过表达ULK3介导GLi1对人膀胱癌细增殖、迁移、侵袭的影响及机制

Overexpression of ULK3 mediates the effects of Gli1 on the proliferation,migration,and invasion of bladder cancer cells and its mechanisms

目的探讨过表达Un51样激酶3(ULK3)介导胶质瘤相关癌基因同源基因1(Gli1)对膀胱癌细胞增殖、迁移与侵袭的影响及其机制。方法培养人膀胱癌T24细胞分为对照组、ULK3过表达组(ULK3+组)、Gli1过表达组(Gli1+组)、ULK3和Gli1过表达组(ULK3+/Gli1+组)、ULK3过表达和Gli1敲低组(ULK3+/Gli1-组),分别使用阴性对照试剂、过表达ULK3质粒、过表达Gli1质粒、shRNA敲低Gli1质粒对相应各组进行细胞转染。收集转染后的各组细胞,采用细胞计数试剂盒(CCK)8检测细胞的增殖能力,划痕实验检测细胞运动能力,Transwell实验检测细胞的迁移、侵袭能力,Western blotting检测LC3A/B、P62蛋白表达情况,荧光蛋白标记技术检测自噬小体LC3B蛋白表达情况。结果(1)对照组、ULK3+组、Gli1+组、ULK3+/Gli1+组、ULK3+/Gli1-组细胞培养0 h时OD值差异无统计学意义(P=1.000),培养24、48、72 h时,ULK3+组、Gli1+组、ULK3+/Gli1+组、ULK3+/Gli1-组的OD值均大于对照组,差异均有统计学意义(F=60.98、56.53、112.70,P值均<0.001)。(2)对照组、Gli1+组、ULK3+组、ULK3+/Gli1+组、ULK3+/Gli1-组细胞迁移数分别为(292.3±28.5)、(376.7±8.1)、(365.7±8.1)、(505.7±22.4)、(301.3±26.7)个,细胞侵袭数分别为(127.7±10.0、(185.0±11.5)、(191.3±8.0)、(278.3±11.0)、(128.7±4.0)个,24 h细胞迁移率分别为8.8%±0.2%、18.8%±0.3%、18.8%±0.4%、24.6%±0.4%、13.8%±0.3%,48 h细胞迁移率分别为14.9%±0.3%、30.3%±2.1%、29.8%±1.6%、50.1%±3.7%、24.9%±1.7%。ULK3+组、Gli1+组、ULK3+/Gli1+组、ULK3+/Gli1-组细胞迁移、侵袭数目,以及24、48 h细胞迁移率均大于对照组,差异均有统计学意义(F=50.82、126.80、112.20、106.70,P值均<0.001)。(3)对照组、Gli1+组、ULK3+组、ULK3+/Gli1+组、ULK3+/Gli1-组细胞LC3A/B蛋白相对表达量分别是0.18±0.01、0.41±0.02、0.41±0.03、0.63±0.03、0.25±0.03,P62蛋白相对表达量分别为1.06±0.07、0.88±0.01、0.87±0.02、0.53±0.02、0.98±0.04;ULK3+组、Gli1+组、ULK3+/Gli1+组、ULK3+/Gli1-组LC3A/B蛋白的相对表达量均高于对照组,P62蛋白的相对表达量均低于对照组,差异均有统计学意义(F=152.80、72.48,P值均<0.001)。(4)ULK3+组、Gli1+组、ULK3+/Gli1+组、ULK3+/Gli1-组自噬小体LC3蛋白荧光表达量均多于对照组。结论过表达ULK3可以增强人膀胱癌T24细胞增殖、迁移及侵袭能力,其机制可能是通过介导Gli1诱导了细胞自噬反应的增强。

Objective This study aims to investigate the effect of overexpression of Un51-like kinase 3(ULK3)on the proliferation,migration,and invasion of bladder cancer cells and its mechanism.Methods Cultured human bladder cancer T24 cells were divided into control group,ULK3 overexpression group(ULK3+group),Glioma-associated oncogene homolog 1(Gli1)overexpression group(Gli1+group),ULK3 and Gli1 overexpression group(ULK3+/Gli1+group),and ULK3 overexpression and Gli1 knockdown group(ULK3+/Gli1−group).The cells were transfected with a negative control reagent,overexpression of ULK3 plasmid,overexpression of Gli1 plasmid and shRNA knockdown of Gli1 plasmid,respectively.After transfection,the cell proliferation was detected by cell counting kit(CCK)-8,the cell movement ability was detected by scratch test,the migration and invasion abilities were detected by Transwell test,the expression levels of LC3A/B and p62 proteins were detected by Western blotting,and the expression of autophagosome LC3B protein was detected by fluorescent protein labeling.Results(1)No significant difference was found in the optical density(OD)values among the control,ULK3+,Gli1+,ULK3+/Gli1+,and ULK3+/Gli1−groups at 0 h culture(P=1.000).At 24,48,and 72 h cultures,the OD values of the ULK3+,Gli1+,ULK3+/Gli1+,and ULK3+/Gli1−groups were all higher than those of the control group,and the differences were statistically significant(F=60.98,56.53,112.70,all P values<0.001).(2)The numbers of cell migration in the control,Gli1+,ULK3+,ULK3+/Gli1+,and ULK3+/Gli1−groups were 292.3±28.5,376.7±8.1,365.7±8.1,505.7±22.4,and 301.3±26.7,respectively;the numbers of cell invasion were 127.7±10.0,185.0±11.5,191.3±8.0,278.3±11.0,and 128.7±4.0,respectively;the 24 h cell migration rates were 8.8%±0.2%,18.8%±0.3%,18.8%±0.4%,24.6%±0.4%,and 13.8%±0.3%,respectively;and the 48 h cell migration rates were 14.9%±0.3%,30.3%±2.1%,29.8%±1.6%,50.1%±3.7%,and 24.9%±1.7%,respectively.The cell migration,invasion number,and 24 and 48 h cell migration rates in the ULK3+,Gli1+,ULK3+/Gli1+,and ULK3+/Gli1−groups were significantly higher than those in the control group(F=50.82,126.80,112.20,and 106.70,all P values<0.001).(3)The relative expression levels of LC3A/B protein in the control,Gli1+,ULK3+,ULK3+/Gli1+,and ULK3+/Gli1−groups were 0.18±0.01,0.41±0.02,0.41±0.03,0.63±0.03,and 0.25±0.03,respectively,and those of p62 protein were 1.06±0.07,0.88±0.01,0.87±0.02,0.53±0.02,and 0.98±0.04,respectively.The relative expression of LC3A/B protein in the ULK3+,Gli1+,ULK3+/Gli1+,and ULK3+/Gli1-groups was higher than that in the control group.Meanwhile,the relative expression of p62 protein was lower than that in the control group,and the difference was statistically significant(F=152.80,72.48,all P values<0.001).(4)The fluorescent expression of LC3 protein in the autophagosome of the ULK3+,Gli1+,ULK3+/Gli1+,and ULK3+/Gli1−group was higher than that of the control group.Conclusion Overexpression of ULK3 can enhance the proliferation,migration,and invasion of human bladder cancer T24 cells,and its mechanism may be enhanced activation of autophagy reaction by induced Gli1.