Analysis Of Long Non-coding RNA Profiles And Preliminary Bioinformatics Study In L02/HBx Cells

OBJECTIVE:To investigate the long non-coding RNAs（lncRNAs） profiles in L02/HBx and L02/pcDNA3.0cells and conduct preliminary bioinformatics study among differentially expressed genes.METHODS:The cell lines of L02/HBx（experiment group） and L02/pcDNA3.0（control group） are the experimental subjects. Total RNA was extracted from each cell line and purified. Its quality and integrity was checked. And then the RNA was reversely transcribed into double-strand cDNA, and biotinylated cRNA probes were synthesized. Utilize lncRNAs hybridization-based microarray technology to get the lncRNA and mRNA expression profiles between experiment group and control group, and screen out the significantly differential expressed lncRNAs and mRNAs after pretreatment and homogenization of raw data. Four over-regulated lncRNAs:TCONS₀0006195, ENST00000557524, NR₀37597, ENST00000539975, and three under-regulated ENST00000508424, ENST00000447433,uc0011va.4from the differentially expression profiles were validated by RT-PCR using SYBRGREENI in two cell lines, using GAPDH as inter reference. Use GO and KEGG pathway to conduct preliminary bioinformatics study via database from NCBI RefSeq, UCSC, RNAdb, lncRNAs,etc.RESULTS:1.Compared with differentially expressed lncRNAs level of control group,323lncRNAs of experiment group are up-regulated and421lncRNAs are down-regulated whose P value<0.05 and fold change≥2. Seven hundred and forty-two mRNAs of experiment group are up-regulated and501mRNAs are down-regulated whose P value<0.05and fold change≥2.2. Four over-regulated lncRNAs and three under-regulated lncRNAs were validated by RT-PCR（SYBRGREENI method） in L02/HBx and L02/pcDNA3.0cells. The results demonstrated that TCONS₀0006195, ENST00000557524, NR₀37597, ENST00000539975are over-regulated; and ENST00000508424, ENST00000447433, uc0011va.4are under-regulated in the L02/HBx cell with control group, in accordance with lncRNA microarray.3.GO analysis reveals that, among diferentially expressed mRNAs,526up-regulated and680down-regulated mRNAs are included in biological process,68up-regulated and59down-regulated mRNAs are included in cellular component,77up-regulated and86down-regulated mRNAs are included in molecular function.4.Fifty-two pathways are involved in up-regulated mRNAs and21pathways are involved in down-regulated mRNAs according to pathway analysis.CONCLUSIONS:1. The expression lncRNA profile of L02/HBx cell changes significantly in comparison with control group. TCONS₀0006195, ENST00000557524, NR037597, ENST00000539975are over-regulated; and ENST00000508424, ENST00000447433, uc0011va.4are under-regulated in the L02/HBx cell with control group.2. Preliminary bioinformatic analysis reveals that differentially expressed mRNA was involved in growth,differentiation, apoptosis and cell cycle. Involved pathways include signaling pathway, apoptosis pathway, cell cycle pathway, etc.