沉默神经菌毛素-1对宫颈鳞状细胞癌SiHa细胞生长、自噬和移植瘤裸鼠存活的影响

Effects of silencing NRP-1 on the growth and autophagy of cervical squamous cell carcinoma SiHa cells and the survival of tumorbearing nude mice

目的:探究沉默神经菌毛素-1(NRP-1)对宫颈鳞状细胞癌SiHa细胞生长、自噬和移植瘤裸鼠存活的影响。方法:构建NRP-1慢病毒载体转染至SiHa细胞,将细胞分为4组:Control组、shRNA-NC组、NRP-1-shRNA1组和NRP-1-shRNA2组。RTqPCR法检测NRP-1 mRNA水平,克隆形成实验检测细胞增殖,流式细胞仪检测细胞凋亡,Westernblotting法检测NRP-1、Bax、Bcl-2、Caspase-3、c-Myc、Beclin1、ATG7、微管相关蛋白1轻链3(LC3)I及LC3II蛋白表达水平。将转染NRP-1-shRNA1的SiHa细胞皮下注射裸鼠,将裸鼠随机分为Control组和NRP-1-shRNA1组,检测肿瘤重量、裸鼠存活率、凋亡和自噬蛋白表达。结果:与Control组和shRNA-NC组比较,NRP-1-shRNA1组、NRP-1-shRNA2组NRP-1 mRNA和蛋白表达水平显著降低,克隆形成率显著降低,细胞凋亡率显著升高,Bax/Bcl-2、cleaved Caspase-3/Caspase-3比值显著升高,c-Myc蛋白水平显著降低,ATG7蛋白水平和LC3II/LC3I比值显著升高(均P<0.05)。裸鼠实验结果表明,NRP-1-shRNA1组肿瘤重量显著降低,裸鼠存活率、细胞凋亡率、Caspase-3阳性表达率和LC3Ⅱ/LC3Ⅰ比值显著升高(均P<0.05)。结论:沉默NRP-1能抑制宫颈鳞状细胞癌Si-Ha细胞增殖,促进细胞凋亡和自噬,并促进移植瘤裸鼠存活。

Objective:To investigate the effects of silencing neuropilin-1(NRP-1)on the growth and autophagy of cervical squamous cell carcinoma SiHa cells and the survival of tumorbearing nude mice.Methods:NRP-1 lentiviral vector was constructed and transfected into SiHa cells.The cells were randomly divided into Control group,shRNA-NC group,NRP-1-shRNA1 group and NRP-1-shRNA2 group.RT-qPCR was used to detect the level of NRP-1 mRNA.Cell proliferation was detected by clone formation assay.Cell apoptosis was detected by flow cytometry.The protein expression levels of NRP-1,Bax,Bcl-2,Caspase-3,c-Myc,Beclin1,ATG7,LC3I and LC3II were detected by Western blotting.Tumor-bearing nude mice(injected subcutaneously with SiHa cells)were randomly divided into Control group and NRP-1-shRNA1 group.The tumor weight and survival rate of nude mice were detected.The apoptosis and autophagy in vivo were determined as well.Results:The NRP-1 mRNA and protein levels,c-Myc protein level and clone formation rate in NRP-1-shRNA1 and NRP-1-shRNA2 groups were significantly lower than those in Control group and shRNA-NC group,while the ATG7 protein level,cell apoptosis rate,Bax/Bcl-2 ratio,cleaved Caspase-3/Caspase-3 ratio,and LC3II/LC3I ratio were notably higher(P<0.05).The in vivo experimental results showed a decrease in tumor weight and increases in the survival rate of nude mice,TUNEL-positive cells,Caspase-3 expression level,and LC3II/LC3I ratioin NRP-1-shRNA1 group(P<0.05).Conclusion:Silencing NRP-1 can inhibit the proliferation of SiHa cells,promote apoptosis and autophagy,and promote the survival of the nude mice bearing the SiHa cervical squamous cell carcinoma xenograft.