Differential Expression And Clinical Significance Of Serum MiR-1228-3p And MiR-181a-5p In Non-small Cell Lung Cancer

Objectives:In order to obtain the differential miRNAs of non-small cell lung cancer,we obtained serum/plasma miRNAs chips through the literature of the databases.By analyzing the biological analysis of the differential miRNAs,the predicted target genes and their GO and Pathways analyses were obtained.By collecting serum and clinical data of non-small cell lung cancer patients and healthy people,the difference expression of candidate miRNAs was verified and the relationship between miRNAs and clinical indicators was analyzed.Methods:1.Literature were searched via Pubmed,GEO and Array Express databases.The miRNA chips in the literature was collected.MiRNAs were classified according to up-regulation and down-regulation.The RRA(FDR<0.05)was applied by R language to obtain the up-regulated and down-regulated miRNAs.2.Target genes were predicted by screening the differential miRNAs in TargetScan,PicTar,miRDB,miRanda and Tarbase databases.3.To conduct enrichment analyses,the GeneCodis web tool was used for GO and Pathways analyses.4.Blood samples of non-small cell lung cancer patients and healthy normal control people were collected,and the serum miRNAs were extracted,cDNA was obtained by reverse transcription,qRT-PCR was performed,and the relative expression of each miRNAs was finally obtained.5.The TNM stage of non-small cell lung cancer group was analyzed by Spearman correlation of respiratory tumor markers.6.The different expression of candidate miRNAs in non-small cell lung cancer group and normal control group was analyzed,and the relationship between differential miRNAs and pathological type,degree of differentiation,tumor size,lymph node metastasis and TNM stages were analyzed.7.ROC analysis was performed on the differential miRNAs.8.The difference miRNAs were used for K-M survival analysis.9.Cox regression analysis of the differential miRNAs was carried out.Results:1.According to the inclusion criteria,12 independent full-text studies were retrieved from public databases(Pubmed,GEO,and ArrayExpress).2.Five up-regulated miRNAs and twentyseven down-regulated miRNAs were screened by RRA.Based on the size of the FDR value,4 miRNAs were selected from 27 down-regulated miRNAs.Five up-regulated miRNAs(miR-1228-3p,miR-1228-5p,miR-133a-3p,miR-1273f,miR-545-3p)and four down-regulated miRNAs(miR-181a-5p,miR-26b-5p,miR-351-5p,miR-130a-3p)were used for subsequent target genes prediction,GO analyses,Pathways analyses and quantitative real-time PCR.3.Nine miRNAs were predicted for target gene.8110 target genes were obtained where 108 of the target genes were repeated,resulting in 8002 target genes.4.8002 predicted target genes were analyzed GO pathways analyses,Panther and Kegg pathways analyses.The GO pathway analysis is divided into biological processes,cellular component and molecular functions.Enrichment of target genes in these three parts are regulation of transcription,DNA-dependent,Nucleus and Protein binding.Kegg pathway analysis and Panther pathway analysis with the largest concentration of target genes were Pathways in cancer,Wnt signaling pathway.5.The TNM stage of non-small cell lung cancer group was analyzed by Spearman correlation of CEA(P=0.003),SCC(P=0.038),CYFRA21-1(P=0.000),CA125(P-O.001)and SA(P=0.001).The correlation coefficient were 0.408,0.294,0.481,0.436 and 0.474.There were a positive correlation between the TNM staging of NSCLC and CEA,SCC,CYFRA21-1,CA125,SA.6.The candidate miRNAs were verified by qRT-PCR,miR-1228-3p(P =0.006),miR-1228-5p(P =0.8678),miR-133a-3p(P =0.0431),miR-1273f(P =0.0996),miR-545-3p(P =0.0473),miR-181a-5p(P = 0.0298),miR-26b-5p(P = 0.5542),miR-361-5p(P= 0.0066)and miR-130a-3p(P = 0.5880).There are 5 differential miRNAs:miR-1228-3p,miR-133a-3p,miR-545-3p,miR-181a-5p and miR-361-5p.7.Analysis carcinoma group and healthy controls the expression of nine miRNAs,the results showed that miR-1228-3p(P = 0.0092),miR-181a-5p(P = 0.0315),miR-361-5p(P = 0.0287).In other words,miR-1228-3p,miR-181a-5p and miR-361-5p are adenocarcinoma differential miRNAs.8.The analysis of squamous cell carcinoma and healthy control group showed that the P value of miR-545-3p was 0.0315,while the P values of other miRNAs were all greater than 0.05.MiR-545-3p is the only differential miRNA of squamous cell carcinoma.9.MiR-181a-5p in the healthy control group and TNM I staging of NSCLC group(P:=0.0279),that is,miR-181a-5p appeared in the early stage of NSCLC expression changes.10.In the ROC analysis of 5 different miRNAs,the largest area of AUC in single miRNA was miR-1228-3p(AUC=0.685).The largest area of the two types of miRNA is miR-1228-3p combined miR-181a-5p,and its AUC area is 0.711 11.Fifty NSCLC patients were treated with 5 different miRNAs K-M survival analyses.The P values of up regulated miR-1228-3p and down regulated miR-181a-5p were 0.0407.The median survival time of NSCLC patients with high expression of miR-1228-3p and low expression of miR-181a-5p was shorter.12.Hazard ratio of miR-1228-3p was 1.487,suggesting that high expression of miR-1228-3p is a risk factor for the median survival time of NSCLC patients.Conclusion:1.MiR-181a-5p play an important role in the early diagnosis of NSCLC.MiR-1228-3p combined miR-181a-5p have certain diagnostic efficacy for NSCLC.2.The median survival time of NSCLC patients with high expression of miR-1228-3p and low expression of miR-181a-5p are shorter.MiR-1228-3p is a risk factor for the median survival time of NSCLC patients.