二磷酸腺苷核糖化因子鸟苷酸激酶1对甲状腺乳头状癌细胞自噬的调节作用

Regulation of autophagy in papillary thyroid carcinoma cells by adenosine di-phosphate-ribosylation factor guanosine triphosph-ase activating protein 1

目的观察二磷酸腺苷(ADP)核糖化因子鸟苷酸激酶1(ASAP1)在调节甲状腺乳头状癌细胞自噬中的作用。方法通过免疫荧光检测郑州大学第一附属医院60例甲状腺乳头状癌组织和癌旁组织中ASAP1以及自噬标志蛋白微管相关蛋白1轻链3(LC3)的表达水平,分析60例标本中ASAP1的表达水平与患者临床病理特征之间的关系,用慢病毒CRISPR/Cas9技术敲除ASAP1来构建稳定细胞系,通过免疫荧光和蛋白质印迹法(Western blot)检测ASAP1敲除组与对照组之间自噬水平的差异,各变量比较采用χ^2检验和t检验。结果甲状腺乳头状癌组织中ASAP1蛋白平均荧光强度为276.33,癌旁组织中ASAP1蛋白平均荧光强度为74.67,两组比较差异有统计学意义(P<0.01);甲状腺乳头状癌组织中LC3蛋白平均荧光强度为74.67,癌旁组织中LC3蛋白平均荧光强度为290.67,两组差异有统计学意义(P<0.01);在甲状腺乳头状癌细胞中,ASAP1敲除组LC3Ⅱ蛋白的表达量高于对照组,两组比较差异有统计学意义(MDA-T32细胞:0.463±0.081,P<0.05;MDA-T85细胞:0.750±0.050,P<0.01)。结论ASAP1能够调节甲状腺乳头状癌细胞的自噬,这可能是ASAP1参与甲状腺乳头状癌细胞转移调控的机制之一。

Objective To observe the role of adenosine di-phosphate(ADP)-ribosylation factor guanosine triphosph(GTP)-ase activating protein 1(ASAP1)in regulating autophagy of thyroid papillary carcinoma cells.Methods The expression levels of ASAP1 and autophagy marker protein microtubule-associated protein 1 light chain 3(LC3)in 60 cases of papillary thyroid carcinoma and adjacent tissues were detected by immunofluorescence.The relationship between the expression level of ASAP1 and the clinicopathological characteristics of 60 patients was analyzed.Knockout ASAP1 gene using lentivirus CRISPR/Cas9 technology to construct stable cell lines.The difference of autophagy level between ASAP1 knockout group and control group was detected by immunofluorescence and Western blotting.Results The average fluorescence intensity of ASAP1 protein in papillary thyroid tissue is 276.33,and the average fluorescence intensity of ASAP1 protein in adjacent tissues is 74.67.The difference between the two groups is statistically significant(P<0.01).The average fluorescence intensity of LC3 protein in papillary thyroid tissue is 74.67,and the average fluorescence intensity of LC3 protein in adjacent tissues is 290.67.The difference between the two groups is statistically significant(P<0.01).The expression of LC3Ⅱprotein in ASAP1 knockout group was higher than that in control group.The difference between the two groups was statistically significant(MDA-T32 cells:0.463±0.081,P<0.05;MDA-T85 cells:0.750±0.050,P<0.01).Conclusion ASAP1 can regulate autophagy in papillary thyroid carcinoma.ASAP1 might regulate papillary thyroid carcinoma cells metastasis by modulating autophagy.