LncRNA2676 Targets BUB1 Via MiR-221-3p To Regulate As₂O₃-Induced Chicken Liver Apoptosis

Arsenic and many of its compounds are powerful naturally occurring toxins and can be widely spread across the globe by polluting water,food and soil.It is not only used in medicine,but also in various fields such as agriculture,animal husbandry,electronics,industry and metallurgy.In addition,arsenic is the most effective hepatotoxic factor.The liver is an important organ for metabolism and the main site of arsenic methylation.Previous studies have shown that there is obvious apoptosis in chicken liver under the action of arsenic trioxide(As2O3),but the specific mechanism remains to be studied.Related research reports indicate that lncRNA can regulate miRNA and affect target gene to form a competitive endogenous RNA(ceRNA)regulatory network,which can cause changes in downstream pathways.Preliminary research in the laboratory proved the targeting relationship between lncRNA2676/miR-221-3p/BUB1.In this study,an As2O3-induced chicken liver poisoning model was established,and real-time fluorescence quantitative technology(q-PCR)detection showed that the expression of lncRNA2676 and BUB 1 increased,and the expression of miR-221-3p decreased.In addition,the application of pathological observation and antioxidant level detection(GSH,GSH-PX,H2O2,NO)to determine the liver toxicity of As2O3.Ultrastructural observation,q-PCR,western blot and other techniques were used to detect the occurrence of apoptosis and cell cycle arrest in the As2O3-induced liver poisoning model.In vitro,use chicken LMH cells to replicate the As2O3 poisoning model,and establish the knockdown model of lncRNA2676 and the knockdown/overexpression models of miR-221-3p and BUB1,and conduct rescue experiments to study the mechanism of lncRNA2676/miR-221/3p-BUB1 in arsenic-induced hepatotoxicity.The results show:1.Feed chickens with a diet containing 30 mg/kg of As2O3 for 90 days.Histopathological observations revealed that the experimental group’s liver was damaged.Electron microscopy observations showed severe liver apoptosis,accompanied by oxidative stress.Western blot and q-PCR experiments showed that the transcription and translation levels of pro-apoptotic genes were significantly up-regulated,and the level of G2/M marker genes increased significantly,which means the occurrence of apoptosis and cycle arrest.2.The MTT experiment determined that the half inhibitory concentration(IC50)was 23.24μM.At the same time,an increase in apoptosis level and G2/M phase block were detected,and the detection of reactive oxygen species(ROS)means that oxidative stress has occurred.It shows that the in vitro model of As2O3-induced liver apoptosis has been successfully established.perform rescue experiments.The results show that BUB1 promotes G2/M phase arrest and apoptosis under the action of arsenic.MiR-221-3p inhibits arsenic-induced apoptosis and is attenuated by the overexpression of BUB 1.The knockdown of lncRNA2676 inhibits arsenic-induced apoptosis,which is attenuated by the knockdown of miR-221-3p.In summary,As2O3 caused pathological damage of chicken liver tissue and induced oxidative stress.In addition,AS2O3 also increased the level of lncRNA2676,which in turn increased the expression of BUB 1 by inhibiting miR-221-3p,leading to cell G2/M phase arrest and eventually apoptosis.This study lays the foundation for the ceRNA network to regulate the mechanism of AS2O3 poisoning,and provides a theoretical basis for the prevention and control of poultry arsenic poisoning.