NF-Y调控KLF4转录对乳腺癌细胞增殖、迁移的影响

Effects of NF-Y regulating KLF4 transcription on proliferation and migration of breast cancer cells

目的探讨核因子Y(NF-Y)调控Kruppel样因子4(KLF4)转录对乳腺癌细胞增殖、迁移的影响。方法从TCGA数据库中下载乳腺癌的RNA-seq数据集,调取乳腺癌组织NF-Y、KLF4 mRNA表达数据,并分析二者的相关性。选择乳腺癌MCF-7细胞,分别采用shNF-Y#1、shNF-Y#2、shCON慢病毒沉默NF-Y表达,检测其NF-Y、KLF4 mRNA和蛋白表达,采用5-溴-2-脱氧尿嘧啶(EdU)插入法和磺酰罗丹明B(SRB)实验检测细胞增殖能力,采用划痕实验检测细胞迁移能力。采用染色质免疫共沉淀(ChIP)实验验证NF-Y能够结合到KLF4启动子上,采用双荧光素酶报告基因实验验证NF-Y能够激活KLF4启动子。结果Pearson相关分析显示,乳腺癌组织NF-Y mRNA表达与KLF4 mRNA表达呈正相关关系(P<0.05)。MCF-7-shNF-Y#1、MCF-7-shNF-Y#2细胞NF-Y、KLF4 mRNA和蛋白表达均低于MCF-7-shCON细胞(P均<0.05),而MCF-7-shNF-Y#1细胞与MCF-7-shNF-Y#2细胞比较P均>0.05。EdU插入法、SRB实验和划痕实验结果显示,MCF-7-shNF-Y#1、MCF-7-shNF-Y#2细胞增殖和迁移能力均低于MCF-7-shCON细胞(P均<0.05),而MCF-7-shNF-Y#1细胞与MCF-7-shNF-Y#2细胞比较P均>0.05。ChIP实验结果显示,Input组、Histone3组、Sp1组、NF-Y组均检测到相应的PCR产物,而IgG组未检测到相应的PCR产物。双荧光素酶报告基因实验结果显示,荧光素酶活性PGL3-Basic+NF-Y组高于PGL3-Basic+vector组,PGL3-KLF4p+NF-Y组高于PGL3-KLF4p+vector组,PGL3-KLF4p+NF-Y组高于PGL3-Basic+NF-Y组(P均<0.05);PGL3-Basic+vector组与PGL3-KLF4p+vector组荧光素酶活性比较P>0.05。结论NF-Y能够通过调控KLF4转录促进乳腺癌细胞的增殖和迁移。

Objective To investigate the effects of nuclear factor Y(NF-Y)regulating Kruppel-like factor 4(KLF4)transcription on proliferation and migration of breast cancer cells.Methods The RNA-seq data set of breast cancer was downloaded from TCGA database,the expression data of NF-Y and KLF4 in breast cancer tissues were collected,and the correlation between them was analyzed.The expression of NF-Y was silenced by shNF-Y#1,shNF-Y#2,and shCON lentiviruses in breast cancer MCF-7 cells,and the expression levels of NF-Y and KLF4 were detected.The cell proliferation was measured by 5-bromo-2 deoxyuracil(EdU)insertion and sulfonyl rhodamine B(SRB)assay.Cell migration was measured by cell scratch test.Chromatin immunoprecipitation(ChIP)assay was used to verify that NF-Y could bind to KLF4 promoter.Double luciferase reporter gene assay was used to verify that NF-Y could activate KLF4 promoter activity.Results Pearson correlation analysis showed that there was a positive correlation between the expression of NF-Y and KLF4 in breast cancer(r=0.444,P<0.05).The expression of NF-Y and KLF4 in the MCF-7-shNF-Y#1 and MCF-7-shNF-Y#2 cells was lower than that in the MCF-7-shCON cells(P<0.05),with statistically significant difference between the MCF-7-shNF-Y#1 cells and MCF-7-shNF-Y#2 cells,P>0.05.EdU insertion,SRB and scratch test showed that the proliferation and migration abilities of MCF-7-shNF-Y#1 and MCF-7-shNF-Y#2 cells were lower than those of MCF-7-shCON cells,with no statistically significant difference between MCF-7-shNF-Y#1 cells and MCF-7-shNF-Y#2 cells(P>0.05).ChIP results showed that the corresponding PCR products were detected in Input,Histone 3,Sp1 and NF-Y groups,but not in the IgG group.The results of double luciferase reporter gene experiment showed that the luciferase activity of PGL3-Basic+NF-Y group was higher than that of PGL3-Basic+Vector group,PGL3-KLF4p+NF-Y group was higher than PGL3-KLF4p+Vector group,and PGL3-KLF4p+NF-Y group was higher than PGL3-Basic+NF-Y group(P<0.05);no significant difference was found between the PGL3-Basic+vector group and PGL3-KLF4p+vector group(P>0.05).Conclusion NF-Y promotes proliferation and migration of breast cancer cells by regulating KLF4 transcription.