Study On The Role Of The Exosomal MiR-487a/miR-487b Cluster In The Process Of Aflatoxin B1-induced The Malignant Transformation Of BEAS-2B Cells

Objective: Screening of critical exosomal miRNAs clusters associated with aflatoxin B1(AFB1)-induced malignant transformation of BEAS-2B cells stably expressing CYP2A13;Exploring the role and mechanism of exosomal miR-487a/miR-487 b cluster in AFB1-induced malignant transformation of B-2A13 cells.Methods:1.The differentially expressed miRNAs were screened using Affymetrix miRNA microarray to detect the miRNAs expression profiles between P50 B-2A13 cells and P50 B-Vector cells.2.OmicsBean software was used to analyze the gene pathways involved in the target genes of the differentially expressed miRNAs,and the miRbase database was used to search the selected miRNAs one by one to screen out clustered miRNAs.3.The SBI kit was used to extract the exosomes from cell supernatant.The relative expression levels of the miRNAs in the P50 B-2A13 and B-2A13 cells and exosomes were analyzed by RT-qPCR.The synchronized miR-487a/miR-487 b clusters serve as key exosome miRNAs clusters.4.The expression of miR-487 a and miR-487 b were separately and jointly inhibited in P50 B-2A13 cells by transient transfection with mi RNA inhibitors.The effect of miR-487 a and mi R-487 b inhibitors on cell proliferation,scratch healing,plate colony formation,migration,and invasion in P50 B-2A13 cells were evaluated through cell proliferation,scratch test,plate colony formation assay,Transwell migration and invasion assay.5.RT-qPCR was used to detect the expression changes of miR-487 a and miR-487 b in B-2A13 cells and exosomes treated with different generations of AFB1.Cell proliferation assay,scratch healing,plate colony formation,Transwell migration and invasion assay,and Western blot assays were used to evaluate the role of exosomal miR-487a/miR-487 b clusters in AFB1-induced malignant transformation of cells.Results:1.There were 59 dysregulated miRNAs were identified,36 of which were up-regulated and 23 were down-regulated.2.Preliminary screening out 3 groups of miRNAs cluster,miR-337-5p/miR-127-3p/miR-432 cluster,miR-379/miR-299-3p/miR-323a/miR-494 cluster and miR-654-3p/miR-381/miR-487b/miR-487a/miR-382 cluster.3.Among the 3 miRNAs clusters,only mi R-487a/miR-487 b cluster were relatively synchronous in P50 B-2A13 and B-2A13 cells and exosomes.4.Compared with miR-Control,miR-487 a inhibitor and miR-487 b inhibitor can reduce the expression of mi R-487 a and miR-487 b in P50 B-2A13 cells and exosomes.5.Inhibiting the expression of miR-487 a and/or miR-487 b in P50 B-2A13 cells can significantly inhibit the proliferation,migration,invasion,and colony formation of P50 B-2A13 cells,and can reverse the expression levels of E-cadherin and N-cadherin of malignant transformed P50 2A13 cells.The inhibitory effect of combined inhibitors did not show a significantly stronger effect than that of a single miRNA inhibitor.6.Compared with P0 2A13,the expression of miR-487 a and miR-487 b was down-regulated in P20 2A13 cells and exosomes.However,after the 30 th generation,the expression level gradually increased.7.P0 2A13 and P20 2A13 cells are relatively weak in proliferation,migration,invasion,and colony formation,but as AFB1 exposure continues.Then the cell proliferation,migration,invasion,and Both colony formation ability and EMT protein markers showed a tendency to increase with prolonged exposure time,and the expression level of E-cadherin gradually decreased.The expression level of N-cadherin gradually increased.Conclusions:1.The miR-487a/ miR-487 b clusters that were relatively synchronously expressed in B-2A13,P50 B-2A13 cells and exosomes were initially screened as candidate exosomal miRNAs clusters through differential expression profiling,pathway analysis,and detection of miRNAs expression in cells and exosomes.2.Inhibition of mi R-487 a and mi R-487 b expression either alone or in combination inhibits proliferation,migration,invasion,scratch healing,and colony formation of P50 B-2A13 cells.,suggesting that the exosomal miR-487a/miR-487 b cluster may play an important role in AFB1-induced malignant transformation of B-2A13 cells.3.In the process of malignant transformation of B-2A13 cells induced by AFB1,the miR-487a/miR-487 b cluster expression levels in cells and exosomes of different treatments increased significantly after P30,it is highly consistent with cell proliferation,migration,invasion,scratch healing and colony formation after P30.On the one hand,it was shown that the malignant transformation of B-2A13 cells may occur in about 30 generations of AFB1 treatment.On the other hand,it was further demonstrated that the mi R-487a/miR-487 b cluster played a key regulatory role in the AFB1-induced malignant transformation of B-2A13 cells.