6）个，提示敲低Sox2表达，显著降低了A549／DDP细胞的克隆形成能力，两组间比较差异有统计学意义，t=7．343，P〈0．0001；在siRNA—Sox2作用下，抑制了A549／DDP细胞的微球体形成能力。流式细胞仪凋亡分析发现，DDP浓度为0和10μmol／L的情况下，siRNA对照组的凋亡百分比分别为（3．9±0．5）％和（5．3±1．0）％；siRN—Sox2组分别为（13．8±1．9）％和（25．9±3．2）％；抑制Sox2表达，增加了A549／DDP细胞对DDP的化疗敏感性，F=133．7，P=0．001。结论Sox2在部分人肺腺癌A549／DDP获得性耐药株中高表达，具有维持A549／DDP细胞干样特征的作用，抑制Sox2表达可以逆转A549／DDP细胞对DDP的化疗抵抗。

OBJECTIVE Emerging evidences that Sox2 has an important role in tumourgenesis in multi-types canc- ers,including lung cancer. This study aimed to explore Sox2 effect on the cisplatin resistant cells A549/DDP stem-like properties in lung adenocarcinoma. METHODS Cell counting kit-8 （CCK-8）, colony forming assay,microsphere formation assay,Western blot and flow cytometry were used to investigate the function of Sox2 on A549/DDP cells stem-like signa- ture,and GraphPad Prism 5 software was used to analyze the related data. RESULTS The induced A549/DDP eisplatin- resistant cell lines showed the stem-like properties including clone formation and microsphere formation. The results of cellular immunofluorescence and Western blot assay showed that Sox2 was highly expressed in the nuclei of part A549/ DDP cells, hut not in A549/parent cells. The number of clone formation was 16.8±1.5 in siRNA-Sox2 group,and 35.3±1.6 in siRNA control group, suggesting that knockdown of Sox2 expression significantly decreased the colone formation a- bility of A549/DDP cells, and the difference was significant （t = 7. 343, P〈 0. 000 1）. Under the treatment of siRNA- Sox2,the mierosphere formation of A549/DDP cells was decreased. Furthermore, flow cytometry analysis showed that with cisplatin concentration of 0 and 10μmol/L,the percentage of apoptotic cells were （3.9±0.5）% and （5.3± 1.0）% in siRNA control group and （13. 8 ± 1. 9）% and （25. 9 ± 3. 2）% in siRN-Sox2 group, respectively. Showing thatinhibition of the expression of Sox2 significantly increased the chemosensitivity of A549/DDP cells to cisplatin,which re suited in more A549/DDP apoptosis （F=133.7,P=0. 001）. CONCLUSION High expression of Sox2 in human lung ad enocarcinoma A549/DDP cisplatin-resistant cells can remain A549/DDP cell stem-like characteristics,and inhibition of the expression of Sox2 can enhance the A549/DDP chemosensitivity to cisplatin.