ENST00000501520 Identification And Function In Malignant Transformation Of BEAS-2B Cells

ObjectiveENST00000501520 potential function was explored in CTPE-induced malignant transformation of BEAS-2B cells model.Possible target genes of ENST000000501520 were searched using bioinformatics and verified experimentally.This study aims to provide theoretical support for exploring the mechanism of malignant transformation from the LncRNAs angel.Methods1.UCSC database was performed to learn location,sequence,length,adjacent gene,exon and intron of gene transcripting ENST00000501520.CPC(Coding potential calculator),CPAT(Coding Potential Assessment Tool)were used to predict ENST00000501520 potential protein encoding ability.The cells were partitioned into nuclear and cytoplasmic fractions to detect ENST00000501520 expression and calculate nuclear-cytoplasmic ratio,which determined subcellular localization.ENST00000501520 expression were detected of different cell algebras.2.Small interfering RNA(siRNA)interfered ENST00000501520 with the help of transfection reagent.The optimal interference concentration was determined by the interference efficiency of ENST00000501520.Taking an observation of the CTPE30 cells migration ability,proliferation,cell cycles and apoptosis.3.CNC(Coding-non-coding Gene Co-expression Network)predicted ENST00000501520 target genes.Target genes transcription and protein expression were detected after ENST00000501520 interference.SBK1 and SOCS3 expression were analysised of lung cancer patients form UALCAN database.Lung cancer patients from Kaplan-Meier Plotter database were preformed survival analysis according to SBK1 and SOCS3 expression.Results1.The basic information and encoding protein ability of ENST00000501520.The gene transcripting ENST00000501520 was located in Chr 16(p12.1)according to UCSC database,contained 2 exons and 1 intron,and was adjacent to encoding gene SBK1.ENST00000501520 length was 2481bp.CPC predited ENST00000501520 encoding protein prediction ability score was-0.73,and ENST00000501520 contained 1 partial ORF.CPAT predicted ENST00000501520 potential protein encoding ability score was 0.05.The two softwares tagged ENST00000501520 no or weak protein encoding ability.2.ENST00000501520 subcellular localization and expression whin different cell algebras.nuclear-cytoplasmic ratio of ENST00000501520 were 80.647%and 89.067%(respectively:β-actin,U6),which indicated that ENST00000501520 mainly locate in nucleus.ENST00000501520 expression of CTPE30 group was higher than that of 15,20,25(all P<0.001),and there was no significant difference between CTPE35 and CTPE30 groups(P=0.804).3.SiRNA transfection concentration determination.After 48h transfection,interference efficiency of 50nM,100nM and 150nM groups were 52.284%,69.371%and 25.734%.The optimum concentration of siRNAs was 100nM.Interference efficiency of transfection reagent(MOCK group)and siRNA negative control(NC group)were no significant difference compared with CTPE30 group(MOCK P=0.718,NC P=0.723).4.Cells function changes after ENST00000501520 interfered.At different time points,siRNA group cells migration detected via Scratch assay were significantly decreased(all P<0.001)compared with the CTPE30 group.siRNA group cells proliferation tested by MTT assay were significantly decreased(all P<0.001)compared with the CTPE30 group at different points.Compared with CTPE30 group,the percentage of S phase cells in siRNA group significant decreased(P<0.001),the percentage of G1 phase cells in siRNA group significant increased(P<0.001);cells apoptosis rate significantly increased within siRNA group(P=0.016)compared with CTPE30 group.5.CNC results and target genes expression after ENST00000501520 interfered.CNC analysis predicted ENST00000501520 target genes.SBK1,CLTB,TAP2,RIPK2 and SOCS3 were selected for futher research according to the Pearson coefficient.SBKI,CLTB,TAP2,SOCS3 transcription decreased percents of siRNA group were 86.508%,47.336%,7.066%and 61.375%after ENST00000501520 interfered,which were significant differences(all P<0.001)compared with the CTPE30 group.There was no significant difference(P=0.054)of RIPK2 transcription between siRNA and CTPE30 groups;SBK1 and SOCS3 were selected to detected protein level due to transpription level decreasing more than 50%,and protein levels of siRNAgroup were significant decreased compared with CTPE30 group after ENST00000501520 interfered(all P<0.001).6.SBK1 and SOCS3 expression in lung cancer and survival analysis.The expression of SBK1 was significantly increased in LUAD(Lung adenocarcinoma)and LUSC(Lung squamous)patients from UALCAN database(all P<0.001),yet SOCS3 expression was significantly induced(all P<0.001).Lung cancer patients from Kaplan-Meier Plotter database were preformed survival analysis.The results showed that down-regulated SBK1 correlated with poor survival rate of LUAD(HR=0.51,95%CI:0.4-0.66,logrankP<0.001),while there was no statistical significance in LUSC(HR=1.32,95%CI:0.96-1.8,logrankP=0.082).Up-regulated SOCS3 correated with poor survival rate og LUAD(HR=1.44,95%CI:1.13-1.84,logrankP=003),but there was no statistical significance in LUSC(HR=0.85,95%CI:0.6-1.21,logrankP=0.3631).ConclusionENST00000501520 is up-reguated in malignant transformation of BEAS-2B cells.Interfering ENST00000501520 inhibites malignant transformation of BEAS-2B cells.SBK1,SOCS3 expression are significantly decreased after siRNA processing.There is a positive correlation between ENST00000501520 and SBK1,SOCS3.