转录因子E2F-3靶向FOSB对结直肠癌细胞增殖、迁移和侵袭的影响

Effects of transcription factor E2F-3 targeting FOSB on proliferation,migration and invasion of colorectal cancer cells

目的探究转录因子E2F-3对结直肠癌细胞增殖、迁移和侵袭的影响。方法在体外培养人的结直肠癌细胞系RKO、SW480、DLD1、LOVO和人的结肠正常上皮细胞NCM460,并用反转录实时荧光定量聚合酶链反应(RT-qPCR)检测结肠上皮细胞NCM460和结直肠癌细胞SW480、RKO、DLD1、LOVO中E2F-3的转录水平。分别将E2F-3的siRNA-E2F3和siRNA-NC阴性对照转染于细胞系RKO中,设为实验组(si-E2F3)与阴性对照组(NC)。采用平板克隆实验细胞与细胞计数试剂盒(CCK-8)检测细胞增殖活性。Transwell迁移和侵袭实验与细胞划痕实验用来检测细胞的迁移及侵袭能力。RT-qPCR和蛋白质印迹法(Western blot)分别可以检测E2F-3靶向蛋白FOSB的表达水平。两样本间对比采取t检验,多组间差异采用单因素方差分析。结果miR-210-3p在结直肠癌细胞系中表达均高于人正常结肠上皮细胞,其中以RKO中升高最为明显,相较NC[(2.349±0.095)倍比(1.000±0.013)倍,t=65.82,P<0.01],差异有统计学意义。转染si-E2F3后,敲降组si-E2F3表达水平显著降低[(0.394±0.010)倍比(1.000±0.013)倍,t=66.38,P<0.01],差异有统计学意义。CCK-8实验表明si-E2F3组在不同时间段吸光度值均低于NC组[(0.720±0.110)倍比(1.210±0.015)倍,t=19.65,P<0.01],差异有统计学意义。细胞克隆实验结果显示,si-E2F3组细胞克隆形成能力低于NC组[(171.000±4.000)倍比(317.000±8.000)倍,t=19.75,P<0.01],差异有统计学意义。划痕愈合实验结果表明,si-E2F3组划痕愈合百分比低于NC组[(0.163±0.004)倍比(0.244±0.013)倍,t=9.39,P<0.01],差异有统计学意义。Transwell侵袭实验结果显示,si-E2F3组细胞中穿出的细胞数低于NC组[(53.667±4.667)倍比(161.67±4.333)倍,t=30.89,P<0.01],差异有统计学意义。Transwell迁移实验结果表明:si-E2F3组细胞迁移细胞数低于NC组[(52.333±5.333)倍比(99.333±5.333)倍,t=11.79,P<0.01],差异有统计学意义。通过生物信息学分析筛选出E2F-3靶蛋白FOSB,Western blot结果表明转染了si-E2F3后,结直肠癌细胞中FOSB表达量显著降低。结论E2F-3在人的结直肠癌细胞系中显著高表达,其中以RKO最为显著,并可能通过靶向FOSB促进结直肠癌细胞的增殖、迁移和侵袭。

Objective To explore the effects of transcription factor E2F3 on the proliferation,migration and invasion of colorectal cancer cells.Methods Human colorectal cancer cell lines RKO,SW480,DLD1,LOVO and human normal colon epithelial cells NCM460 were cultured in vitro.The above cell lines were purchased from the cell bank of the Chinese Academy of Sciences,verified by STR,and are now frozen in the Medical Science Research Center of Zhongnan Hospital,Wuhan University.quantitative real‐time polymerase chain reaction,(RT-qPCR)was used to detect E2F3 transcription levels in colon epithelial cells NCM460 and colorectal cancer cells SW480,RKO,DLD1 and LOVO.siRNA-E2F3 and siRNA-NC negative controls of E2F3 were transfected into cell line RKO,respectively,and were set as experimental group(si-E2F3)and negative control group(NC).cell proliferation activity was detected by cell counting kit-8(CCK-8)and plate cloning experimental cells.Transwell migration and invasion assay and cell scratch assay were used to detect the migration and invasion ability of cells.RT-qPCR and Western blot can detect the expression level of the E2F3-targeting protein FOSB,respectively.T test was used for comparison between two samples,and one-way analysis of variance was used for differences between multiple groups.Results The expression of miR-210-3p in colorectal cancer cell lines was higher than that in human normal colon epithelial cells,and the increase was most obvious in RKO[(2.349±0.095)times vs.(1.000±0.013)times,t=65.82,P<0.01],After transfection with si-E2F3,the expression level of si-E2F3 in knockdown group was significantly decreased[(0.394±0.010)times vs.(1.000±0.013)times,t=66.38,P<0.01],and the difference was statistically significant.CCK-8 experiment showed that the absorbance of si-E2F3 group in different time periods was lower than that of NC group[(0.720±0.110)times vs.(1.210±0.015)times,t=19.65,P<0.01],and the difference was statistically significant.The results of cell cloning experiment showed that the cell cloning capacity of si-E2F3 group was lower than that of NC group[(171.000±4.000)times vs.(317.000±8.000)times,t=19.75,P<0.01],and the difference was statistically significant.The results of scratch healing experiment showed that the percentage of scratch healing in si-E2F3 group was lower than that in NC group[(0.163±0.004)times vs.(0.244±0.013)times,t=9.388,P<0.01],and the difference was statistically significant.The results of Transwell invasion experiment showed that the number of perforated cells in si-E2F3 group was lower than that in NC group[(53.667±4.667)times vs.(161.67±4.333)times,t=30.89,P<0.01],and the difference was statistically significant.The results of Transwell migration experiment showed that the number of cells migrated in si-E2F3 group was lower than that in NC group[(52.333±5.333)times vs.(99.333±5.333)times,t=11.79,P<0.01],the difference was statistically significant.The target protein FOSB of E2F3 was screened by bioinformatics analysis,and the results of Western Blot showed that the expression of FOSB in colorectal cancer cells was significantly decreased after transfection with si-E2F3.Conclusion E2F3 is significantly overexpressed in human colorectal cancer cell lines,most notably RKO,and may promote proliferation,migration and invasion of colorectal cancer cells by targeting FOSB.