The Role Of Rassf4 In Growth Inhibition Toxicity Induced By T-2 Toxin

Mycotoxins are highly diverse secondary metabolites produced in nature by a wide variety of fungus which causes food contamination,resulting in mycotoxicosis in animals and humans.Among them,T-2 toxin is trichothecenes mycotoxin with the widest pollution range and the greatest toxicity.T-2 toxin pollutes barley,wheat,corn,oats,cereals such as soybeans and their processed products,causing huge economic losses.Long term low-dose intake of T-2 toxin contaminated food by humans or animals can lead to vomiting,food refusal,gastric necrosis,intestinal injury,reproductive disorder,immune dysfunction and neurotoxicity,and young animals are more vulnerable to T-2 toxin than adult animals.Research shows that T-2 toxin can cause weight loss and growth retardation of dairy cows,goats,pigs,chickens,ducks,shrimps and crabs,which can significantly reduce feed utilization and feed return,and bring huge economic losses to animal husbandry.Exposure to T-2 toxin during pregnancy can also lead to neonatal dysplasia,which has a great impact on human health.Therefore,the pollution of T-2 toxin in food and feed is a worldwide problem,which will cause the health problem of blocked growth in humans and animals,hinder the development of animal husbandry and cause serious economic losses.Although researchers have found various purification technologies to reduce the concentration of T-2 toxin in feed and agricultural products,due to its extremely stable physical and chemical properties,it is impossible to degrade 100%;Moreover,T-2 toxin accumulates in animal tissues and can enter the pituitary through the blood-brain barrier.Therefore,finding effective drugs to antagonize the toxicity of growth disorders caused by T-2 toxin has become a food safety and urgent problem to be solved.With the continuous development of molecular biology technology,screening based on a combination of target and phenotype is the primary method of drug discovery in the pharmaceutical industry.Sufficient evidence shows that inflammatory response,apoptosis and growth hormone(GH)deficiency are important molecular mechanisms of T-2 toxin leading to animal and cell growth retardation.It was found that T-2 toxin significantly increased the expression of Ras association domain family member 4(RASSF4),and DNA methylation regulated the expression of RASSF4;At the same time,other studies have shown that RASSF4 is closely related to growth and apoptosis,but the role of RASSF4 in the growth inhibition caused by T-2 toxin is unclear;The upstream mechanism of methylation in the promoter region of RASSF4 gene is also unknown;It is worth exploring which transcription factors in the promoter region of RASSF4 can regulate RASSF4;It is also completely unclear whether targeting RASSF4 can antagonize the cytotoxicity of cell or animal growth retardation caused by T-2 toxin.Based on the above background and purpose,this study explores from the cellular level(GH3)and animal level.The following results were achieved:1.RASSF4 is a key target of growth inhibition induced by T-2 toxinWestern blot,flow cytometry and Ed U cell proliferation analysis showed that T-2 toxin significantly increased the expression of pro-inflammatory cytokines and pro-apoptotic proteins,and decreased the expression of anti-apoptotic proteins and GH,IGF-1 and IGFALS,thus inhibiting cell growth.RT-q PCR,Western blot and immunofluorescence showed that T-2 toxin promoted the gene and protein expression of RASSF4.The results of overexpression and si RNA interference with RASSF4 showed that RASSF4 exacerbated the inflammatory response,apoptosis and inhibition of cell growth caused by T-2 toxin by activating P38 and JNK signaling pathways and inhibiting PI3K-Akt-m TOR and ERK pathways;The silencing of RASSF4 can inhibit the activation of these death signal pathways by T-2 toxin,relieve the inhibition of T-2 toxin on cell survival signal pathway,reduce the degree of inflammatory reaction,apoptosis and S-phase arrest of cell cycle caused by T-2 toxin,and reverse the inhibition of cell growth caused by T-2 toxin.This fully proves that RASSF4 is the key target of growth arrest caused by T-2 toxin.2.DNA demethylation and transcription factor Sp1 mediate T-2 toxin-induced RASSF4 expressionThe results of colorimetry showed that exposure to T-2 toxin reduced the level of genomic 5-m C.Methylation specific-q PCR further showed that T-2 toxin reduced the methylation level of RASSF4 promoter;The results of bisulfite sequencing(BSP)showed that T-2 toxin reduced the methylation level of some Cp G sites in the Cp G island region;In vitro methylase induction combined with Dual-Luciferase Report Assay showed that hypermethylation of RASSF4 promoter inhibited its transcriptional activity and demethylation activated its transcriptional activity;In addition,DNA methylase inhibitor DAC or DAC combined with T-2 toxin further reduced the methylation level of RASSF4 promoter region.These results showed that T-2 toxin promoted RASSF4 expression through demethylation of RASSF4 promoter region.The results of chromatin immunoprecipitation(Ch IP)-q PCR showed that T-2 toxin increased the H3K9 ac modification level of TSSs region of RASSF4 gene;Histone deacetylase inhibitor TSA significantly promoted the protein expression of RASSF4,indicating that histone acetylation can also promote the expression of RASSF4.DAC combined with TSA significantly promoted the expression of RASSF4.This proves that DNA methylation and histone acetylation mediate T-2 toxin induced RASSF4 expression.This study also found that IL-6 significantly promoted the expression of DNMT1/3A,thereby reducing the expression of RASSF4;Dexamethasone,as a scavenger of IL-6,significantly reduced DNMT1/3A and promoted the expression of RASSF4,while overexpressed IL-11 had no effect on the expression of DNMT1/3A,indicating that IL-6 is one of the stimulating factors of RASSF4 methylation.Further,after si RNA interference with DNMT1/DNMT3 A and treatment with T-2 toxin or IL-6,it was found that only the silencing of DNMT1 significantly increased the expression of RASSF4;Compared with si NC+IL-6 group,si DNMT1+IL-6 increased the expression of RASSF4,indicating that IL-6-DNMT1 decreased the expression of RASSF4.IL-6 can significantly increase the methylation level of RASSF4 promoter region(-463~-348),while DNMT1 knockdown significantly reduces the methylation level of this region.These results show that IL-6-DNMT1 can reduce the expression of RASSF4 to a certain extent by increasing the methylation level of some RASSF4 promoter regions.Further predict the transcription factor of RASSF4 promoter region in PROMO and other databases,and select the transcription factor Sp1 with Cp G site at the binding site.Dual-Luciferase Report Assay with different truncates in RASSF4 promoter region and the eukaryotic expression vector of Sp1 were constructed,and then the core activation region of transcription factor was detected by Dual-Luciferase Report Assay.The results showed that the transcription activation region of RASSF4 promoter was located in-500~-1,and there were four binding sites between Sp1 and RASSF4 promoter region,three of which were located in this region.Ch IP and mutation tests showed that Sp1 bound to three GC boxes at the distal end of RASSF4 promoter region,and demethylation of these sites increased the binding activity of Sp1 to RASSF4.Ch IP-q PCR results showed that T-2 toxin could promote the binding of Sp1 to RASSF4 promoter region and improve the transcriptional activity of RASSF4.It was further found that T-2 toxin promoted the protein expression of transcription factor Sp1,and the test of overexpression and interference with Sp1 found that Sp1 could positively regulate the expression of RASSF4.Interestingly,IL-6 can significantly increase the expression of Sp1.This indicates that under the exposure of T-2 toxin,although IL-6-DNMT1 can reduce the expression of RASSF4 to some extent;However,IL-6 can promote the production of Sp1,significantly increase the expression of RASSF4,and finally increase the expression of RASSF4.Importantly,the silencing of transcription factor Sp1 makes it difficult for T-2 toxin to induce inflammation,apoptosis and cell cycle arrest,so as to relieve cell growth inhibition.3.Inhibitors of RASSF4 reduce the toxicity of growth retardation induced by T-2 toxinIn order to screen the inhibitors of RASSF4 protein to reduce the growth inhibitory toxicity of T-2 toxin,firstly,I-TASSER website modeled the RASSF4 protein and selected the crystal structure model with the highest score.SYBYL-X 2.0 software carries out molecular docking between RASSF4 crystal protein and the prepared small molecule database,and selects three compounds with high scores: AD-310 > AE-562 > AQ-086.The results of CCK8 showed that AD-310 and AE-562 compounds had low cytotoxicity to cells,AQ-086 had relatively high cytotoxicity to GH3 cells,and the three compounds could improve the viability of cells exposed to T-2 toxin.It was further found that AD-310 and AE-562 could specifically inhibit the expression of RASSF4 and reduce the RASSF4 induced by T-2 toxin exposure.At the cellular level,only AD-310 and AE-562 in a certain concentration range can alleviate the inflammatory response induced by T-2 toxin or LPS,reduce the apoptosis rate caused by T-2 toxin,and reverse the S-phase arrest of cell cycle caused by T-2 toxin,so as to increase the protein expression of GH/IGF growth axis and reduce the cell growth inhibition toxicity caused by T-2 toxin.Cellular thermal shift assay(CETSA)and Drug affinity responsive target stability(DARTS)showed that only AD-310 had binding potential with RASSF4 protein.The results of amino acid mutation showed that Gln136 and Arg139 may be the key sites of AD-310 binding to RASSF4 protein.AE-562 decreased the protein expression of Sp1 in a concentration dependent manner;DualLuciferase Report Assay and Ch IP-q PCR showed that AE-562 inhibited the enrichment level of Sp1 in RASSF4 promoter region,indicating that AE-562 inhibited RASSF4 expression at the transcriptional level.At the animal level,AD-310(6 mg/kg b.w.)can reduce the degree of pituitary injury in rats caused by T-2 toxin(2 mg/kg b.w.),reduce the high expression of RASSF4 in the pituitary induced by T-2 toxin,reduce the inflammatory and apoptotic toxicity of pituitary cells in rats caused by T-2 toxin,and increase the low level of GH in the pituitary induced by T-2 toxin,so as to alleviate the phenomenon of low food intake in rats caused by T-2 toxin and quickly recover the body weight of rats.Therefore,RASSF4 protein inhibitor AD-310 can effectively reduce the growth inhibition toxicity caused by T-2 toxin.In conclusion,this study first verified the importance of RASSF4 target,then explored the molecular mechanism of RASSF4 expression induced by T-2 toxin,and finally targeted RASSF4 to find an antidote to reduce the toxicity of animal growth retardation caused by T-2 toxin,which will provide important strategies and guarantee for human health and the development of animal husbandry.