Screening And Study On Differentially Expressed LncRNA In Malignant Transformation Of 16HBE Cells Induced By Glycidyl Methacrylate

Glycidyl methacrylate (GMA) is a derivative of glycidyl aldehyde, harboring functional groups of C=C and-CH(O)CH-, which could take part in not only the ionic type, but also free radicals reaction, and GMA is high in reactivity. As a kind of synthetic resin monomer, GMA is mainly used for chemical materials, such as resin, coating, bonding agent, and plastic. It’s the molecular structure of GMA that makes chemical materials have ultraviolet protection, water resistant and heat resistant properties. Previous studies showed that, GMA was of low toxic, a number of mutagenicity test including Ames, in vitro mammalian chromosome aberration test and mammalian erythrocyte micronucleus test were positive. GMA had genotoxicity, could make fibroblasts of normal human fetal lung, CHL cell,16HBE and other cells malignantly transformed, which haboured potentially carcinogenic.Long non-coding RNA (LncRNA) is currently defined as transcripts greater than 200bp without known protein-coding function, which have been reported to be involved in all aspects of gene regulation, including epigenetic regulation, transcription levels and post-transcriptional level et al. Currently, research on targets and mechanism of LncRNA action is unclear, and the network that the regulation of gene expression by LncRNA has been confirmed importantly and complicated. Research shows that LncRNA is closely associated with human tumors, and abnormal expression of LncRNA exists in the development process of a variety of tumors. Specific LncRNA are differentially expressed in different tumors, so some LncRNA could be a tumor-biological markers. As a consequence, tumor-associated specific LncRNA have been hotspot for looking for early detection and diagnosis-related biomarkers of tumors. However, tumors are developed with the malignant transformation of cells, and are the result of the proliferation of malignant cells. Abnormal expression of LncRNA exists in the process of malignant transformation of cells. Therefore, that dentifing malignant transformation-associated specific LncRNA is not only beneficial to reveal the molecular mechanisms of environmental chemicals causing cancer cells, but also be used as potential molecular markers of environmental chemicals making cells cancerous.Based on study of related gene expression profiles, methylation gene and silencing gene screening,8μg/ml GMA was administered to 16HBE cells 72h 3 times with 24h interval to induce 16HBE cells malignant transformed, meanwile DMSO was administered to vehicle-control. ConA test and soft-agar colony formation test were used for biological identification of malignant transformed cells. Cells that malignant transformation 16HBE cells (8 μg/mL GMA) and solvent control group cells were all of the 30th generation were harvested. High throughput LncRNA microarrays was used to detect the difference of expression profile of LncRNA between two groups of the 30th generation, so as to screen differentially expressed LncRNA and LncRNA-associated mRNA; function of differentially expressed LncRNA were investigated by GO and Pathway analysis to provide reliable experimental data in order to explore the mechanism of LncRNA; target LncRNA was screened through strategies such as fold change and neighboring genes analysis. Opon verification and analysis of real-time quantitative PCR (qPCR), specific LncRNA related with GMA-induced malignant transformed cells was defined; gene chip were applied to measure the expression levels of target gene to investigate specific biomarkers involved in malignant transformation 16HBE cells induced by glycidyl methacrylate.1 Screening on differentially expressed LncRNA of malignant transformed cells induced by GMACompared with cells of DMSO control group, LncRNA/mRNA expression profile in malignant transformed cells induced by GMA were significantly expressed. We screen out 2664 differentially expressed LncRNA including 896 upregulated with foldchange of 2.0～198.8,1768 downregulated with foldchange of 2.0-262.1. Simultaneously,2216 differentially expressed LncRNA-associated mRNA were screened out, including 706 upregulated with foldchange of 2.0-642.0, and 1510 downregulated with foldchange of 2.0-74.9.2 Analysis of differentially expressed LncRNA of malignant transformed cells induced by GMAResults of GO analysis of LncRNA-associated mRNA showed,166 GO terms about molecular function aspect were found, involving bingding, protein bingding, DNA binding, signal transducer activity, molecular transducer activity and receptor bingding, et al.869 GO terms about biological process aspect were found, involving cellular process, metabolic process, cellular process and signal-organism process, et al. 20 GO terms about cellular component aspect were found, involving cell part, intracellular part, membrane, membrane part, and intrinsic component of membrane, et al.Results of Pathway analysis of LncRNA-associated mRNA showed,25 and 17 signaling pathways involved in these up-regulated genes and down-regulated genes, respectively, mismatch repair, DNA replication, protein digestion and absortion, neuroactive ligand-receptor interaction and so on.3 Analysis of specific LncRNABased on the result of LncRNA microarrays, the expressions of LncRNA PC A3, CTBP1-AS 1 and CDKN2B-AS1 were up-regulated by 7.17,2.20 and 5.39 fold change, respectively. qPCR showed that the levels of LncRNA PC A3 and CTBP1-AS 1 expression increased in the malignant transformation 16HBE cells induced by glycidyl methacrylate in accordance with the result of LncRNA microarrays. According to the biological information and relevant literature, PRUNE2 and CTBPl were the target gene of target gene, respectively. The expressions of PRUNE2 was down-regulated by 2.54, CTBP1 up-regulated by 1.26, in line with the result of Gene chip.Therefore, LncRNA PCA3 and CTBP1-AS 1 could regulate the expression of target gene and could be considered as the stable and specific biomarkers involved in malignant transformation 16HBE cells induced by glycidyl methacrylate.In conclusion,16HBE cells could be induced malignantly transformed by a low concentration of 8μg/ml GMA with malignant characteristics. Compared with cells of DMSO control group, LncRNA/mRNA expression profile in malignant transformed cells induced by GMA were significantly expressed. We screened out a number of abnormal expression of LncRNA correlated with malignant transformation 16HBE cells induced by glycidyl methacrylate. We explored many GO and Pathway terms by GO and Pathway analysis of LncRNA-associated mRNA for providing reliable experimental data in order to explore the mechanism of LncRNA. Rresults of LncRNA microarrays, qPCR and gene chip shows, specific LncRNA PC A3 and CTBP1-AS1 to prove the accuracy of LncRNA microarrays results, so as to provide a basis for specific biomarkers involved in malignant transformation 16HBE cells induced by glycidyl methacrylate.