E74样因子5过表达抑制结肠癌细胞生物学行为的体内外实验研究

In vivo and in vitro Experiment of E74-Like Factor 5 Overexpression Inhibiting the Biological Behavior of Colon Cancer Cells

目的探讨E74样因子5(ELF5)过表达对结直肠癌细胞的生长和侵袭能力以及裸鼠成瘤的影响。方法人结直肠癌SW480和HT-29细胞分为5组:LV-GFP组,转染空载体LV-GFP慢病毒;LV-ELF5组,转染重组LV-ELF5慢病毒;shRNA-NC组,转染空载体shRNA-NC慢病毒;shRNA-ELF5组,转染重组shRNA-ELF5慢病毒;对照组,未转染任何载体。转染72 h后,取含有慢病毒的细胞上清液,实时荧光定量PCR(RT-qPCR)检测各组细胞中ELF5的mRNA表达水平;Western blot检测ELF5、凋亡相关的cleaved Caspase-3/Caspase-3和cleaved Caspase-9/Caspase-9、侵袭相关的E-cadherin和Ncadherin的蛋白表达水平;CCK-8检测细胞活力;克隆形成实验检测克隆形成率;流式细胞术检测细胞凋亡;Transwell实验检测细胞侵袭;TUNEL实验检测组织中细胞凋亡;免疫组化检测组织中E-cadherin、N-cadherin的表达。将20只BALB/c裸鼠分为4组,每组5只:LV-GFP组、shRNA-NC组、LV-ELF5组、shRNA-ELF5组,于裸鼠皮下注射含重组慢病毒的SW480细胞上清液,构建裸鼠成瘤模型,监测移植瘤体积变化。第30天取裸鼠移植瘤组织,测量肿瘤质量,Western blot检测移植瘤ELF5蛋白的表达,TUNEL染色检测移植瘤凋亡,免疫组化检测移植瘤中N-cadherin阳性表达。结果与对照组细胞比较,两个细胞系LV-GFP组和shRNA-NC组细胞的各指标差异无统计学意义。Western blot结果与RT-qPCR结果表明,两个细胞系LV-ELF5组的ELF5 mRNA和蛋白水平均上调(P<0.05,与LV-GFP组比较),shRNA-ELF5组的ELF5 mRNA和蛋白水平均下调(P<0.05,与shRNA-NC组比较),ELF5过表达体系和干扰体系构建成功。与LV-GFP组比较,LV-ELF5组降低了细胞活力和克隆形成率(P<0.05),促进SW480细胞凋亡,并上调cleaved Caspase-3/Caspase-3和cleaved Caspase-9/Caspase-9蛋白表达(P<0.05),抑制细胞侵袭,并上调E-cadherin蛋白表达,下调N-cadherin蛋白表达(P<0.05);ELF5干扰后,细胞的上述表现呈相反趋势(P<0.05,shRNA-ELF5组与shRNA-NC组比较)。体内实验结果表明,ELF5过表达缩小裸鼠肿瘤体积和降低肿瘤质量(P<0.05),促进组织中细胞凋亡(P<0.05),ELF5蛋白表达增加,抑制N-cadherin蛋白表达(P<0.05)。当EFL5表达抑制,上述实验结果呈相反趋势。结论体内外实验表明ELF5过表达可促进结直肠癌细胞凋亡,抑制结直肠癌细胞的生长和侵袭。

Objective To investigate the effect of E74-like factor 5(ELF5)overexpression on the growth and invasion ability of colorectal cancer cells and its effect on tumor formation in nude mice.Methods Human colorectal cancer SW480 and HT-29 cells were divided into 5 groups:the lentivirus(LV)-GFP group transfected with empty vector LV-GFP,the LV-ELF5 group transfected with recombinant LV-ELF5,the shRNA-NC group transfected with empty vector shRNA-NC,the shRNA-ELF5 group transfected with recombinant shRNA-ELF5,and the control group,not transfected with any vector.Seventy-two h after transfection,the cell supernatant containing lentivirus was collected.The mRNA expression level of ELF5 in each group was examined by real-time fluorescent quantitative PCR(RT-qPCR).The protein expression levels of ELF5,apoptosis-related cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9,and invasion-related E-cadherin and N-cadherin were checked with Western blot.CCK-8 was used to check cell viability.Colony formation experiment was done to evaluate colony formation rate.Flow cytometry was used to assess cell apoptosis.Transwell migration assay was used to examine cell invasion.TUNEL assay was used to examine the apoptosis of tissues cells.Immunohistochemistry test was done to determine the expression of E-cadherin and N-cadherin in tissues.20 BALB/c nude mice were put into 4 groups(5 in each group):LV-GFP group,shRNA-NC group,LV-ELF5 group,and shRNA-ELF5 group.Recombinant lentiviral SW480 cell supernatants were subcutaneously injected into nude mice to construct nude mice tumorigenesis models and the volume changes of transplanted tumors were monitored.On the 30 th day,transplanted tumor tissues from the nude mice were extracted and the tumor mass was measured.Western blot was done to measure the expression of ELF4 protein in the transplanted tumors.TUNEL staining was used to check cell apoptosis in the tissues,and the positive expression of N-cadherin in the transplanted tumor was measured by immunohistochemical tests.Results Compared with the control group,there was no statistically significant difference in the indicators of the two cell lines in the LV-GFP group and shRNA-NC group.The results of Western blot and RT-qPCR showed that the ELF5 protein and mRNA of the LV-ELF5 group of the two cell lines were up-regulated(P<0.05,compared with those of the LV-GFP group),and the ELF5 protein and mRNA of the shRNA-ELF5 group were downregulated(P<0.05).The ELF5 overexpression system and interference system were successfully constructed.Compared with the LV-GFP group,data from the LV-ELF5 group showed that cell viability and colony formation rate(P<0.05)were reduced,SW480 and HT-29 cell apoptosis was promoted,cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 protein expression was up-regulated(P<0.05),cell invasion was inhibited,and the expression of E-cadherin protein was up-regulated while the expression of N-cadherin protein was down-regulated(P<0.05).After ELF5 interference,the above-mentioned expression of cells demonstrated an opposite trend(P<0.05,comparing shRNA-ELF5 group with shRNA-NC group).In vivo experimental results indicated that ELF5 overexpression reduced tumor volume and tumor mass(P<0.05),promoted cell apoptosis in tissues(P<0.05),and inhibited N-cadherin protein expression(P<0.05).When ELF5 expression was inhibited,the above mentioned experimental results showed the opposite trend.Conclusion In vivo and in vitro experiments showed that ELF5 overexpression could promote the apoptosis of colorectal cancer cells and inhibit the growth and invasion of colorectal cancer cells.