Preliminary Study On The Screening And Roles Of CircRNA-miRNA-mRNA In Aflatoxin B1-induced The Malignant Transformation Of B-2A13 Cells

Objective:The study aimed to screen the circRNAs,miRNAs and mRNAs associated with AFB1 induced malignant transformation of P50 B-2A13 cells and to explore the target regulation of circRNA-miRNA-mRNA in AFB1-induced the malignant transformation of B-2A13 cells.Methods:Dysregulated miRNAs,mRNAs and circRNAs were identified by miRNA microarray,mRNA microarray and circRNA microarray between P50 B-2A13 cells and P50 B-Vector cells.Target genes of miRNAs were predicted with TargetScan software.The common genes were reserved after comparing the target gene of miRNAs and differentially expressed mRNAs.Then the target circRNAs of the screened miRNAs were found in the circRNA-miRNA library.Last but not least,combining the pathway and disease enrichment analysis,the regulation relationship of the circRNA-miRNA-mRNA were preliminarily screened and established,which was validated by reverse transcription quantitative real time polymerase chain reaction?RT-qPCR?.The proteins of mRNA expression level were detected by western blot.Transient transfection of siRNAs were used to inhibit the expression of ID3 and FGF5,and the effect of them on cell proliferation and migration in P50 B-2A13 cells were evaluated using colony formation assay and scratch healing assay.Results:?1?There were 37 upregulated and 23 downregulated miRNAs,771 upregulated and 243 downregulated mRNAs,1496 upregulated and 2812 downregulated circRNAs identified in P50 B-2A13 cells.?2?Three circRNA-miRNA-mRNA regulation relationships based on the gene of GJA1,ID3 and FGF5 were screened by target correlation analysis in the differentially expressed circRNAs,miRNAs and mRNAs,i.e.hsa_circ₀002842-miR-23c-GJA1.?3?RT-qPCR validation of mRNAs and miRNAs suggested a high correlation between the expression and microarray data.The consistency of circRNAs between expression and microarray data was 80%.?4?Knockdown of ID3 could inhibit the proliferation of P50 B-2A13 cells,but FGF5 had few effect.The scratch healing assay showed that either knockdown of ID3 or FGF5 could suppress the migration of P50 B-2A13 cells.The wound closure of blank control,knockdown of ID3 and knockdown of FGF5 were 53.9%,51.1%,38.8%and 30.6%respectively.Conclusion:?1?Three circRNA-miRNA-mRNA regulation relationship based on the gene of GJA1,ID3 and FGF5 were screened by target correlation analysis in differentially expressed circRNAs,miRNAs and mRNAs.?2?Knockdown of ID3 could inhibit the proliferation and migration of P50 B-2A13 cells,but knockdown of FGF5 only suppresssed the migration of cells and had no significant effect on the preliferation of P50 B-2A13 cells.