| Lung cancer,whose treatment has not been effectively controlled and improved for many years,is the most common cancer with the highest worldwide mortality.And,lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)are two of most common histological type of lung cancer.MicroRNA(miRNA)plays an important role in carcinogenesis through its targeted regulatory function.However,the role and mechanism of miR-452-5p in lung cancer remains unclear.A systematic study of this disorder will therefore be valuable for its diagnosis and treatment of lung cancer.Objective1.Demonstrate expression and clinical significance of miR-452-5p in LUAD.2.Demonstrate expression and clinical significance of miR-452-5p in LUSC.3.Exploring and illuminate potential targeting mechanism of miR-452-5p.Methods1.For the purpose of analyzing the relationships between miR-452-5p expression and diagnosis,prognoses,and clinical pathological characteristics of LUAD,the current study used the quantitative real time polymerase chain reaction(RT-qPCR)to detect the expression levels of miR-452-5p in paraffin imbedded samples from 101 patients with LUAD.Moreover,The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases were utilized to confirm the expression levels of miR-452-5p in LUAD.2.The Cancer Genome Atlas(TCGA)was utilized to demonstrate the expression levels of miR-452-5p in LUSC and its relationship with diagnosis,prognosis,and clinical pathological characteristics of LUSC.Combined with TCGA and GEO databases,the expression levels of miR-452-5p in LUSC were also confirmed by us.3.Fourteen online prediction tools were used to screen the target genes of miR-452-5p,including miRWalk,TargetScan,miRanda,Pictar2,RNAhybrid,miRDB,RNA22-HAS,Targetminer,EMBL-EBI,DIANA-microT,mirbridge,miRMap,miRNAMap and PITA.Combined with DAVID and GSEA software,gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses of the miR-452-5p target genes were performed to explore the molecular mechanisms of lung cancer.STRING database was utilized for the selection of hub genes which would probably play a key role inthe protein-protein interaction(PPI)network.The expression correlation between miR-452-5p and its hub genes were verified via analyzing TCGA sequence data.Moreover,the targeting relationship between miR-452-5p and the hub gene CDKN1 B was further confirmed by Luciferase reporter assay.Results1.The expression of miR-452-5p and its clinical significance in LUAD.The expression of miR-452-5p in LUAD was lower than that in adjacent lung tissues(2.2952±1.4518 vs.5.3319±3.0569,P< 0.001).The AUC was 0.855[95% confidence interval(CI): 0.805 ~ 0.906,P< 0.001],with a sensitivity of85.1%,specificity of 76.2%,and diagnostic threshold value of 2.80.And the expression level of miR-452-5p was negatively correlated with the tumor size(r=-0.243,P= 0.014),lymph node metastasis(r=-0.214,P= 0.032),and TNM stage(r=-0.209,P= 0.036)of LUAD patients.Via the analysis of TCGA data,the results showed the expression of miR-452-5p in LUAD was lower than that in adjacent lung tissues(8.3539±1.40738 vs.8.7608±2.06101,P< 0.001)and the AUC of miR-452-5p expression for LUAD was 0.678(95% CI: 0.587 ~ 0.769,P< 0.001).Moreover,combined with the results from GEO database,we confirmed the low expression of miR-452-5p in LUAD [standard mean deviations(SMD)=-0.393,95% CI:-0.774 ~-0.011,P= 0.044].2.The expression of miR-452-5p and its clinical significance in LUSC.The results analyzed with TCGA data showed the expression of miR-452-5p in LUSC was higher than that in adjacent lung tissues(7.1525±1.39063 vs.6.0885±0.35298,P< 0.001)and the AUC of miR-452-5p expression for LUSC was 0.778(95% CI: 0.734 ~ 0.821,P< 0.001).In addition,the expression level of miR-452-5p was negatively correlated with the age(r=-0.187,P= 0.001)and TNM stage(r=-0.119,P= 0.028)of LUSC patients.Finally,combined with the results from GEO database,we confirmed the high expression of miR-452-5p in LUSC(SMD= 0.372,95% CI: 0.020 ~ 0.724,P=0.038)by applying meta-analysis.3.The potential function and mechanism of miR-452-5pA total of 17,094 candidate target genes of miR-452-5p were screened by using a total of 14 prediction programs.The positive candidates verified by at least seven programs or validated by experiments were further analyzed.Thus,a total of 249 target genes of miR-452-5p were selected and subjected to GO and pathway analyses.The GO analysis showed that the miR-452-5p target genes were mainly enriched in 17 items of biological process(BP)pathway(P< 0.01),and three most significant pathways enriched by target genes of miR-452-5p were as follows: positive regulation of transcription from RNA polymerase II promoter(GO:0045944,P< 0.001),commissural neuron axon guidance(GO:0071679,P<0.001)and transcription,DNA-templated(GO:0006351,P< 0.001);In 7 items of cellular components(CC)pathway(P< 0.01),three most significant pathways enriched by target genes of miR-452-5p were as follows: nucleoplasm(GO:0005654,P< 0.001),nucleus(GO:0005634,P< 0.001)and cytoplasmic vesicle membrane(GO:0030659,P= 0.001);In 14 items of molecular function(MF)pathway(P< 0.01),three most significant pathways enriched by target genes of miR-452-5p were as follows: protein binding(GO:0005515,P< 0.001),chromatin binding(GO:0003682,P< 0.001)and zinc ion binding(GO:0008270,P< 0.001).The KEGG signal pathway analysis showed that the miR-452-5p target genes were mainly enriched in 10 items of signal pathway(P< 0.05),and three most significant pathways enriched by target genes of miR-452-5p were as follows: signaling pathways regulating pluripotency of stem cells(hsa04550,P=0.002),oocyte meiosis(hsa04114,P= 0.003)and cell cycle(hsa04110,P=0.005).Ten hub genes were identified and screened from the PPI network.Among these ten hub genes,five hub genes,including SMAD4,SMAD2,CDKN1 B,YWHAE and YWHAB,were significantly enriched in the cell cycle pathway.Utilizing the RNA sequencing data from TCGA,Spearman’s correlation analyses demonstrated that miR-452-5p was negatively correlated with CDKN1 B in LUSC(r=-0.163,P= 0.003).Additionally,the results of luciferasereporter assay showed that miR-452-5p could regulate the expression of CDKN1 B.ConclusionThis study demonstrated miR-452-5p might be a tissue specific molecule expressed in non-small cell lung cancer(NSCLC).Moreover,miR-452-5p was down-regulated in LUAD tissue,and negatively correlated with the clinicopathological parameters of LUAD patients including tumor size,lymph node metastasis and TNM stage.However,in LUSC tissue,miR-452-5p was up-regulated and negatively correlated with the age and TNM stage of patients with LUSC.MiR-452-5p might play a critical role in LUSC by targeting CDKN1 B involved in cell-cycling.Based on the results of our research,further verifications including in vitro and in vivo experiments are still needed to elucidate the targeting function and mechanism of miR-452-5p in NSCLC.As a result,it may provide a new clue for the development of early diagnosis and molecular targeted therapy of NSCLC. |
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