Study On The Mechanism Of Inorganic Arsenic Mediating Cell Apoptosis By Regulating DHX9

Objective（s）:Based on the statistical data of the population exposed to occupational arsenic,human lung adenocarcinoma cell A549 and human bronchial epithelial cell 16HBE were selected as experimental cells in this study to explore the effects of inorganic arsenic and its main metabolites on the expression of DHX9 gene in vitro and in vivo,as well as the specific molecular mechanism of DHX9’s involvement in apoptosis regulation,so as to provide more scientific reference for the explanation of the carcinogenic mechanism of arsenic.Methods:1.Population:Blood and urine samples of occupational arsenic exposed people and control group were collected.Urinary arsenic level was detected to reflect occupational exposure level,and Q-PCR was used to detect DHX9 gene relative expression level in blood samples.2.Cells:A549 and 16HBE cells were selected as experimental cells.A549 and16HBE cells were treated with 0,20,40,60μM and 0,1.5,3,4.5μM sodium arsenite（NaAsO₂）,respectively.Total protein and totalRNA were extracted.Quantitative real-time PCR（Q-PCR）and Western blot were used to detect the relative expression levels of DHX9 mRNA and protein.At the same time,A549（60μM）and 16HBE（4.5μM）cells were infected with monomethyl Arsonic acid（MMA）and dimethyl Arsonic acid（DMA）,respectively,and the relative expression levels of DHX9 mRNA and protein were also detected.A549 and 16HBE cells infected with 40μM NaAsO₂and 3μM NaAsO₂were collected for protein co-precipitation（CO-IP）to detect the changes of DHX9 binding ability to MDM2 protein.After siRNA interfered with DHX9,CCK-8 was used to detect cell viability,JC-1 fluorescent probe was used to detect mitochondrial membrane potential,and Hochest33342/PI fluorescence double-staining experiment showed early and late cell apoptosis patterns.In addition,total proteins were extracted from cells,and the relative expression levels of P53pathway proteins P53,PUMA,MDM2,BAX,BIK,Cyt C and P21;the relative expression levels of NF-κB pathway protein P65,IKBα,C-MYC,E2F1,CFLAR,Bcl-2,cyclin D1 and Bcl-X proteins;the relative expression levels of apoptosis effector proteins Caspase3 and 7;P53 protein ubiquitination level were detected by Western blot.After siRNA was used to interfere with DHX9,totalRNA was extracted from cells and the relative expression levels of circRNAs hsa＿circ＿0027479,hsa＿circ＿0003599,hsa＿circ＿0027478 and hsa＿circ＿0027477 were detected by Q-PCR.RNA binding protein co-precipitation（RIP）was used to detect whether DHX9 was bound to P65,P53,P21,PARP1 and Bcl-X mRNA,and CO-IP was used to detect whether DHX9 was bound to the proteins translated by the above genes.A549 and 16HBE cells infected with 40μM NaAsO₂and 3μM NaAsO₂were collected for CO-IP and RIP,and the ability of DHX9 to bind to the mRNA and protein of the above target genes was detected by inorganic arsenic exposure.Results:1.Population statistics:The contents of inorganic arsenic（iAs）,MMA and DMA increased in the urine of people exposed to occupational arsenic,while the relative expression level of DHX9 mRNA decreased.The relative expression level of DHX9mRNA was negatively correlated with the contents of iAs,MMA,DMA and MMA%,and positively correlated with DMA%and secondary methylation index（SMI）,but was not correlated with iAs%and primary methylation index（PMI）.2.DHX9 mRNA and protein relative expression levels after exposure to inorganic arsenic and its metabolites in vitro:in A549 and 16HBE cells,DHX9mRNA relative expression level decreased and protein relative expression level increased after exposure to NaAsO₂.The CO-IP experiment showed that the binding ability of DHX9 protein to MDM2 protein was weakened after NaAsO₂exposure.The relative expression levels of DHX9 mRNA and protein in A549 and 16HBE cells were not affected by MMA and DMA exposure.3.Exploration of downstream cell pathway:The results of cell viability assay,mitochondrial membrane potential assay and Hochest33342/PI fluorescence double-staining experiment show that cell vitality and mitochondrial membrane potential decreased,but early apoptotic cells and late apoptotic cells increased in DHX9 siRNA interference group.Western blot results showed that in DHX9 siRNA interference group,the expression of Caspase3 and 7 protein increased,the expression of P53 pathway proteins P53,PUMA,BAX,BIK and Cyt C increased,the expression of P21 and MDM2 protein decreased,and the ubiquitination level of P53 protein detuned.The expressions of NF-κB pathway proteins P65,IKBα,C-MYC,E2F1,CFLAR,Bcl-2 and cyclin D1 were decreased,while the expressions of Bcl-Xs were increased.4.Exploration of upstream cell pathway:Compared with the control group,RIP results showed that DHX9 protein was bound to P65,P53,P21,PARP1 and Bcl-X mRNA,and the difference was statistically significant（P<0.05）;Compared with the control group,the CO-IP results showed that DHX9 protein was bound to protein P65,P53 and PARP1.5.Gene regulation induced by inorganic arsenic:A549 and 16HBE cells infected with 40μM NaAsO₂and 3μM NaAsO₂were collected for CO-IP and RIP.RIP results showed that arsenic exposure resulted in increased binding of DHX9 protein to P65mRNA in both A549 and 16HBE cells.Arsenic exposure resulted in reduced DHX9protein binding to P53 mRNA in A549 cells and increased DHX9 protein binding to P53 mRNA in 16HBE cells.In addition,in both A549 and 16HBE cells,arsenic exposure resulted in decreased binding of DHX9 protein to the mRNA of P21,PARP1,and Bcl-X genes downstream of the pathway.CO-IP assay showed that the interaction between DHX9 protein and P65 protein was enhanced in both A549 and 16HBE cells due to arsenic exposure.Arsenic exposure resulted in decreased binding of DHX9protein to P53 protein in A549 cells and increased binding of P53 protein in 16HBE.6.DHX9 regulates circRNAs:After siRNA was used to interfere with DHX9,the relative expression levels of several circRNAs were up-regulated.Among them,the relative expression levels of four circRNAs,hsa＿circ＿0027479,hsa＿circ＿0003599,hsa＿circ＿0027478 and hsa＿circ＿0027477 were significantly increased compared with the control group（P<0.05）.Conclusion（s）:1.The relative expression level of DHX9 mRNA in vivo and in vitro was down-regulated under arsenic exposure.In vivo,it was correlated with iAs,MMA and DMA,but in vitro MMA and DMA had no significant effect on the relative expression level of DHX9.The up-regulated relative expression of DHX9 protein in vitro is due to the decreased ubiquitination degradation of DHX9 protein dependent on MDM2.2.Functional mechanism of DHX9:two regulatory axes are DHX9/P65/Bcl-X and DHX9/P53/P21.One plays the role ofRNA helicase to bind P53,P65 mRNA and its downstream target genes P21,PARP1 and Bcl-X mRNA to promote gene translation.The other is to directly bind to P53,P65 proteins and downstream protein PARP1 to feedback and regulate the protein accumulation brought by their translation promotion.3.Mechanism of apoptosis induced by siRNA interference with DHX9:After siRNA interference with DHX9,the level of DHX9 protein is down-regulated.In the first,P65 mRNA translation was weakened,P65 protein was down-regulated,and downstream proteins such as IKBαand C-MYC were decreased,and NF-κB pathway was inhibited.Secondly,although the promotion of P53 mRNA translation is weakened,the ubiquitination level of P53 protein is maladjusted,and eventually the P53 protein is up-regulated,resulting in the increased expression of downstream proteins such as PUMA,BAX and BIK,and the activation of apoptosis pathway.The synergistic effect of NF-κB pathway and P53 pathway mediates apoptosis.4.DHX9 regulatory response under inorganic arsenic induction:NaAsO₂exposure enhances DHX9 protein’s promotion of P65 mRNA translation and leads to P65 protein accumulation.The interaction between DHX9 protein and P53 mRNA under inorganic arsenic induction was down-regulated in A549 cells and up-regulated in 16HBE cells,and the DHX9/P53 regulatory axis was differentially played between normal cells and cancer cells.