TGM2对甲状腺癌干细胞紫杉醇化疗敏感性的影响及作用机制  

Effect and mechanism of TGM2 on chemosensitivity of paclitaxel for thyroid cancer stem cells

作　　者：林琳[1] 王承芳[1] 洪志军[2] LIN Lin;WANG Cheng-Fang;HONG Zhi-Jun(Department of Health and Physical Examination,the First Affiliated Hospital of Dalian Medical University,Dalian 116021,China)

机构地区：[1]大连医科大学附属第一医院普外科,大连116021 [2]大连医科大学附属第一医院外科,大连116021

出　　处：《中国免疫学杂志》2020年第13期1594-1599,共6页Chinese Journal of Immunology

基　　金：辽宁省科学技术计划项目(No:2018010782-301)资助

摘　　要：目的:探究转谷氨酰胺酶2(TGM2)对甲状腺癌干细胞(TCSC)紫杉醇化疗敏感性的影响及作用机制。方法:收集甲状腺癌患者新鲜肿瘤组织,分离培养原代甲状腺癌细胞,经无血清培养成悬浮肿瘤细胞球时,流式细胞仪筛选出CD133+表型的TCSC进行后续实验。Western blot检测TCSC中干性基因Nanog、Sox2蛋白表达水平。小干扰RNA技术转染TCSC,分为si-TGM2组和si-NC组,MTS检测TGM2 siRNA转染的TCSC对紫杉醇的敏感性;qRT-PCR法检测紫杉醇处理TCSC后细胞TGM2 mRNA表达。软琼脂克隆实验检测TGM2 siRNA转染对紫杉醇处理的TCSC肿瘤球形成能力的影响;Tunnel凋亡和流式细胞仪检测TGM2 siRNA转染对紫杉醇处理的TCSC凋亡的影响。裸鼠实验检测TGM2对紫杉醇处理TCSC敏感性的影响。Western blot检测TGM2 siRNA转染对紫杉醇处理的TCSC凋亡蛋白caspase-3、Bcl-2表达的影响。结果:流式细胞仪分选出CD133+细胞占全部细胞的1.21%,CD133+细胞中Nanog、Sox2蛋白表达水平显著高于CD133-细胞;沉默TGM2表达可显著提高TCSC对紫杉醇的敏感性;紫杉醇处理的TCSC中TGM2 mRNA表达水平显著高于未经紫杉醇处理的TCSC;沉默TGM2表达显著抑制紫杉醇处理的TCSC肿瘤球形成能力,显著增加紫杉醇处理的TCSC凋亡,提高TCSC对紫杉醇的敏感性,提高紫杉醇处理的TCSC凋亡蛋白caspase-3、Bax表达(P<0.05)。结论:TGM2基因可能通过抑制凋亡信号通路,降低TCSC的紫杉醇化疗敏感性。Objective:To investigate effect and mechanism of transglutaminase 2(TGM2)on chemosensitivity to paclitaxel of thyroid cancer stem cells(TCSC).Methods:Fresh tumor tissues of thyroid cancer patients were collected,and primary thyroid cancer cells were isolated and cultured.When serum-free cultured into suspension tumor cell spheres,CD133+phenotype TCSC were screened by flow cytometry for subsequent experiments.Western blot was used to detect expression levels of stem genes Nanog and Sox2 in TCSC.Small interfering RNA technology were transfected into TCSC,which were divided into si-TGM2 group and si-NC group.MTS was used to detect sensitivity of TGM2 siRNA transfected TCSC for paclitaxel.qRT-PCR was used to detect expression of TGM2 mRNA in TCSC treated with paclitaxel.Soft agar cloning assay was used to detect effect of TGM2 siRNA transfection on paclitaxel-treated TCSC tumor spheroid formation.Tunnel and flow cytometry were used to detect the effect of TGM2 siRNA transfection on paclitaxel-treated TCSC apoptosis.Nude mice were tested for effect of TGM2 on sensitivity of TCSC to paclitaxel.Effect of TGM2 siRNA transfection on expressions of caspase-3 and Bcl-2 in paclitaxel-treated TCSC were detected by Western blot.Results:Flow cytometry sorted CD133+cells to 1.21%of all cells.Expression levels of Nanog and Sox2 in CD133+cells were significantly higher than those in CD133-cells.Silencing of TGM2 expression significantly increased sensitivity of TCSC to paclitaxel.Expression level of TGM2 mRNA in paclitaxel-treated TCSC was significantly higher than that without paclitaxel-treated TCSC;Expression of silencing TGM2 significantly inhibited ability of paclitaxel-treated TCSC tumor globule formation,and significantly increased apoptosis of paclitaxel-treated TCSC,and increased sensitivity of TCSC to paclitaxel,and significantly increased expressions of caspase-3 and Bax in paclitaxel-treated TCSC.(P<0.05).Conclusion:TGM2 gene may reduce chemosensitivity of TCSC to paclitaxel by inhibiting apoptotic signaling pathway.

关 键 词：谷氨酰胺转胺酶2 甲状腺癌 肿瘤干细胞 紫杉醇 化疗敏感性 

分 类 号：R736.1[医药卫生—肿瘤]