| Objectives: NUAK1 was highly expressed in tumors,and promoted their invasion and metastasis.This study used gene chip technology to explore the downstream target genes of NUAK1 based on the high expression level of NUAK1 in gastric cancer to better understand the relationship between NUAK1 and gastric cancer.Methods:(1)The cDNA expression profiles related-gastric cancer was screened and downloaded from GEO database;(2)Western Blot and immunohistochemistry were performed to determine the expression of NUAK1 protein in gastric cancer with different degrees of differentiation;(3)The gastric cancer cells SGC-7901 were interfered by shRNA-NUAK1 and profiled using AffymetrixPrimeView Human Gene Expression Array;(4)DEGs were identified by R language Bioconductor related packages and followed by functional enrichment analyses of GO and KEGG using DAVID,then a protein-protein interaction(PPI)network was constructed by Cytoscape software;(5)qRT-PCR experiment detected fifteen DEGs(CDK2,BMP2,MAP4K4,UBE2 N,JAK2,MDM2,CDKN2 B,JUN,PMAIP1,IL18,MAP2K6,PERP,CCNG1,FAS and CDKN1A)expression in the shRNA-NUAK1(KD)and(Negative control)NC groups;(6)Kaplan-meier and TCGA database validated respectively selected DEGs(UBE2N,CDK2,MAP4K4,BMP2,IL18,PERP,PMAIP1 and MAP2K6)that the relationship of expression with patients survival rate and expression in normal group compared with gastric cancer.Results: 1.(1)The data set results downloaded in the GEO database showed that NUAK1,a member of the AMPK family,is highly expressed in gastric cancer,and gastric cancer patients with high expression of NUAK1 exist a lower survival rate;(2)The results of Western Blot and immunohistochemistry showed that the expression of NUAK1 was higher in low differentiation of gastric cancer tissues for different degrees of differentiation(P<0.05);(3)840 differentially expressed genes including 264 upregulated and 576 down-regulated genes were obtained from gene chip data analysis.The firstly five down-regulated genes were INSL4,MREG,ALPPL2,GPR180 and TRAK2,and the firstly five up-regulated genes were STC2,SERPINE1,CSTA,IGFBP1 and NUPR1;(4)GO analysis showed that the differentially expressed genes of GO-MF mainly enriched in protein Binding,receptor binding and cytokine receptor activity,etc.,the KEGG results show pathway enriched in metabolic pathways,P53 pathways and MAPK pathways;2.(1)The up-regulated genes in the 15 differentially expressed genes verified by qRT-PCR were PMAIP1 and IL18,while down-regulated genes were CDK2,BMP2,MAP4K4,UBE2 N,MAP2K6,PERP,CCNG1,FAS,CDKN1 A,JAK2,MDM2,CDKN2 B and JUN;(2)The Kaplan-Meier results showed that the genes with low expression and low survival rate of gastric cancer patients were BMP2,IL18,MAP2K6,PERP,PMAIP1 and UBE2 N.The genes with high expression and low survival rate of gastric cancer patients were CDK2 and MAP4K4;(3)TCGA database showed that genes(CDK2,IL18,MAP2K6,MAP4K4,PERP,PMAIP1,and UBE2N)were more highly expressed in gastric cancer than normal,while the expression of BMP2 was lower in gastric cancer than normal.Conclusions: The expression of NUAK1 was positively correlated with the malignancy of gastric cancer and negatively correlated with patient survival.NUAK1 may promote gastric cancer progression through its downstream target genes CDK2 and MAP4K4. |
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