Screening and validation of the prostate cancer promoting gene NUDT11 based on GEO database
吴宇 1王升通 2徐卫强1
扫码查看
点击上方二维码区域，可以放大扫码查看
作者信息
1. 蚌埠医科大学第二附属医院
2. 蚌埠医科大学,安徽 蚌埠 233000
折叠
摘要
目的:探讨影响前列腺癌进展的差异表达基因,为其诊治提供新思路.方法:⑴采用生物信息学和SVM-RFE 机器学习方法筛选出影响前列腺癌进展的差异表达基因(Different Express Genes,DEGs),使用"clusterProfiler"R包对差异表达基因进行基因本体(Gene Ontology,GO)富集分析和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,并筛选关键基因.⑵RT-PCR验证前列腺癌细胞PC3和LNCaP NUDT11 mRNA表达.⑶将前列腺癌细胞LNCaP、PC3分为si-NC组和si-NUDT11组,采用脂质体法进行瞬时转染,转染成功后进行细胞克隆实验、EdU实验和细胞凋亡实验观察NUDT11对细胞增殖及凋亡的影响.⑷构建稳定低表达NUDT11细胞株进行裸鼠皮下成瘤实验,观察NUDT11对前列腺癌细胞成瘤能力的影响.结果:⑴在GEO数据集GSE46602中,共筛选187个DEGs.与正常前列腺组织相比,前列腺癌组织中76个基因表达上调,111个基因表达下调.对差异表达基因进行GO富集分析发现:生物学过程(Biological Process,BP),DEGs主要富集在上皮细胞增殖、上皮细胞增殖的调节、跨膜受体蛋白丝氨酸/苏氨酸、细胞外基质组织、细胞外结构组织、外部封装结构组织、上皮细胞对生长因子刺激反应的调节;细胞学组分(Cytological Components,CC),DEGs主要富集在含胶原的细胞外基质、基底膜、突触囊泡、外囊泡;分子生物学功能(Molecular Biological Functions,MF),DEGs在硫化物结合、肝素结合、细胞外基质结构成分、寡肽结合中富集.对差异表达基因的KEGG富集分析发现DEGs在PI3K-Akt信号转导通路、人乳头瘤病毒感染、癌症中的转录错调、动脉粥样硬化和流体剪切应力、前列腺癌富集.使用SVM-RFE机器学习方法和随机森林(Random forest,RF)特征选择筛选基因,得到共同关键基因为NUDT11和GGTLC1.⑵与前列腺上皮细胞RWPE-1相比,前列腺癌细胞PC3和LNCaP中的NUDT11 mRNA表达量增加.⑶平板克隆实验结果显示与si-NC组相比,si-NUDT11组细胞克隆形成数量均显著下降(P＜0.05);EdU实验结果显示:与si-NC组相比,si-NUDT11组细胞EdU阳性细胞数量显著下降(P＜0.05);与si-NC组相比,si-NUDT11组细胞凋亡率均显著增加(P＜0.05).⑷与sh-NC组相比,sh-NUDT11组细胞成瘤能力显著下降,瘤体重量变小(P＜0.05).结论:生物信息学和SVM-RFE机器学习方法筛选出与前列腺癌进展相关的基因NUDT11和GGTLC1,NUDT11在前列腺癌细胞系中高表达,干扰NUDT11表达后抑制前列腺癌细胞增殖、促进前列腺癌细胞凋亡,提示NUDT11可调控前列腺癌进展.
Abstract
Objective:To explore the differentially expressed genes that affect the progression of prostate cancer,and to provide new ideas for its diagnosis and treatment.Methods:⑴ Bioinformatics and SVM-RFE machine learning were used to screen out differentially expressed genes affecting the progression of prostate cancer.⑵ RT-PCR verified the expression of PC3 and LNCaP in prostate cancer cells.⑶ After transfection with si-NUDT11,the effect of NUDT11 on cell proliferation and apoptosis was observed by cell function experiment.⑷ A stable and low expression NUDT11 cell line was constructed for subcutaneous tumor formation experiment in nude mice to observe the effect of NUDT11 on tumor formation ability of prostate cancer cells in vivo.Results:⑴ In GEO dataset GSE46602,differentially expressed genes(DEG,187 DEGs)were screened out between prostate cancer and corresponding normal tissues.Compared to normal prostate tissue,in prostate cancer tissue 76 gene expression were up-regulated,111 down-regulated.GO enrichment analysis for differentially expressed genes:Biological Process(BP),DEGs was mainly enriched in epithelial cell proliferation,epithelial cell proliferation,transmembrane receptor protein serine/threonine,the extracellular matrix,tissue extracellular structure,external package structure tissue,regulation of the response of epithelial cells to growth factors stimulation,cytological components,DEGs was mainly enriched in collagen extracellular matrix,basement membrane,synaptic vesicle,external vesicle,molecular biological functions;DEGs enriched in sulfide binding,heparin binding,extracellular matrix structure,oligopeptide binding.KEGG enrichment analysis for differentially expressed genes:DEGs was enriched in PI3K-Akt signal transduction pathway,a human papillomavirus infection,transcriptional dysregulation in cancer,shear stress in arteriosclerosis and fluids,and prostate carcinoma.Further bioinformatics studies based on the data from gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)showed that these differentially expressed genes were closely related to the progression of prostate cancer.SVM-RFE machine learning method and Random forest(RF)were used to select the screened genes,and they were drawn into Wayne diagram,and the intersection genes NUDT11 and GGTLC1 were obtained.⑵ NUDT11 had a significantly high expression in the prostate cancer cell line PC3 and LNCaP.⑶ The results of plate cloning experiment of the two types of cells after interfering NUDT11 showed a significant decrease in the number of cell clones compared with the control group(P＜0.05);The EdU test results showed that the number of EdU positive cells was significantly decreased compared with the control group(P＜0.05),and the apoptosis rate was significantly higher than that of the control group(P＜0.05).⑷ After interfering NUDT11 expression in PC3 cells,the tumorigenic ability of nude mice decreased and the tumor volume decreased(P＜0.05).Conclusion:Bioinformatics and SVM-RFE machine learning methods screen out NUDT11 and GGTLC1 genes,which are related to the progression of prostate cancer,and verify that NUDT11 is highly expressed in prostate cancer cell lines,which can inhibit cell proliferation and promote apoptosis after interfering with NUDT11 expression,suggesting that NUDT11 can regulate the progression of prostate cancer.
维普
万方数据