The Inflammation Of 16HBE Cells And MicroRNAExpression Profile By Beryllium Sulfate

Objective:To investigate the inflammation of human bronchial epithelial cells?16HBE?induced by beryllium sulfate?BeSO₄?and its possible mechanisms.Methods:1.Human bronchial epithelial cells?16HBE?were used as the research models and were treated with BeSO₄ at different concentrations?0,100,150,200,250,300?mol/L?for 48 h.Cell viability was detected by MTT assay,and the expression of Interleukin-10?IL-10?,tumor necrosis factor-alpha?TNF-??,interferon-??IFN-??,and cyclooxygenase-2?COX-2?,and inducible nitric oxide synthase?iNOS?was measured by ELISA and western blot.2.The profile of miRNA in 16HBE cells treated with BeSO₄ was detected by small RNA sequencing,and differentially expressed miRNAs were screened with P<0.05 and|log₂?fold change?|>1.Some miRNAs were predicted by using TargetScan,miRanda,and PiTa.Gene Ontology?GO?analysis and the Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway enrichment analysis of genes was performed.Cytoscape software was used to construct the miRNA-gene regulatory network,and qRT-PCR was used to verify small RNA sequencing.3.A miRNA associated with inflammation was select for functional verification.miR-663b mimics and inhibitor were constructed.16HBE cells were treated by liposome transfection technology and then treated with BeSO₄.The expression of IL-10,TNF-?,IFN-?,COX-2,and iNOS was measured by ELISA and western blot.The aim to investigate the role of increasing or decreasing miRNA in BeSO₄-induced 16HBE cells inflammation.Target genes of miR-663b were predicted by using miRanda,TargetScan,DIANA Tools,and miRwalk.And the results of mRNA sequencing?preliminary sequencing results of research group?were taken.The Venn diagram was drawn.The potential binding site of miR-663b in the LIF mRNA 3'UTR was analyzed by TargetScan.Results:1.Compared with the control group,the cell viability of BeSO₄-induced 16HBE cells was inhibited in a dose-dependent relationship,the differences were statistically significant?P<0.05?.The half-inhibitory concentration of BeSO₄ treated cells for 48 h was 260?mol/L.The treatment of BeSO₄ at different concentrations increased the expression of IL-10,TNF-?,IFN-?,COX-2,and iNOS?P<0.05?.2.179 differentially expressed miRNAs were found in 16HBE cells exposed to 150?mol/L beryllium sulfate for 48 h,including 88up-regulated and 91 down-regulated,in which 93 known miRNAs and 86unnamed miRNAs.3.There are 28 miRNAs conforming to the differential expression screening criteria,including 18 up-regulated miRNAs and 10down-regulated miRNAs.Target gene prediction of 28 miRNAs revealed that 23 miRNAs predicted a total of 27,160 target gene transcripts?5,140genes?,and the other 5 miRNAs did not predict a common target gene.The target genes were primarily related to transcription,signal transduction,protein binding,and so on.The results of qRT-PCR showed that the expression of miR-1306-5p,miR-193b-5p,miR-4485-3p,and miR-877-5p was up-regulated,and the expression of miR-23b-5p and miR-663b was down-regulated,which was consistent with the sequencin.4.Compared with the control group,miR-663b mimics significantly inhibited the expression of IL-10,TNF-?,IFN-?,COX-2,and iNOS.And it reduced the inflammation of BeSO₄-induced 16HBE cells.The miR-663b inhibitor increased the expression of IL-10,TNF-?,IFN-?,COX-2,and iNOS of 16HBE cells.5.A total of 271 target genes of miR-663b were predicted,and 65common target genes were obtained by overlapping with mRNA sequencing.The 3'UTR of LIF may have the potential binding site of miR-663b,which is a potential target gene of miR-663b.LIF is primarily involved in the JAK-STAT signaling pathway.Conclusions:1.BeSO₄ reduces the viability of 16HBE cells and induces inflammation damage.2.BeSO₄ can up-regulate 88 miRNAs and down-regulate 91miRNAs in 16HBE cells..3.LIF is a potential target gene of miR-663b.Overexpression of miR-663b reduces the inflammation of BeSO₄-induced 16HBE cell.And down-regulation of miR-663b aggravated the inflammation of BeSO₄-induced 16HBE cell.