| Objective:Tumor microenvironment(TME)plays important roles in different cancers.In recent years,the hypothesis of ceRNA network has provided new and broader biological functions for coding protein genes and non coding genes.Our study aimed to identify CDK2-related prognostic molecules and construct relevant Nomogram,immune model,ceRNA in LUAD.Methods:"GEO2R","limma" R packages were used to identify all differentially expressed mRNAs from Gene Expression Omnibus(GEO)GSE68465 and The Cancer Genome Atlas(TCGA)databases.The function analyses of 250 overlapping mRNAs was showed by DAVID and Metascape databases.UALCAN,Prognoscan,Oncomine,TIMER databases were used to verify the expression level and immune infiltration of CDK2 in LUAD and pan-cancer.TISIDB database was used to explore immune activators and immunosuppressants related to CDK2.cBioProtal database screened the above immune modulator related genes."Survival","survminer","rms" R packages were used to construct a nomogram model of age,gender,stage,T,M,N.Univariate and multivariate Cox regression were used to establish prognosis-related immune forecast model in LUAD.CeRNA network was constructed by various online databases.Results:A total of 250 differentially expressed genes(DEGs)were identified to participate in many cancer-related pathways,such as activation of immune response,cell adhesion,migration,PI3K-AKT signaling pathway.The target molecule CDK2 had prognostic value for the survival of LUAD patients(P=5.8e-15).Through Oncomine,TIMER,UALCAN,PrognoScan databases,the CDK2 expression in LUAD was higher than normal lung tissues.Pan-cancer analysis revealed that the expression,stage and survival of CDK2 in 33 cancers were statistically significant.Through TISIDB database,we selected 13 immunodepressants,21 immunostimulants related to CDK2 and explored 48 genes related to these 34 immunomodulators in cBioProtal database(P<0.05).Gene Set Enrichment Analysis(GSEA)and Metascape indicated that 49 mRNAs were involved in PUJANA ATM PCC NETWORK(ES=0.557,P=0,FDR=0),SIGNAL TRANSDUCTION(ES=-0.459,P=0,FDR=0),immune system process,cell proliferation.Forest map and Nomogram model showed the prognosis of patients with LUAD(Log-Rank=1.399e-08,Concordance Index=0.7).Cox regression showed that four mRNAs(SIT1,SNAI3,ASB2,CDK2)were used to construct the forecast model to predict the prognosis of patients(P<0.05).LUAD patients were divided into two different risk groups(low and high)had an obvious statistical significance(P=6.223e-04).By "survival ROC" R package,the total risk score of this prognostic model was AUC=0.729(SIT1=0.484,SNAI3=0.485,ASB2=0.267,CDK2=0.579).CytoHubba selected ceRNA mechanism medicated by potential biomarkers 6 lncRNAs-7miRNAs-CDK2.Conclusions:1.CDK2 was highly expressed in LUAD tissues and its higher expression was positively correlated with CD4 T cells and macrophage infiltration.Patients with high expression of CDK2 had a poor prognosis.2.CDK2 participated in the PI3K-AKT signaling pathway and affected the proliferation.Objective:We predicted the involvement of CDK2 in the classical cancer pathway of LUAD by bioinformatics.The correlation between expression and prognosis was further verified in vivo,in vitro experiments and clinical specimens.Methods:The expression of CDK2 in normal bronchial epithelial cells(BEAS-2B,16HBE)and LUAD cells(H1299,A549,H1975,PC9)was detected by qRT-PCR and Western Blot.CDK2 expression was knocked down in H1299 cells and H1975 cells,and CDK2 plasmid was overexpressed in A549 cells.The knockdown and over-expression efficiency were detected by qRT-PCR and Western Blot.Cell proliferation toxicity test(CCK8),scratch test,invasion test,migration test and cell proliferation imaging(EDU)were used to explore cell functions.The position of CDK2 expression in cells was detected by immunofluorescence.The overexpression of CDK2 lentivirus was constructed,and the over-expression efficiency in A549 cells was verified by qRT-PCR and Western Blot.The stable transformed A549 strain with successful overexpression was used for subcutaneous tumorigenesis experiment in nude mice.The tumor volume was measured every 3 days.When there was significant difference in the long and short diameter of the tumor between the experimental group and the control group,the tumor dissected after killing the nude mice was embedded,sliced and immunohistochemical,The expression position and relative expression of Ki-67 and CDK2 were observed.Fresh adenocarcinoma tissues and adjacent normal lung tissues from LUAD patients were collected.The CDK2 expression in cancer tissues and adjacent tissues of patients was detected by qRT-PCR and Western Blot.The age,sex,tumor size and TNM stage of 15 LUAD patients were analyzed.Results:CDK2 expression in LUAD cells(H1299,A549,H1975)was significantly higher than normal bronchial epithelial cells(BEAS-2B,16HBE).The CDK2 expression was the highest in H1299 cells(P=0.0006),followed by H1975 cells(P=0.0030),and lower in A549 cells(P=0.002).The knockdown efficiency of CDK2 in H1299 cells was interference sequence 1:P=0.0026;Interference sequence 2:P=0.0107.The knockdown efficiency of CDK2 in H1975 cells was interference sequence 1:P=0.0003;Interference sequence 2:P=0.0011.CCK8 experiment showed that after knockdown of CDK2 in H1299 cells,the OD value and proliferation ability decreased significantly after 48 hours and 72 hours.After knockdown of CDK2 in H1975 cells,the proliferation ability decreased significantly after 72 hours.Scratch test showed that the migration ability of H1975 cells decreased significantly in 12 hours and 24 hours(P<0.001).Both migration and invasion experiments showed that the ability of H1299 and H1975 cells decreased significantly(P<0.001),EDU experiment showed that the number of proliferating cells decreased significantly after knockdown of CDK2 in H1299 cells(P<0.01).The efficiency of over-expression of CDK2 plasmid in A549 cells was detected by qRT-PCR and Western Blot(P<0.01).CCK8 experiment showed that after over-expression of CDK2 in A549 cells,the proliferation ability of A549 cells increased significantly at 24 hours,48 hours and 72 hours.Western Blot showed that the expression of P-I3K,P-AKT decreased significantly after knockdown of CDK2 in H1975 cells.After over-expression of CDK2 in A549 cells,the expression of P-I3K and P-AKT increased significantly,and there was no significant change in total 13K and total AKT.The expression of N-Cad and vimentin decreased and the expression of E-Cad increased after knockdown of CDK2 in H1299 and H1975 cells.After over-expression of CDK2 in A549 cells,the expression of N-Cad increased and the expression of E-Cad and catenin decreased.The subcutaneous tumorigenesis experiment in nude mice showed that the tumor volume after over-expression of CDK2 was significantly higher than that in the control group.Immunohistochemistry showed that expressions of proliferation related index Ki67 and CDK2 in over-expression CDK2 group were significantly higher than those in control group(P<0.05).qRT-PCR and Western Blot showed that the CDK2 expression in LUAD patients was significantly higher than that in adjacent normal lung tissues.Among the 15 LUAD patients,73%were women and 27%were men.The maximum tumor volume was 3×2.2×2.0cm and TNM stage was mostly stage Ⅱ.Conclusion:1.CDK2 is highly expressed in LUAD and promotes proliferation,invasion and metastasis.2.CDK2 promotes the proliferation of LUAD through PI3K-AKT signaling pathway,affects the process of EMT,mediates the invasion and metastasis. |
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