Functional And Mechanistic Investigation Of Circ＿0000745 In The Malignant Transformation Of Human Bronchial Epithelial Cells Induced By Benzo [a] Pyrene

Background and object Benzo [a] pyrene is the most representative environmental chemical pollutant of polycyclic aromatic hydrocarbons,which is not easily degraded due to its stable nature and can be absorbed by the human body through ingestion or inhalation and so on,thereby causing irreversible damage to human health.Airborne benzo [a] pyrene is mainly derived from combustion of industrial production,traffic exhaust,incineration of waste,cooking,and tobacco smoke,etc.toxicological and epidemiological studies have reported that benzo [a] pyrene causes adverse effects on the body’s respiratory,circulatory,and nervous systems,etc.As early as 2008,benzo [a] pyrene was classified as a class of carcinogens by the international agency for research on cancer,and acute exposure to benzo [a] pyrene can cause oxidative stress and cellular DNA damage,and long-term exposure leads to accumulation of gene mutations in cells,which ultimately activate the cancer process.There have been many studies on the association between benzo [a] pyrene exposure and lung cancer,but the molecular mechanisms involved remain to be further investigated.Among epigenetic regulation,the regulatory role of non coding RNAs（nc RNAs）is the current research hotspot.Among them,circrnas are a class of non coding RNAs formed by back splicing without 5 ’cap and 3’ tail in a closed circular structure,which are stably expressed in eukaryotes.In recent years,a large number of studies have shown that circrnas play important regulatory roles in the exposure of exogenous chemicals.Benzo [a] pyrene can induce carcinogenesis in cells or animals,and previous studies have also shown that circrnas are aberrantly expressed in tumor cells,but current studies on the regulation of the malignant transformation process of benzo [a] pyrene have rarely reported.This study explored the function of circrnas in the malignant transformation of human bronchial epithelial cells caused by benzo [a] pyrene by detecting aberrantly expressed circrnas in malignant transformed cells,and further enriched the mechanism of benzo [a] pyrene carcinogenesis by analyzing the mechanism of action of circrnas.MethodsThe differentially expressed circrnas and m RNAs in 16HBE-T cells were obtained by high-throughput sequencing of circrnas,and the circrnas screened were validated by QRT PCR.After treatment with added rnaser,QRT PCR was performed to verify the circular nature of circrnas.QRT PCR products were examined by agarose gel electrophoresis experiments to further determine the tolerance of circrnas to rnaser.QRT PCR was utilized to detect the expression of circrnas to determine their subcellular localization by separating cytoplasmic RNA and nuclear RNA.The functions of circrnas in cells were investigated by constructing knockdown / overexpression cell lines of circrnas,detecting the proliferation ability of cells by CCK-8 and edu experiments,detecting the migration ability of cells by scratch assay,and detecting the malignant proliferation ability of malignant transformed cells by plate clonogenic assay.MiRNAs potentially bound to circrnas were analyzed by bioinformatics prediction and validated by QRT PCR.In addition,miRNAs were reversely predicted by analyzing the expression levels of m RNAs,forming a circrna miRNA m RNA network regulatory mechanism.Results1.Aberrant circrna expression in benzo [a] pyrene malignant transformed cellsWhole transcriptome sequencing results showed that 17484 circrnas were aberrantly expressed in benzo [a] pyrene malignant transformed cells compared with control cells,among which 8700 circrnas were downregulated and 8784 circrnas were upregulated in 16HBE-T cells.The expression levels of 8 circrnas were found to be consistent with the sequencing results by screening out 10 circrnas for QRT PCR validation,and the sequencing data were basically credible this time.2.circ₀000745 underexpression in benzo [a] pyrene induced malignant transformation 16HBE-T cellsAfter validation by multiple QRT PCR,we found that circ₀000745 was downregulated in 16HBE-T cells.Circrnas were also observed in non malignantly transformed cells chronically infected with benzo [a] pyrene for 30 passages_Downregulation of 0000745.Circrnas were detected by examining their expression in cancer cells₀000745 was down regulated in A549,H460 and H1299 lung cancer cells,we further found by acute exposure to benzo [a] pyrene that circ₀000745 presented low expression throughout the course of malignant transformation of 16 HBE cells by benzo [a] pyrene exposure.3.circ₀000745 tolerates linear enzymatic treatmentBy adding rnaser to the extracted RNA,we found that circ₀000745 is tolerant to rnaser treatment,whereas the linear gene actin and its parental genes are intolerant to rnaser treatment.Agarose gel electrophoresis demonstrated that products amplified from the circrna reverse primer were tolerant to rnaser treatment and could no longer be amplified in genomic DNA.By Sanger of the amplified products,we determined the splice sites of circrnas and confirmed by nucleocytoplasmic fractionation experiments that circrnas are predominantly expressed in the cytoplasm.4.circ₀000745 inhibits cell proliferation and migrationTo investigate the regulatory function of circ₀000745 in benzo [a] pyrene induced malignant transformation of 16 HBE cells,by knocking down the expression level of circ₀000745 in 16HBE-T cells,it was found that compared to the NC control group,the siRNA knockdown group showed significantly higher cell viability and proliferation ability of 16HBE-T cells,and similar results were also observed in A549 and H460 cells.By performing acute exposure of benzo [a] pyrene following knockdown and overexpression circ₀000745 in 16 HBE,we found that the cell viability of 16 HBE cells in the siRNA knockdown group was higher than that in the control group,while the cell viability of the OE overexpression group was lower than that in the control group.Subsequently,we found by scratch assay that the migration ability of 16HBE-T cells was elevated after circ₀000745 knockdown,consistent with the interference efficiency of circrnas.In addition,the results of plate let clonogenic assay showed that knockdown circ₀000745 could increase the malignant proliferation ability of 16HBE-T cells.5.circ₀000745 may be involved in the malignant transformation of cells by binding miRNAs to regulate the expression of GSTO2,dlg4 and pik3cdThe basic characteristics of circ₀000745 suggested that it might function as a Cerna,and we predicted circ₀000745 potentially binding miRNAs through three websites: circbank,circinteractome and encori.The predicted 262 miRNAs were intersected,resulting in 21 miRNAs,from which seven miRNAs were randomly selected for validation,and the results showed that all seven were downregulated in 16HBE-T cells.By bioinformatics analysis of the m RNA differential expression results,we found that the differentially expressed genes were mostly enriched in cancer pathways.We picked out 3 genes from them,predicted their possible binding miRNAs,and constructed circ₀000745 network analysis diagram that function as cernas.circ₀000745 may be involved in the overexpression of benzo [a] pyrene induced malignant transformation of 16 HBE cells by modulating the expression of miRNA target genes GSTO2,dlg4 and pik3 cd through competitive binding with miRNAsConclusions1.circ₀000745 in benzo [a] pyrene induced malignant transformation 16HBE-T cells low table.2.circ₀000745 inhibits proliferation and migration of malignantly transformed 16HBE-T cells.3.circ₀000745 may regulate GSTO2,dlg4 and pik3 cd gene expression through a ce RNA mechanism in combination with miRNAs.