Targeting The Relationship Between MiR-520e And PRAP1 Based On TCGA Data Analysis And Verification In A549 Cells

Objective:To rapidly and efficiently screen genes that may have co-expression relationships with microRNAs(miRNAs)in lung cancer tissues by analyzing TCGA(The Cancer Genome Atlas)database for non-small cell lung cancer(NSCLC)-related high-throughput sequencing data.We also used the A549 lung cancer cell line to further verify the expression and targeting relationship between the screened genes and miRNAs in the in vitro experiments,as well as the effects of their differential expression on the proliferation and migration of lung cancer cells,providing more reference and basis for the study of the mechanism of action of these genes and miRNAs in lung cancer.Materials and Methods:1.Analysis of all NSCLC-related high-throughput sequencing data from the TCGA project to obtain differentially expressed(DE)genes and miRNAs using the R language and the edgeR and DESeq2 packages.Then screening of target genes and miRNAs potentially co-expressed with them using the WGCNA method in combination with known annotation information.2.The experimental lung cancer cell line was A549,were divided into 11 groups according to the transfected components:the untreated cells and which were transfected reagents only were set up as the Mock group and NC group,the-PRAP1,PRAP1-NC,siRNA,mimics and inhibitor groups were added according to their respective names.Four additional co-transfected groups,PRAP1+mimics group,PRAP1+inhibitor group,siRNA+mimics group,and siRNA+inhibitor group,based on components co-transfected thus vector/siRNA and miRNA mimics/inhibitor.3.Relative quantification of target gene(PRAP1)and miRNA(miR-520e)nucleic acids and target gene protein expression in each cell group using qPCR and Western blot,respectively;verification of miRNA targeting to the 3' untranslated region(UTR)of target gene mRNA using dual-luciferase reporter gene assays;proliferative activity,migration capacity and cell cycle in each cell group using CCK-8,Transwell and flow cytometry,respectively.4.After using the Shapiro-Wilk test and Bartlett test for data normality and homogeneity of variance,respectively.The t-test was used to compare the mean between the two groups,one-way ANOVA or Kruskal-Wallis test was used to statistically analyze the between-group differences in the results,and the Tukey's HSD test was used for further comparison and correction.Results:TCGA lung cancer-associated transcriptome sequencing data came from 1145 tissue samples and miRNA sequencing results from 1090 samples.After DE and co-expression analysis,and then based on the length of the 3' UTR,the target genesand miRNAs were finally screened for PRAP1 and miR-520e,respectively,and in vitro experiments were performed to verify the results as follows:1.Cells co-transfected with the PRAP1 over-expressing plasmid and miR-520e mimics showed significantly higher relative quantification of PRAP1 mRNA(1093.08±345.96)than the PRAP1 group(303.79 ± 26.35,P<0.001).The protein expression of PRAP1-transfected group(concentration,?g/?L:0.96±0.13)was significantly lower than that of the control group(1.61±0.23,P=0.003).2.Cells over-expressing PRAP1 had significantly higher migration capacity(cell counts:2334.33±106.57)than cells transfected with siRNA group(161.33±39.15)and controls(135.00±18.25,P<0.001),and the proportion of G1-phase cells in the PRAP1-transfected group(61.33±1.14)was significantly higher than controls(49.81 ± 1.43).3.Cells transfected with miR-520e mimics showed lower migration capacity(1005.00±153.86)than the PRAP1 group and the inhibitor group(2869.33±109.77,P<0.001).4.The proliferative activity of cells co-transfected with PRAP1 plasmid and miR-520e mimics was lower than the proliferation of the two groups co-transfected PRAP1 siRNA with mimics/inhibitor and lower than that of the mimics transfected group at 15min,30min,and 60min post-transfection(P values are all less than 0.05).5.The relative luciferase activity of the PRAP1 wild-type plasmid with miRNA mimics group(WT+mimics)(0.61±0.16)was lower than that of mutant control group(MUT+mimics,0.95±0.12,P=0.049).Conclusions:1.miR-520e has a possible targeting relationship to the PRAP1 gene;up-regulation of miR-520e promotes PRAP1 transcription in A549 cells.2.Up-regulation of miR-520e inhibits A549 cell migration;repression of PRAP1 mRNA increases the proliferative activity of A549 cells,however,over-expression of PRAP1 arrests A549 cells at the G1 phase.