The Aberrant Expression Profiles Of Long Non-coding RNAs And Micro RNAs In Malignant Transformation Of BEAS-2B Cells

ObjectiveLong non-coding RNAs（lnc RNAs） regulate the biological functions in a certain degree. Aberrant expressions of lnc RNAs have been considered potential biomarkers of tumors. Micro RNAs（mi RNAs） can play a negative role in gene expression regulation. Furthermore, the interactions of mi RNAs and lnc RNAs contribute to biological processes. The purpose of this research was to detect the role of aberrant expression of lnc RNAs and mi RNAs in the process of malignant transformation of BEAS-2B cells which were treated with carcinogens and to provide basis for further mechanism study.Methods1. CTPE（coal tar pitch extracts） and CSC（cigarette smoke condensate） were utilized as test substances to induce the BEAS-2B cells malignant transformation model in vitro.As a positive control, the exposure concentration of benzo（a）pyrene [B（a）P] was5μg/m L. DMSO（terminal concentration: 2‰） was used as solvent control. Two experimental groups, treated with CTPE and CSC, the exposure concentrations were2.4μg/m L and 25μg/m L, respectively. Morphological observation, wound healing assay, colony formation assay, MTT assay and cell apoptosis analysis were performed to evaluate the malignant transformation of BEAS-2B cells.2. Microarray analysis-based lnc RNA/m RNA and micro RNA expression profiles was performed through the hybridization between BEAS-2B cells samples with Human Lnc RNA Microarray V3.0 and Exiqon’s micro RNA arrays respectively.3. The differentially expressed lnc RNAs and mi RNAs were analyzed by Bioinformatics methods and validated by real time-PCR.Results1. The establishment of the malignant transformation model of BEAS-2B cells in vitroThe substance CSC that the median inhibitory concentration（IC50） was about0.084mg/m L was indicated by CCK-8 assay treating BEAS-2B cells. Among three treatment groups, the passage twenty（P20） and passage thirty（P30） cells appeared morphological changes after B（a）P, CTPE, and CSC stimulation respectively, such as unclear cell configuration, extensive cytoplasmic vacuolization, cell swelling and irregular nuclear.Wound healing assay showed an increased migration distance in three treatment groups, B（a）P, CTPE and CSC, compared to the blank control and DMSO group（P<0.05）. The results of colony formation assay indicated a higher number in three poisoned groups（P<0.05）. Meanwhile, at the passage of 30 th, three treatment cells showed a higher focus counting than the cells of passage 20th（P<0.05）. MTT assay exhibited similar results that had a higher growth rate in the groups of B（a）P, CTPE and CSC treatment and with the increase of passage, the activity of proliferation rose（P<0.05）. Flow cytometry analysis determined a lower apoptosis rate（P<0.05） which was found in three treatment cells compared to the controls. The passage of 30 th cells showed a lower apoptosis rate than the cells of passage 20th（P<0.05）.2. Lnc RNA/m RNA expression profiles detected by microarray analysisThe expression profiles of 263 aberrant expressed lnc RNAs were indicated between B（a）P 30 cells and blank control cells samples, whereas CTPE 30 samples were detected 707 differentialy expressed lnc RNAs（Fold change≥1.5, P<0.05）.Among B（a）P groups, 159 were up-regulated and 104 were down-regulated. Among CTPE groups, the number of up-regulated and down-regulated lnc RNAs was 408 and299 respectively. Based on the positional relationship with their adjacent coding genes, the categories of antisense and intergenic had a high proportion in aberrant expressed lnc RNAs.The expression profiles of 159 different expression m RNAs were detected in B（a）P 30, and the number of CTPE 30 group was 569（Fold change≥1.5, P<0.05）,compared to blank control cells respectively. Among B（a）P group, 99 were up-regulated and 60 were down-regulated. Among CTPE group, the number was 357 and 212 respectively.3. Micro RNA assays expression profiles detected by microarray analysisThe expression profiles of 127 abnormal expressed mi RNAs were evaluated between B（a）P 30 and blank control cells, whereas CTPE 30 cells were detected 78 aberrant expressed mi RNAs（Fold change≥2.0, P<0.05）. Among B（a）P groups, 29 were up-regulated and 98 were down-regulated. Among CTPE groups, 52 were up-regulated and 26 were down-regulated.4. Real time-PCR validationCompared to blank and solvent controls, the expression profiles of lnc RNAs and mi RNAs were consistent with the results of real time-PCR in B（a）P, CTPE and CSC treatment groups. Among lnc RNAs expression profiles, the expressions of ENST00000501520, ENST00000556926 and ENST00000535078 were significantly higher in B（a）P groups than in controls（P<0.05）, while TCONS₀0011820 was significantly low-expression（P<0.05）. In mi RNAs expression profiles, the expressions of hsa-mi R-2115-3p and hsa-mi R-4521 were significantly down-regulated（P<0.05）.ConclusionsAs exogenous chemicals, CTPE and CSC had the ability to stimulate the BEAS-2B cells malignant transformation, which could induce morphological changes,enhance the ability of migration, proliferation, and change the expression profiles of lnc RNAs and mi RNAs. With the increase of passage, the degree of malignancy aggravated. Lnc RNA-ENST00000556926 expression was up-regulated and the expression of hsa-mi R-4521 was down-regulated which were involved in cell cycle regulation. Lnc RNA-ENST00000501520 was over-expression and hsa-mi R-2115-3p was low-expression which were involved in cell proliferation regulation.