ZNF750对子宫内膜癌细胞生物学行为的影响及机制研究

Effect of ZNF750 on the biological behavior of endometriaI lcancer cells and its mechanism

目的探讨锌指蛋白750(ZNF750)在子宫内膜癌细胞的增殖、侵袭和迁移中的作用并筛选其调控的潜在靶基因方法通过实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质印迹法检测子宫内膜癌细胞系Ishikawa细胞转染ZNF750过表达质粒(OE-ZNF750组)和ZNF750干扰片段(si-ZNF750组)后ZNF750的mRNA和蛋白质表达水平;细胞计数试剂盒8(CCK8)检测细胞增殖能力,Transwell实验检测细胞的侵袭和迁移能力;基因表达微阵列分析结合生物信息学方法筛选ZNF750调控的候选靶基因。设置对照组(NC组),组间比较采用t检验和非参数检验。结果上调ZNF750基因第72h,CCK8实验结果显示,与NC组(5.79±0.20)相比,OE-ZNF750组(4.70±0.10)Ishikawa细胞的增殖能力降低,t=10.571,P<0.001;Transwell实验结果显示,NC组细胞侵袭数为(156.44±7.84)个,迁移数为(125.55±19.69)个,OE-ZNF750组细胞侵袭数为(103.44±19.21)个,迁移数为(49.33±14.15)个,差异均有统计学意义,t值分别为7.660和9.428.均P<0.001。下调ZNF750基因第72h.CCK8实验结果显示,与NC组(5.21±0.07)相比,si-ZNF750组(5.59±0.12)Ishikawa细胞的增殖能力升高,t=-5.876,P<0.001;Transwell实验结果显示,NC组细胞侵袭数为(159.11±32.91)个,迁移数为(84.88±11.47)个,si-ZNF750组细胞侵袭数为(314.77±24.06)个,迁移数为(181.11±18.01)个,差异均有统计学意义,t值分别为-11.454和-13.518.均P<0.001。通过基因表达微阵列分析筛选ZNF750的下游靶基因,结果显示,上调ZNF750后,有414个差异表达基因,下调ZNF750后,有50个差异表达基因,结合生物信息学筛选出与癌症相关的基因有RAC2、KLF4、FN1、IGF2和MAGEA2。qRT-PCR结果显示,上/下调ZNF75O后,与NC组相比,KLF4、RAC2、MAGEA2和IGF2的mRNA表达水平显著升高/降低,FN1的mRNA表达水平显著降低/升高,差异均有统计学意义,均P<0.05。结论在子宫内膜癌细胞中,ZNF750作为抑癌基因发挥作用,抑制子宫内膜癌细胞增殖、侵袭和迁移,RAC2、KLF4、FN1、IGF2和MAGEA2为ZNF750的潜在候选靶基因。

Objective To study the role of zinc finger protein 750(ZNF750)in the proliferation,invasion and migration of endometrial cancer cells and to screen out its potential target genes.Methods Real time fluorescence quantitative polymerase chain reaction(qRT-PCR)and western blotting were used to detect the mRNA and protein expression levels of ZNF750 in endometrial cancer cell line Ishikawa after transfection of ZNF750 overexpression plasmid(OE-ZNF750 group)and ZNF750 interference fragment(si-ZNF750 group).Cell counting kit 8(CCK8)was used to detect cell proliferation ability,while Transwell assay was used to detect cell invasion and migration ability.Gene expression microarray analysis combined with bioinformatics methods to screen candidate target genes regulated by ZNF75o.Set up a control group(NC group),and compare between groups by using t-tests and non parametric tests.Results At 72 hours of upregulation of the ZNF750 gene,the CCK8 experiment showed that compared with the NC group(5.79±0.20),the proliferation ability of Ishikawa cells in the OE-ZNF750 group(4.70±0.10)decreased.t=10.571,P<0.001.Transwell experiment showed that the number of cell invasions and migrations in the NC group was(156.44±7.84)and(125.55±19.69),while the number of cell invasions and migrations in the OE-ZNF750 group was(103.44±19.21)and(49.33±14.15),with statistically significant differences.The t-values were 7.660 and 9.428,respectively,both P<0.001.After downregulating the ZNF750 gene for 72 hours,the CCK8 experiment results showed that compared with the NC group(5.21±0.07),the proliferation ability of Ishikawa cells in the si-ZNF750 group(5.59±0.12)was increased,t=-5.876,P<0.001.Transwell experiment results showed that the invasion number of cells in the NC group was 159.11±32.91,migration number was 84.88±11.47,and the invasion number of cells in the si-ZNF750 group was 314.77±24.06,migration number was 181.11±18.01,and the differences were statistically significant,with t-values of-11.454 and-13.518,respectively,both P<0.001.Through gene expression microarray analysis,downstream target genes of ZNF750 were screened.The results showed that after upregulating ZNF750,there were 414 differentially expressed genes,and after downregulating ZNF750,there were 50 differentially expressed genes.Combined with bioinformatics,cancer related genes were screened,including RAC2,KLF4,FN1,IGF2,and MAGEA2.The qRT-PCR results showed that after up/down regulation of ZNF750,compared with the NC group,the mRNA expression levels of KLF4,RAC2,MAGEA2 and IGF2 significantly increased/decreased,while the mRNA expression levels of FN1 significantly decreased/increased,with statistical significance(all P<0.05).Conclusion In endometrial cancer cells,ZNF750 acts as a tumor suppressor gene,inhibiting the proliferation,invasion and migration of endometrial cancer cells.RAC2,KLF4,FNl,IGF2 and MAGEA2 are potential candidate target genes for ZNF750.