摘要
目的:探究转录因子SOX2在胰腺癌干细胞干性维持及吉西他滨化疗耐药性中的作用及机制。方法:悬浮肿瘤球形成实验培养人胰腺癌细胞系PACN-1细胞,流式细胞仪筛选出CD133^+的胰腺癌干细胞(PCSC)进行后续实验。Western blot检测CD133^+的PCSC细胞中干性相关基因Oct4、Nanog的表达水平。构建SOX2敲除的稳转PCSC细胞株,MTT检测Sh-SOX2稳转的PCSC对吉西他滨的敏感性。软琼脂克隆实验检测SOX2对胰腺癌肿瘤球形成能力的影响;流式细胞仪检测SOX2对细胞凋亡的影响。Western blot检测SOX2对Caspase-3及Caspase-9蛋白表达的影响。结果:CD133^+细胞中Oct4、Nanog蛋白的表达水平显著高于CD133-细胞;体外实验显示敲除SOX2后的PCSC增殖活性及肿瘤球的形成能力显著降低;细胞的凋亡率显著升高;Caspase-3及Caspase-9蛋白的表达升高(P<0.05)。结论:SOX2基因可能通过调控细胞凋亡蛋白Caspase-3、Caspase-9表达促进细胞增殖,维持胰腺癌细胞的干性,降低吉西他滨治疗的灵敏性。
Objective:To investigate role and mechanism of transcription factor SOX2 in stemness maintenance of pancreatic cancer stem cells and resistance of gemcitabine.Methods:Human pancreatic cancer cell line PACN-1 cells were cultured by suspension tumor sphere formation.CD133^+pancreatic cancer stem cells(PCSC)were screened by flow cytometry for subsequent experiments.Western blot was used to detect expression levels of stemness related genes Oct4 and Nanog in CD133^+PCSC cells.SOX2 knockout stable PCSC cell line was constructed,and sensitivity of Sh-SOX2-stable PCSC to gemcitabine was detected by MTT.Soft agar clone assay was used to detect effect of SOX2 on tumor formation ability of pancreatic cancer.Effect of SOX2 on apoptosis was detected by flow cytometry.Effect of SOX2 on expression of Caspase-3 and Caspase-9 protein was detected by Western blot.Results:Expression of Oct4 and Nanog protein in CD133^+cells were significantly higher than those in CD133-cells.Proliferation activity of PCSC after knocking out SOX2 and ability to from tumor spheres was significantly decreased in vitro.Apoptotic rate of cells was significantly increased;expressions of Caspase-3 and Caspase-9 protein were increased(P<0.05).Conclusion:SOX2 may promote cell proliferation,maintainin stemness of pancreatic cancer cells,and reduce resistance of gemcitabine by regulating expression of Caspase-3 and Caspase-9.
作者
陶洪
吴福道
张小静
胡芳
TAO Hong;WU Fu-Dao;ZHANG Xiao-Jing;HU Fang(Department of Oncology,Dazhou Central Hospital,Dazhou 635000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第11期1380-1383,1386,共5页
Chinese Journal of Immunology
