The Role Of Aurora B In DNA Damage Response Under T-2 Toxin Treatment In HeLa Cells

T-2 toxin is a type A Trichothecenes that produced by Fusarium,which mainly contaminates food crops,feed materials such as grains,and seriously affects the health of livestock and human.It has been reported that T-2 toxin can induce DNA damage in many cells,but the molecular mechanism remains unclear.Histone modification-mediated changes in chromosomal spatial structure play an important role in DNA damage response.Aurora B is a serine/threonine kinase that is abundantly expressed and activated during cell division,and regulates chromatin structure by phosphorylating histones H3S10 and H3S28,thereby regulating chromosome segregation in the middle and late stages of cell division and cytoplasmic separation at the end of cell division.Wait for the process.The function and mechanism of Aurora B-mediated histone phosphorylation in DNA damage response has not been reported.In this study,Hela cells were used as model cells to study the function and mechanism of Aurora B in the process of DNA damage induced by T-2 toxin.The main results were presented as follows:1、T-2 toxin induces DNA damage in HeLa cellsIn order to clarify the DNA damage effect of T-2 toxin on Hela cells,the cytotoxicity of T-2 toxin on HeLa cells was firstly analyzed by MTT assay.The results showed that the toxicity of T-2 toxin on HeLa cells was dose-effect,and the median lethal concentration was 15.2 n M.HeLa cells were treated with 20 n M T-2 toxin,and the level of DNA damage was measured by different detection means.Those results showed that after treatment with T-2 toxin for 3 h in Hela cells,obvious comet tail appeared in comet assay;the level of DNA damage marker γH2AX was significantly increased;DNA damage repair response protein 53BP1(p53 binding protein1)gathered in the nucleus.The above experiments showed that T-2 toxin can significantly induce DNA damage in HeLa cells.2、T-2 toxin inhibits Aurora B expression,phosphorylation and phosphorylation of histone targetsTo study the DNA damage response mechanism under the action of T-2 toxin,HeLa cells were treated with 20 n M T-2 toxin for 15 min,30 min,1 h,3 h,6 h,and Western Blot was used to detect the changes of histone modification.It was found that H3S10 ph and H3S28 ph decreased significantly after 1 h of T-2 toxin treatment.Further analysis showed that the expression and phosphorylation of histone kinase Aurora B were also significantly reduced.Changes in Aurora B and H3S10 ph are often thought to correlate with changes in cell cycle.To determine whether the down-regulation of Aurora B under T-2 toxin treatment was related to cell cycle changes,cell cycle was detected by flow cytometry.The results showed that there was no significant change in HeLa cell cycle after treatment with T-2 toxin for 3 h and 6 h.Further,the cell cycle was synchronized to G1/S,G2/M,and M,and then treated with T-2 toxin to detect H3S10 ph.The results showed that T-2 toxin still significantly inhibited H3S10 ph after cell cycle synchronization.The above experiments showed that T-2 toxin significantly inhibited Aurora B expression,phosphorylation and phosphorylation of histone targets H3S10 and H3S28.3、Overexpression of Aurora B reduced DNA damage induced by T-2 toxinIn order to clarify the function of Aurora B in the process of DNA damage induced by T-2 toxin,the DNA damage level of HeLa cells after Aurora B overexpression was further analyzed under the exposure of T-2 toxin.The results showed that after overexpression of Aurora B,the level of γH2AX and DNA damage induced by T-2 toxin significantly decreased,indicating that overexpression of Aurora B can effectively reduce DNA damage induced by T-2 toxin in HeLa cells.4、Knockdown PP1 and PP2 A reduced DNA damage induced by T-2 toxinPP1(Protein serine/threonine phosphatase 1)or PP2A(Protein serine/threonine phosphatase 2A,protein phosphatase 2A)is phosphatase of Aurora B,and inhibits the activity of Aurora B by dephosphorylating Aurora B.Further,the method of si RNA was used to analyze the level of DNA damage of cells with decreased expression of PP1γ and PP2 A under T-2 toxin treatment.The results showed that si PP1γ and si PP2 A significantly increased the phosphorylation levels of Aurora B,H3S10 and H3S28,and attenuated the DNA tailing degree and γH2AX level under the exposure of T-2 toxin.In summary,this study first explored the molecular mechanism of T-2 toxin-induced DNA damage,and found that phosphorylation of Aurora B and its target sites H3S10 and H3S28 was significantly reduced by T-2 toxin,and T-2 Toxin-induced DNA damage is closely related.The relevant results have important theoretical significance for understanding the molecular toxicology of T-2 toxin.