Screening and verification of mRNA expression profile in a stable miR-21 knockdown human neuroblastoma cell line (SK-N-SH1381)

Abstract

Objective To explore mRNA expression profiling in a stable miR-21 knockdown human neuroblastoma cell line (SK-N-SH1381) for screening potential downstream target genes of miR-21. Methods Human neuroblastoma cell line (SK-N-SH1381) with stable miR-21 knockdown via lentiviral transfection was screened for changes in mRNA levels using Agilent Human Gene Expression Chips in comparison with the same cell line transfected with nonsense sequence (SK-N-SHlv3-nc). At the same time, we conducted in silico miR-21 targeting gene prediction, enrichment function of genes screened by chips and literature review to aid the selection of candidate genes. Then fluorescent quantitative polymerase chain reaction (PCR) was performed for verifying the results of chips at cellular level. Results Using mRNA chips, we identified 4985 mRNAs with a change of >2 folds (P<0.05). There were 2734 up-regulated and 2251 down-regulated genes. Based on the function of miRNAs, only the up-regulated genes were selected. After Venny analysis combining the results of chip and 398 potential target genes as predicted in silico, 64 candidate genes were identified as potential targets of miR-21. After further bioinformatic analysis and literature review, our selection was limited to 6 target genes of EVA1A, TOM1L2, RGMA, LINC-PINT, NACC2 and RPAP3. Quantitative PCR showed that the expressions of these genes were consistent with the findings of mRNA chips. Among 6 genes, EVA1A and TOM1L2 had the highest fold changes of differential expression. Conclusions With a combination of gene chip and bioinformatic analysis, 6 target genes of miR-21, i. e. EVA1A, TOM1L2, RGMA, NACC2, RPAP3 and LINC-PINT, are selected from human neuroblastoma cell line with stable miR-21 knockdown. And EVA1A and TOM1L2 show the highest fold changes of differential expression. Further studies are required for exploring their roles during tumorigenesis. Key words: Neuroblastoma; Gene expression chips; Gene expression profiling