ATG5对甲状腺乳头状癌细胞增殖的影响

Effect of ATG5 on the proliferation of papillary thyroid carcinoma

目的通过模拟肿瘤微环境,观察自噬抑制后自噬相关蛋白5(ATG5)对甲状腺乳头状癌细胞增殖的影响。方法蛋白质印迹法和逆转录-聚合酶链反应(RT-PCR)实验检测甲状腺正常细胞系(Nthy-ori-3-1)和甲状腺乳头状癌K1细胞中ATG5蛋白和mRNA表达差异;培养敲除ATG5的shRNA-486和shRNA-938稳转细胞株,细胞计数试剂盒8(CCK-8)实验和克隆形成实验分别检测在体积分数为10%血清培养条件下和体积分数为0.5%血清培养条件下,shRNA-486和shRNA-938与对照组对K1细胞增殖的影响。结果Nthy-ori-3-1细胞中ATG5蛋白表达量为0.682±0.037,低于K1细胞的1.933±0.010,t=32.330,P<0.001。Nthy-ori-3-1细胞中ATG5 mRNA表达量为0.503±0.017,低于K1细胞的0.902±0.016,t=17.140,P<0.001。转染ATG5-shRNA后,对照组LC3-Ⅰ/LC3-Ⅱ比值(5.521±0.205)低于shRNA-486敲除组(14.490±0.512)和shRNA-938敲除组(9.639±0.558);对照组p62表达量(1.335±0.0754)低于shRNA-486敲除组(2.593±0.081)和shRNA-938敲除组(2.830±0.066)。CCK-8增殖实验中,在10%血清培养条件下,对照组分别与shRNA-486敲除组和shRNA-938敲除组比较,敲除ATG5后促进了K1细胞的增殖,经两因素方差分析,F_(敲除)=38.590,P_(敲除)<0.001,F_(时间)=385.300,P_(时间)<0.001;在0.5%血清培养条件下,对照组分别与shRNA-486敲除组和shRNA-938敲除组比较,敲除ATG5后抑制了K1细胞的增殖,经两因素方差分析,F_(敲除)=14.730,P_(敲除)=0.005,F_(时间)=336.300,P_(时间)<0.001。克隆形成实验10 d后观察发现,在10%血清培养条件下,对照组分别与shRNA-486敲除组和shRNA-938敲除组比较,敲除ATG5后促进了K1细胞的增殖;在0.5%血清培养条件下,对照组与shRNA-486敲除组和shRNA-938敲除组比较,敲除ATG5后反而抑制了K1细胞的增殖,经两因素方差分析,F_(敲除)=76.380,P_(敲除)=0.001,F_(血清浓度)=1106,P_(血清浓度)=0.001。结论自噬对甲状腺癌K1细胞增殖的调控受癌细胞所处微环境的影响,在营养充足时,阻断自噬抑制了甲状腺癌细胞的增殖;在营养剥夺时,阻断自噬促进甲状腺癌细胞的增殖。

Objective To observes the effect of autophagy inhibition of ATG5on the proliferation of papillary thyroid carcinoma cells by simulating tumor microenvironment.Methods Western blot and RT-PCR were used to detect the the difference of ATG5protein and mRNA expression between normal thyroid cell line(Nthy-ori-3-1)and papillary thyroid cancer K1cells.Culture of knockdown ATG5stable cell line(shRNA-486and shRNA-938),CCK-8experiment and clone formation experiments were performed to examine the effect of ATG5knockdown on K1cell proliferation,which were compared with control group under 10%serum culture conditions and 0.5%serum culture conditions.Results The protein expression level of ATG5in Nthy-ori-3-1cells was significantly lower than that in K1cells(0.682±0.037 vs 1.933±0.010,t=32.330,P<0.001).The mRNA level of ATG5in Nthy-ori-3-1cells was significantly lower than that in K1cells(0.503±0.017 vs 0.902±0.016,t=17.140,P<0.001).After transfection of ATG5-shRNA,the LC3-I/LC3-Ⅱratio of the control group(5.521±0.205)was significantly lower than that of the shRNA-486(14.490±0.512)and shR-NA-938(9.639±0.558)knockout groups;the expression of p62in the control group(1.335±0.075)was significantly lower than that in the shRNA-486(2.593±0.081)and shRNA-938(2.830±0.066 1)knockout groups.In the CCK-8proliferation experiment,the control group was compared with the shRNA-486and shRNA-938knockout groups under10%serum culture conditions,and knockdown of ATG5promoted the proliferation of K1cells(F_(knockout)=38.590,P_(knockout)<0.001;F_(time)=385.300,P_(time)<0.001).In the 0.5%serum culture condition,the control group was compared with the shRNA-486and shRNA-938knockout groups,and knockdown of ATG5inhibited the proliferation of K1cells(F_(knockout)=14.730,P_(knockout)=0.005;F_(time)=336.300,P_(time)<0.001).In the colony formation experiment,the control group was compared with the shRNA-486and shRNA-938knockout groups under 10%serum culture conditions,and knockdown of ATG5promoted the proliferation of K1cells;in the 0.5%serum culture condition,the control group was compared with the shRNA-486and shRNA-938knockout groups,and knockdown of ATG5inhibited the proliferation of K1cells(Fknockout=76.380,Pknockout=0.001;Fserum concentration=1 106,Pserum concentration=0.001).Conclusions The regulation of autophagy on the proliferation of thyroid carcinoma K1cells is affected by the microenvironment of cancer cells.When nutrient is sufficient,the blocking autophagy inhibits the proliferation of thyroid cancer cells.In the case of nutrient deprivation,the blocking autophagy promotes the proliferation of thyroid cancer cells.