表皮细胞生长因子样结构域3对SNU-449肝癌细胞增殖、凋亡、转移、侵袭的影响

Effects of epidermal growth factor-like domain-3 on proliferation,apoptosis,migration,and invasion of SNU-449 hepatocellular carcinoma cells

目的观察表皮细胞生长因子样结构域3(EGFL3)基因敲减对SNU-449人肝癌细胞生长增殖、凋亡、迁移、侵袭的影响,探索EGFL3发挥作用的分子机制。方法利用慢病毒介导的小干扰RNA(siRNA)技术构建EGFL3基因敲减的SNU-449细胞系,并利用实时荧光定量聚合酶链反应(RT-qPCR)检测EGFL3基因干扰效率,选出敲减效率最高的一组作为基因敲减组(KD组),同时设阴性对照组(NC组),随后进行噻唑蓝(MTT)实验、细胞克隆形成实验、细胞凋亡实验、Transwell实验以及Transwell小室侵袭实验,以对比两组SNU-449细胞系的生长、增殖、凋亡、侵袭和转移的能力,最后通过RT-qPCR技术检测两组SNU-449细胞系中转化生长因子-β1(TGF-β1)、SMAD基因的相对表达水平。组间样本比较采用独立样本t检验。结果相较于NC组,KD组SNU-449细胞系中EGFL3基因的敲减效率为75.7%(t=13.730,P<0.01),KD组SUN-449细胞的生长速度和细胞克隆数量明显低于NC组[5.29±0.23比6.30±0.26,t=6.606,P<0.01;(1±1)个比(19±2)个,t=14.690,P<0.01],KD组细胞凋亡率明显高于NC组[(11.06±0.29)%比(3.20±0.13)%,t=-43.280,P<0.01],KD组转移及侵袭的细胞数均明显低于NC组[(104±6)个比(178±3)个,t=20.090,P<0.01;(209.00±0.00)个比(306.00±3.51)个,t=48.170,P<0.01]。RT-qPCR检测结果显示,KD组SNU-449细胞系中的TGF-β1、SMAD2、SMAD3和SMAD4基因的相对表达量高于NC组(t=3.759、5.464、5.979、8.956,P<0.05),SMAD7基因的相对表达量则低于NC组(t=0.368,P>0.05)。结论EGFL3基因可促进SNU-449肝癌细胞系的生长、增殖、侵袭、转移并抑制凋亡,其机制可能与抑制TGF-β1/SMAD信号通路有关。

Objective To investigate the influence of epidermal growth factor-like domain-3(EGFL3)on the proliferation,apoptosis,migration,and invasion of SNU-449 human hepatocellular carcinoma cells,while also delving into the underlying molecular mechanisms.MethodsLentivirus-mediated small interfering RNA technology was used to construct EGFL3 knockdown SNU-449 cell lines,and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)technology was used to detect the interference efficiency of the EGFL3 gene,and the group with the highest knockdown efficiency was selected as the knockdown group(KD group),and the negative control group(NC group)was set up,and then the methyl thiazolyl tetrazolium(MTT)assay,cell clone formation assay,apoptosis assay,Transwell assay and Transwell invasion assay were performed to compare the proliferation,apoptosis,invasion and metastasis of the two SNU-449 cell lines,and finally,the relative expression of transforming growth factor-β1(TGF-β1),SMAD gene relative expression levels were detected.Independent sample t-test was used for comparison of samples between groups.ResultsCompared with the NC group,the knockdown efficiency of EGFL3 gene in the KD group was 75.7%(t=13.730,P<0.01),and the growth rate of SNU-449 cells in the KD group was significantly slowed down(5.29±0.23 vs.6.30±0.26,t=6.606,P<0.01;1±1 vs.19±2,t=14.690,P<0.01),apoptosis rate increased significantly[(11.06±0.29)%vs.(3.20±0.13)%,t=-43.280,P<0.01],and the number of metastasized and infiltrated cells decreased significantly[(104±6)cells vs.(178±3)cells,t=20.090,P<0.01;(209.00±0.00)cells vs.(306.00±3.51)cells,t=48.170,P<0.01].The RT-qPCR assay showed that the relative expression of TGF-β1,SMAD2,SMAD3 and SMAD4 genes was significantly up-regulated in the SNU-449 cell line of the KD group as compared with that of the NC group(t=3.759,5.464,5.979,8.956,and P<0.05),while the relative expression of SMAD7 gene was slightly down-regulated(t=0.368,P>0.05).ConclusionEGFL3 gene promotes growth,proliferation,invasion,metastasis and inhibits apoptosis of SNU-449 hepatocellular carcinoma cells by a mechanism that may be related to the inhibition of TGF-β1/SMAD signaling pathway.