摘要
目的观察卵泡抑素样蛋白1(FSTL1)在结肠癌细胞生长和凋亡中的作用。方法将FSTL1小干扰RNA(siRNA)瞬时转染体外结肠癌DLD1细胞后,细胞计数试剂盒-8(CCK-8)法检测细胞活力;克隆形成实验观察单个DLD1细胞形成细胞克隆的能力;流式细胞术检测细胞周期和凋亡;蛋白质印迹法(Western blot)试验检测细胞周期相关蛋白(Cyclin E、E2F-1、p-Rb、p53和p73)、细胞凋亡相关蛋白[Cytochrome C、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、B细胞淋巴瘤/白血病-2(bcl-2)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和聚腺苷二磷酸核糖聚合酶(PARP)]和细胞通路蛋白[磷酸化细胞外信号调节激酶1/2(p-ERK1/2)、磷酸化c-Jun氨基末端激酶(p-JNK)、磷酸化蛋白激酶B(p-Akt)和κB抑制蛋白(IκB)]的表达。结果空白对照(BC)组、siRNA阴性对照(NC)组和FSTL1 siRNA组细胞吸光度A值分别为1.124±0.157、1.243±0.161和0.708±0.114,组间差异有统计学意义(F=11.172,P<0.01);DLD1细胞经转染FSTL1 siRNA后,细胞克隆形成数目为(42.3±6.5),与BC组(154.7±16.2)或siRNA NC组(183.8±12.6)比较,差异有统计学意义(F=24.694,P<0.001);BC组和siRNA NC组DLD1细胞凋亡率分别为(1.62±0.24)%和(1.84±0.27)%,与FSTL1 siRNA组(12.321.96)%比较,差异有统计学意义(F=19.687,P<0.001);沉默FSTL1的表达后可下调Cyclin E、E2F-1和p-Rb的表达,上调p53和p73的表达(F=5.214、21.953、10.842、22.264、18.062,P<0.05);干扰FSTL1的表达后可促进细胞中Cytochrome C、bax、cleaved-PARP和cleaved-Caspase 3的表达,并抑制bcl-2的表达(F=18.694、31.481、28.043、34.875、39.746,P<0.01);敲除FSTL1的表达后可导致DLD1细胞中p-ERK1/2和p-Akt表达下调(F=35.184、15.274,P<0.01)。结论FSTL1与结肠癌细胞生长和凋亡密切相关。
Objective To investigated the functional role of Follistatin-like protein 1(FSTL1)on the growth and apoptosis of colon cancer cells.Methods The expression of FSTL1 was dyregulated in DLD1 cells by transfection of FSTL1 small interfering RNA(siRNA).Cell counting kit-8(CCK-8)and cell clone formation assays were employed to detect the cell viability and proliferation activity in vitro.The cell cycle was analyzed by flow cytometry.Annexin V/PI apoptosis assay was utilized to explore the apoptosis of DLD1 cells.The cell cycle regulating proteins(Cyclin E,E2F-1,p-Rb,p53 and p73),cell apoptosis related factors[Cytochrome C,B cell lymphoma/leukemia-2 associated X protein(bax),B cell lymphoma/leukemia-2(bcl-2),cysteinyl aspartate-specific protease(Caspase)-3 and poly adenosine diphosphate-ribose polymerase(PARP)]as well as cell signaling pathway factors[phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2),phosphorylated c-Jun N-terminal kinase(p-JNK),phosphorylated protein kinase B(p-Akt)and inhibitoryκB(IκB)]were determined by Western blotting.Results The A value in blank control(BC)group and siRNA negative control(NC)group were 1.124±0.157 and 1.243±0.161 respectively,significantly higher than that of FSTL1 siRNA group(0.708±0.114)(F=11.172,P<0.01).The clones number in BC group and siRNA NC group were(154.7±16.2)and(183.8±12.6),significantly higher than that of FSTL1 siRNA group(42.3±6.5)(F=24.694,P<0.01).The apoptosis rate in BC group and siRNA NC group were(1.62±0.24)%and(1.840.27)%,significantly lower than that of FSTL1 siRNA group(12.32±1.96)%(F=29.687,P<0.01).Knockdown of FSTL1 also increased the levels of Cyclin E,E2F-1 and p-Rb as well as up-regulated the expression of p53 and p73.The F value were 18.694,31.481,28.043,34.875 and 39.746,respectively.The P value were<0.01 and P<0.05,respectively.Silencing of FSTL1 induced DLD1 cell apoptosis through the up-regulation of Cytochrome C and bax as well as the proteolysis of PARP and Caspase 3.The F value were 18.694,31.481,28.043,34.875 and 39.7
作者
骆锴冉
刘岸
Luo Kairan;Liu An(Department of General Surgery,Affilicated Hospita of Shaoxing University,Shaoxing 312000,China;Hunan Institute of Science and Technology,YueYang 414000,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第11期2014-2017,共4页
Chinese Journal of Experimental Surgery
基金
2017年度浙江省医药卫生科技计划项目(2017ZH024)。
