| Glycidyl methacrylate(GMA)is a kind of basic industrial chemical with high yield.It is widely used in the synthesis and processing of polymer materials such as medicine and food packaging due to its cross-linking polymerization properties.In December 2019,the International Agency for Research on Cancer(IARC)classified GMA as a chemical substance that is likely to cause Cancer in humans(Group 2A).However,the carcinogenic mechanism of GMA is still unclear.Therefore,it is important to explore the carcinogenesis mechanism of GMA.Long non-coding RNAs(lncRNAs)can participate in the regulation of multiple signal transduction in cell activities and play an important role in the occurrence of malignant tumors.ObjectivesBased on the establishment of the model of malignant transformation of 16HBE cells induced by repeated exposure to low concentrations of GMA,high-throughput lncRNA microchips were used to screen the differentially expressed lncRNAs related to the malignant transformation process of 16HBE cells induced by GMA to determine the differentially expressed lncRNAs and their related signaling pathways at different stages of the process.The co-expression of specific lncRNAs was analyzed and verified to explore their potential function in the process of GMA-induced malignant transformation of 16HBE cells.Methods1.Cytotoxicity test confirmed that low-concentration repeated exposure to GMA induced malignant transformation of 16HBE cells at a dose of 8?g/mL.16HBE cells in logarithmic growth stage were taken,DMSO was used as the solvent control,and the final concentration of 8?g/mL GMA was used as the treatment group.Each time was exposed to GMA for 72h,and the interval of 24h was followed by repeated exposure for 3 times,which was recorded as the 0 generation.The cells of the 10th,20th and 30th generation in the GMA treatment group and the DMSO control group were harvested,and the biological characteristics of the malignant transformation process were observed by ConA agglutination test and soft AGAR clone formation test.2.The 10th,20th and 30th generation cells of the GMA treatment group and the synchronous DMSO control group were collected,and total RNA was extracted with Trizol reagent.High-throughput lncRNA microarray was used to obtain the expression levels of lncRNAs at different stages,and the differentially expressed lncRNAs at different stages were screened.The screening criteria were set as Fold Chang(Fc)?2.0,differentially expressed lncRNA in a single stage(Fc?2.0,and P<0.05),and no significant change in the other two stages(Fc<2.0 or Fc?2.0 and P>0.05).Cluster method was used to analyze the malignant transformation process of 16HBE cells induced by GMA and the specific differential expression of lncRNA at different stages.According to the dynamic changes in the expression levels of differentially expressed lncRNAs in different periods,the state changes of differentially expressed lncRNAs in different periods were further analyzed by STEM software combined with time series analysis.3.Gene Set Enrichment Analysis(GSEA)of the signaling pathways involved in the differential expression of lncRNAs at different stages of this process.Combined with the results of lncRNA and related mRNA expression levels in different periods,the highly relevant lncRNA-miRNA-mRNA pairs in the bioinformatics database were screened,and the ceRNA network was constructed by Cytoscape software.Based on key gene scores in the ceRNA network and literature evidence,specific lncRNAs related to the malignant transformation of 16HBE cells induced by GMA and their related regulatory axes were predicted,and the expression levels of specific lncRNAs related to the malignant transformation of 16HBE cells were verified by real-time quantitative PCR.Results1.GMA-induced malignant transformation of 16HBE cells and screening of related lncRNA1.1 GMA-induced malignant transformation of 16HBE cellsThe cytotoxicity test of GMA showed that the cell survival rate decreased gradually with the increase of the exposure concentration.The relative survival rate of cells treated with 8 ?g/mL GMA was 93.67%,and the cell survival rate decreased significantly when treated with 8 ?g/mL GMA.Therefore,8 ?g/mL was selected as the dose of low concentration GMA repeated induction of 16HBE cell transformation test.In the process of malignant transformation of 16HBE cells induced by GMA,the ConA agglutination test was observed.In the 10th generation of GMA treated cells,weak agglutination occurred only when the ConA concentration was 200 ?g/mL.In the 20th and 30th generation of GMA treated cells,the degree of cell agglutination increased gradually,but no agglutination occurred in the DMSO control cells at the same period.It was observed in the soft AGAR clone formation test that the 10th and 20th generation of GMA treated cells did not form colonies in the soft AGAR,the 30th generation of GMA treated cells could form colonies in the soft AGAR,while the DMSO control cells did not form colonies at the same time,indicating that the 30th generation of GMA treated cells had the biological characteristics of malignant transformed cells.Based on this,the GMA treated cells of the 10th,20th,and 30th generation were divided into prophase,metaphase,and anaphase of malignant transformation.1.2 Screening of differentially expressed lncRNAs related to the malignant process of 16HBE cells induced by GMAThe expression levels of lncRNAs in 16HBE cells at the 10th,20th and 30th generation of GMA-induced malignant transformation and DMSO control group were detected.In the 10th generation of GMA-treated cells,1789 lncRNAs were differentially expressed,including 642 up-regulated and 1147 down-regulated variations.2601 lncRNAs were differentially expressed in 20th-generation GMA treated cells,of which 1517 were up-regulated and 1084 were down-regulated.There were 1183 IncRNAs in the 30th generation of GMA treatment group,including 574 up-regulated and 609 down-regulated lncRNAs.2 Analysis of differential expression of IncRNA related to the malignant process of 16HBE cells induced by GMA2.1 Analysis of the specific differential expression of IncRNA related to the malignant process of 16HBE cells induced by GMAIn the process of malignant transformation induced by GMA,1117 lncRNAs were differentially expressed in cells of the 10th generation GMA treated group,of which 436 lncRNAs were differentially up-regulated and 681 lncRNAs were down-regulated.1806 lncRNAs were specifically differentially expressed in the 20th generation GMA treatment group,including 974 up-regulated lncRNAs and 832 down-regulated lncRNAs.774 lncRNAs were specifically differentially expressed in cells treated with GMA at the 30th generation,of which 361 were up-regulated and 413 were down-regulated.2.2 Analysis of the status change of differentially expressed lncRNA related to the malignant process of 16HBE cells induced by GMAIn the analysis of the changes of the differentially expressed lncRNAs at different stages during the malignant transformation of 16HBE cells induced by GMA,21 differentially expressed lncRNAs were found in the 10th,20th and 30th generation of GMA-treated cells in the same direction of changes of the cell states,of which 16 were uniformly up-regulated and 5 were uniformly down-regulated.At the 10th generation,cells in the GMA treatment group were down-regulated,while 8 lncRNAs were up-regulated at the 20th and 30th generations.At the 10th and 20th generation,cells in the GMA treated group were downregulated,while at the 30th generation,4 lncRNAs differentially expressed were upregulated.At the 10th generation,cells in the GMA treated group were up-regulated,while at the 20th and 30th generations,4 lncRNAs were differentially down-regulated.At the 10th and 20th generations,cells in the GMA treated group were up-regulated,while at the 30th generation,there were 3 differentially expressed lncRNAs down-regulated.STEM time series analysis software was used to further analyze the status changes of all lncRNAs in the three periods.In the given top 20 time trend models,differentially expressed lncRNAs with up-regulated and down-regulated trend changes in the malignant transformation process of 16HBE cells were screened,among which 35 differentially expressed lncRNAs with up-regulated trend changes were found.There were 30 lncRNAs differentially expressed in down-regulated trend changes.3 Functional analysis of lncRNA differential expression in 16HBE cells during malignant transformation induced by GMA3.1 Analysis of signal pathways related to differential expression of lncRNA in the malignant process of 16HBE cells induced by GMAThe analysis of the signaling pathways involved in the differential expression of lncRNAs in the 10th,20th and 30th generation of 16HBE cells during the GMA-induced malignant transformation revealed that cell damage in the early stage of malignant transformation was characterized by abnormal energy metabolism accompanied by activation of some cancer-related signaling pathways.In the middle stage of malignant transformation,cell cycle-related signaling pathways were significantly enriched,which were also associated with the activation of TGF-?signaling pathway and Wnt signaling pathway.Enrichment item of late malignant cell transformation,and the middle of the malignant cell transformation enrichment results have more consistent,including cell cycle and the mitotic signal enrichment score value to related pathways are definitely higher than,in the middle of the malignant cell transformation also P53 signaling pathway and MYC target cancer related gene set of signaling pathways are activated.3.2 Co-expression analysis and verification of specific lncRNAs related to malignant transformation of 16HBE cells induced by GMALncRNA was selected as the trend of up-regulation and down-regulation of differential expression in the process of GMA-induced malignant transformation of 16HBE cells to construct a ceRNA regulatory network.The results indicated that in the process of GMA-induced malignant transformation of 16HBE cells,Differentially expressed higher trend LncRNA can LncRNA T215985 | miR-372-3 p | SLAMF7,LncRNA G020213 | miR-124-5 p | NRG1,LINC00310 | miR-377-3 p | KIAA1644 regulation axis function.Cut trend is likely to be differentially expressed LncRNA through LncRNA RMST | miR-10522-5 p | ARMCX4,LncRNA AC009542.2 | miR-1197 | GNB1L,LINC01580 | miR-1266-5 p| AP5S1 control axis,play a role in the malignant cell transformation process.Signalling pathway analysis was performed on differentially expressed mRNAs in the ceRNA regulatory network that had a targeted regulation relationship with differentially expressed lncRNAs.The results indicated that the up-regulated and down-regulated IncRNAs tended to be involved in epithelial mesenchymal transformation and cancer-related signal activation in the process of malignant cell transformation.LINC00310 and IncRNA RMST can be used as specific lncRNAs related to the process of malignant transformation induced by GMA in 16HBE cells,and the mechanism of action remains to be further studied.Conclusions1.Repeated exposure to 8 ?g/mL GMA induce the malignant transformation of 16HBE cells.2.In the process of GMA-induced malignant transformation of 16HBE cells,there are multiple differentially expressed lncRNA with specific and state changes at different stages,which can provide clues for the screening of specific lncRNA related to the process of GMA-induced malignant transformation of 16HBE cells.3.In the process of malignant transformation of 16HBE cells induced by GMA.the differentially expressed IncRNA at different stages involved different signaling pathways.Cell damage in the early stage of malignant transformation was mainly caused by abnormal energy metabolism.DNA damage repair were activated in the middle stage of malignant transformation.At the late stage of malignant transformation,such as P53 signaling pathways,were activated,while cell cycle-related signaling pathways were inhibited,prompting the 30th generation of cells in the GMA treated group to complete malignant transformation.4.LINC00310 and lncRNA RMST can be used as specific lncRNA related to the malignant transformation of 16HBE cells induced by GMA,and its mechanism needs to be further studied. |
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