The Roles Of Lnc-ENST00000556926 In Malignant Behaviors Of BEAS-2B Cells Induced By Coal Tar Pitch

ObjectiveThis study aims to explore the effect of Lnc-ENST00000556926 on malignant-transformed immortalized human bronchial epithelial(BAES-2B)cells induced by coal tar pitch extracts(CTPE),which may provide theoretical basis for investigating the mechanism of malignant transformation from Long non-coding RNAs(LncRNAs)level.Methods1.BEAS-2B cells were treated with 2.4 μg/mL of CTPE for 72h.Then passaged,the cells were treated with the CTPE solution for another four times in the same procedure,which named CTPEO.Next,the cells were passaged until passage 30(CTPE30),And the malignant transformation cell model was established.2‰ of Dimethyl sulfoxide(DMSO)was used as control.The relative expression level of Lnc-ENST00000556926 was detected by RT-PCR.2.The basic information of Lnc-ENST00000556926 was analyzed by UCSC database and the protein coding ability of Lnc-ENST00000556926 was obtained by Coding Potential calculator 2(CPC2)and Coding Potential Assessment Tool(CPAT);The subcellular localization of Lnc-ENST00000556926 was confirmed by separating cytoplasmic and nuclear assay.3.The malignant-transformed CTPE30 groups were transfected with 100nmol/L siRNA for Oh,12h,24h and 36h,respectively.Then,the relative expression level of Lnc-ENST00000556926 was detected by RT-PCR.4.The proliferation of cells was analyzed by CCK-8 assay;the cell cycle and apoptosis of cells were detected by flow cytometer.5.The mRNAs in malignant-transformed CTPE30 group and CTPE30+siRNA group were sequenced by Illumina method;differentially expressed genes were detected by DESeq2.The relative expression level of mRNAs were analytized by RT-PCR;6.Statistical analysis:All statistical analysis was performed with SPSS 21.0 software.All data was expressed as mean±standard deviation((?)±s).Comparisons between more than two groups were analyzed by one-way ANOVA.LSD test was used for multiple comparisons between groups.Comparisons between two groups were analyzed by Student’s t-test.The statistically significance was defined as a=0.05.Results1.The relative expression level of Lnc-EN ST00000556926 in malignant-transformed cells.The expression level of Lnc-ENST00000556926 in malignant-transformed CTPE30 group was significantly higher than that in control group(P<0.001).2.The basic information,protein coding ability and the subcellular localization of Lnc-ENST00000556926Lnc-ENST00000556926,located in chrl4q31.3,includes two exons and one intron.And the length of Lnc-ENST00000556926 was 458bp with no or weak protein coding ability.And the Lnc-ENST00000556926 was mainly located in nucleus and less located in the cytoplasm(P<0.05).3.The optimal interference time of Lnc-ENST00000556926Compared with control group,the relative expression levels of Lnc-ENST00000556926 were reduced by 790.08-fold,29.18-fold and 16.98-fold when the transfection time was 12h,24h and 36h,respectively(P<0.05).Therefore,the optimal transfection time of Lnc-ENST00000556926 was confirmed as 12h.4.The influence of cells function after interference of Lnc-ENST00000556926Compared with the malignant-transformed CTPE30 group and CTPE30+NC group,the proliferation of CTPE30+siRNA group significantly decreased(P<0.05).Compared with the CTPE30+siRNA group,the percentage of G0/G1 phases cells were decreased in malignant-transformed CTPE30 group and CTPE30+NC group,while the percentage of G2 phases was significantly increased(P<0.05).The apoptosis rate of CTPE30+siRNA group was significantly higher than that in malignant-transformed CTPE30 group and CTPE30+NC group(P<0.05).5.The transcriptome sequencing and the RT-PCR verificationThe 159 differentially expressed mRNAs were selected based on the screening criterion(Fold change≥1.5 and P<0.05).Among them,62 mRNAs were up-regulated and 97 mRNAs were down-regulated.Compared with the malignant-transformed CTPE30 group,the expressions of TXNDC5 and FOXD1 in CTPE30+siRNA group were down-regulated(P<0.05).ConclusionLnc-ENST00000556926 could regulate the proliferation,cycle and apoptosis of CTPE-induced malignant transformed BEAS-2B cells.And Lnc-ENST00000556926 may have a regulatory relationship with TXNDC5 and FOXD1.