Study On The Mechanism Of Carcinogenesis Of CircSP3 Expression In Inorganic Arsenic

Objective(s):Taking A549(human lung adenocarcinoma cell line)as an example,this study investigated the effects of inorganic arsenic and its methylated main metabolites on the expression of circSP3(circ＿0002642).The role of circSP3 in the process of inorganic arsenic-induced carcinogenesis and its possible molecular mechanisms were explored,providing new ideas for the study of the carcinogenic mechanism of arsenic and preliminary exploration of the potential of circSP3 as a biomarker.Methods:In A549 cells,the total RNA was extracted after treating the cells with different concentrations of sodium arsenite(20,40,60 μmol/L)and methylated arsenic metabolites,including monomethyl arsenic acid(60 μmol/L)and dimethylarsenic acid(60 μmol/L),with a control group set.The relative expression of circSP3 was detected by q RT-PCR experiment.Circ SP3 has interfered specifically with small RNA interference fragments,and CCK-8 assay was used to detect cell viability.Hoechst33342/PI staining and JC-1 mitochondrial membrane potential assay were used to detect the effect of circSP3 with the low expression on cell apoptosis.Western blot experiment was used to further analyze the effects of low expression circSP3 on apoptotic-related proteins(Bcl-2,Bax,Cytc,caspase7,PARP1).The cells were treated with 40μmol/L sodium arsenite and the silenced fragment of circSP3.The changes in cell viability were detected by CCK-8 assay.Hoechst33342/PI staining and JC-1mitochondrial membrane potential test were used to detect cell apoptosis,and the role of circSP3 in inorganic arsenic carcinogenesis was preliminarily discussed.The western blot experiment was used to detect changes in the expression of p65 pathway-related proteins after the downregulation of circSP3.RNA immunoprecipitation(RIP)experiment confirmed the binding of circSP3 with p65 and IκBα,and a protein immunoprecipitation(Co-IP)experiment was performed to detect changes in the binding ability of p65 and IκBα after silencing circSP3.Finally,the binding of circSP3 with p65 and IκBα was detected by RIP experiment after treatment with sodium arsenite(40 μmol/L).Results:1.The effects of inorganic arsenic and its methylated metabolites on circSP3expression: Compared with the control group,different concentrations of sodium arsenite(20,40,60 μmol/L)significantly promoted circSP3 expression,while the expression of circSP3 showed no significant change compared with the control group after exposure to monomethylarsonic acid or dimethylarsinic acid.2.Apoptosis assay of cells after circSP3 knockdown: Specifically knockdown the expression of circSP3 in A549 cells,CCK-8 experiment showed that the cell viability of the silenced circSP3 group(si-circSP3 group)was significantly decreased compared with the control group.The results of JC-1 showed that the mitochondrial membrane potential of the si-circSP3 group was significantly lower than that of the control group.Hoechst33342/PI staining showed obvious apoptosis in the si-circSP3 group.Western blot analysis further confirmed the occurrence of cell apoptosis,showing that the protein expression of Bcl-2 in the si-circSP3 group was significantly decreased,while the protein expression levels of Bax,Cytc,caspase7,Cleaved caspase7,and Cleaved PARP1 were significantly increased compared with the control group.3.The effects of the combined action of sodium arsenite and si-circSP3 on cell apoptosis: after adding 40μmol/L sodium arsenite,the CCK-8 experiment showed that the cell viability decreased more obviously.Both JC-1 and Hoechst33342/PI staining showed that apoptosis was more significant in the si-circSP3 group.4.Mechanism research of the p65 signaling pathway: After knocking down circSP3,Western blot analysis was used to detect changes in p65 and its downstream related proteins.The results showed that the expression of p65,c-IAP1,XIAP,Bclx,and p21 proteins in the si-circSP3 group was significantly decreased,while the phosphorylation of IκBα and its phosphorylation sites S32,S36,and p65 phosphorylation site S536 was also significantly decreased,and p65 ubiquitination was significantly increased.5.The binding of circSP3 with p65 and IκBα: The RIP experiment showed that circSP3 was able to bind with p65 and IκBα proteins.The Co-IP experiment showed that knocking down circSP3 could change the binding of p65 and IκBα proteins,significantly decreasing their binding.6.Changes in the binding of circSP3 with p65 and IκBα after exposure to sodium arsenite: In the RIP experiment conducted with A549 cells exposed to 40 μmol/L sodium arsenite,the results showed that the binding of circSP3 with p65 and IκBα was significantly reduced.Conclusion(s):1.Sodium arsenite promotes the expression of circSP3,while monomethylarsonic acid and dimethylarsinic acid do not cause any changes in the expression of circSP3.There are significant differences in the effects of inorganic arsenic and its methylated metabolites on circSP3 expression.2.Low expression of circSP3 can significantly inhibit the activity of A549 cells,promote cell apoptosis significantly,and cell apoptosis is related to the mitochondrial apoptosis pathway.3.The presence of inorganic arsenic can enhance the effect of low expression of circSP3 on cell apoptosis.4.circSP3 silencing can significantly inhibit the p65 signaling pathway and is closely related to the phosphorylation and ubiquitination of p65.5.circSP3 can interact with p65 and IκBα,and the low expression of circSP3 can inhibit the binding of p65 and IκBα.6.The induction of circSP3 expression by sodium arsenite may be related to its decrease in binding of circSP3 with p65 and IκBα.