摘要
目的观察毛蕊异黄酮(CA)对人甲状腺乳头状癌细胞系TPC-1细胞增殖的影响,并从TFEB-自噬途径的角度探讨其可能的作用机制。方法体外培养甲状腺乳头状癌细胞TPC-1细胞,实验分为对照组、CA组、3-MA组和3-MA+CA组。CA组:用含25、50和100μg/mL CA的培养液分别处理TPC-1细胞24,48和72 h。3-MA组:用含5 mmol/L 3-MA的DMEM培养液培养细胞48 h。3-MA+CA组:先用含5 mmol/L 3-MA的DMEM培养液处理细胞0.5 h,然后再加入100μg/mLCA共同继续培养48 h。每组设5个重复孔(n=5)。细胞的增殖采用CCK-8法进行检测,通过透射电镜观测细胞的超微结构,自噬相关基因Beclinl、LC3和p62的表达以及TFEB总蛋白和TFEB在细胞核中表达通过蛋白质印迹检测。结果与对照组(0.85±0.05和1.13±0.07)比较,CA(50和100μg/mL)处理TPC-1细胞48(0.53±0.04和0.33±0.05)和72 h(0.81±0.06和0.45±0.05)降低了TPC-1细胞的增殖水平,并具有剂量依赖性,差异有统计学意义,F_(浓度)=14.98,F_(时间)=11.41,均P<0.05。与对照组比较,CA(50和100μg/mL)处理48 h组细胞中自噬体的总面积占细胞胞质面积的百分率(分别为1.46%±0.38%、7.91%±0.69%和13.62%±1.46%,F=6.28,P=0.012)、Beclin-l(分别为0.19±0.03,0.45±0.06和0.86±O.07,F=4.82,P=0.005)和LC3-Ⅱ(分别为0.09±0.02、0.35±0.05和0.54±0.06,F=6.74,P=0.007)的蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ的比值(分别为0.25±0.07,0.95±0.03和1.26±0.02,F=5.83,P=0.003)均增加,p62的蛋白表达水平分别为0.57±0.07,0.22±0.03和0.09±0.02,下调并具有剂量依赖性,差异有统计学意义,F=5.74,P=0.023。CA和CA+3-MA组的A值分别为0.35±0.05和0.67±0.09,细胞自噬抑制剂3-MA部分逆转了CA抑制TPC-1细胞增殖的效应,t=5.63,P=O.038。与对照组比较,CA(50和100μg/mL)处理48 h组TFEB在细胞核中的表达(分别为0.17±0.03,0.49±0.06和0.78±0.08,F=6.39,P=0.009)以及TFEB在核中表达与TFEB总蛋白的比值(分别为0.20±0.03、0.57±0.05和0.93±0.06,F=5.43,P=0.016)均增加,并�
Objective To observe the effect of Calycosin(CA)on the proliferation of human papillary thyroid carcinoma cell line TPC-1 cells and explore its possible mechanism from the perspective of TFEB autophagy pathway.Methods TPC-1 cells were cultured in vitro.The experiment was divided into control group,CA Group,3-MA group and 3-MA+CA group.TPC-1 cells in CA group were treated with culture medium containing 25,50 and 100μg/mL CA for 24,48 and 72 h respectively.Cells in 3-MA group were cultured in DMEM medium containing 3-MA(5 mmol/L)for 48 h.Cells in the 3-MA+CA group were treated with DMEM medium containing 3-MA(5 mmol/L)for 0.5 h,then CA(100μg/mL)was added into the medium and cells were continued to co-culture for 48 h.CCK-8 method was used to detect the proliferation level of the cells.The ultrastructures of the cells were observed by transmission electron microscopy.The expressions of autophagy related gene Beclin-1.LC3 and p62/SQSTM1 and expressions of TFEB total protein and TFEB in nucleus were measured by Western Blot.Results Compared with the control group(0.85±0.05 and 1.13±0.07),treatment with CA(50 and 100μg/mL)for 48(0.53±0.04 and 0.33±0.05)and 72 h(0.81±0.06 and 0.45±0.05)reduced the proliferation of TPC-1 cells in a dose-dependent manner,and the difference was statistically significant(F_(concentration)=14.98,F_(time)=11.41,F<0.05).Compared with the control group,the number of autophagy,the percentage of total autophagy area to the cytoplasmic area(1.46%±0.38%,7.91%±0.69%and 13.62%±1.46%,respectively,F=6.28,P=0.012),the protein expressions of Beclin-1(0.19±0.03,0.45±0.06 and 0.86±0.07,respectively,F=4.82,P=0.005)and LC3-Ⅱ(0.09±0.02,0.35±0.05 and 0.54±0.06,respectively,F=6.74,P=0.007),and the ratio of LC3-Ⅱto LC3-Ⅰ(0.25±0.07,0.95±0.03 and 1.26±0.02,respectively,F=5.83,P=0.003)in treatment with CA(50 and 100μg/mL)for 48 h groups were increased,and the protein expression level of p62(0.57±0.07,0.22±0.03 and 0.09±0.02,respectively,F=5.74,P=023)was decreased,and the difference wa
作者
封海岗
程飚
王佑权
曹洪
左俊
FENG Hai-gang;CHENG Biao;WANG You-quan;CAO Hong;ZUO Jun(Second Affiliated Hospital,University of South China,Hengyang 421001,China;Department of Mammary Gland and Thyroid Gland Surgery Southern Theater General Hospital,Guangzhou 440104,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第9期644-649,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
广东省科技计划项目(2015B020233012)。
关键词
毛蕊异黄酮
甲状腺乳头状癌
细胞增殖
转录因子EB
自噬
calycosin
papillary thyroid carcinoma
cell proliferation
transcription factor EB
autophagy
