HIF-1?/ MiR-210 Targeted Regulation Of Homologous Recombinant Repair Factor Rad52 In Nano-Co-induced Cell Transformation

ObjectiveThis study aims to explore the potential carcinogenic effects and molecular mechanisms involved in long-term Nano-Co exposure and provide important information on the safety evaluation of Nano-Co exposure.Immortalized human bronchial epithelial cells BEAS-2B were exposed to low doses of Nano-Co,the effects of long-term Nano-Co exposure on hypoxia injury,DNA double strand break damage,genomic instability and dynamic expression of homologous recombination repair factor,RAD52 were evaluated.The role of HIF-1? / mi R-210 in targeting inhibition of Rad52 in Nano-Co-induced cell transformation was investigated.MethodsThe immunofluorescence staining was used to detect the formation of the DSBs,Western blot method was used to detect the expression of gamma-H2 AX.The effect of long term treatment with low dose Nano-Co on DSBs in BEAS-2B cells was observed.The cytokinesis-block micronucleus cytome assays was used to analyze the Nano-co's long-term effects on the stability of the BEAS-2B cell chromosomes.qPCR,Western blot method was used to detect Rad52 m RNA and protein expression of cells after 15 weeks of treatment with 0.05?g/m L and 0.1?g/m L Nano-co.qPCR,Western blot method was used to detect the expression of mi R-210 and HIF-1? protein.The activation of HIF-1?/mi R-210 pathway by long-term stimulation of low dose Nano-Co was analyzed.mi R-210 mimic and inhibitor plasmid transfection cells were used to regulate the expression level of mi R-210,q PCR and Western blot were used to analyze the expression of Rad52 m RNA and protein in each group.The dual-luciferase reporter gene detection system was used to detect the target relationship between mir-210 and Rad52.ResultsThe results of immunofluorescence staining showed that the level of ?-H2 AX in the experimental group was significantly higher than that in the control group.Western blot results showed that the level of ?-H2 AX in experimental group increased significantly(P < 0.05).The results of cytokinesis-block micronucleus cytome assays showed that the number of micronucleus in the experimental group was significantly higher than that in the control group(P < 0.05),nuclear bud and nuclear bridge are also seen.The results of q PCR,Western blot showed that the levels of Rad52 m RNA and protein in the experimental group that after 15 weeks of treatment with 0.05?g/m L and 0.1?g/m L Nano-Co were significantly lower than those in the control group(P < 0.05).The results of Western blot,q PCR showed that HIF-1? and mi R-210 levels of cells after 15 weeks of treatment with 0.05?g/m L and 0.1?g/m L Nano-Co were significantly higher than those of the control group(P<0.05).Transient transfection of mi R-210 mimics,inhibitor,NC plasmid to transformed cells,q PCR and Western blot were used to detect the m RNA and protein expression levels of Rad52,the results showed that mimics group was lower than the control and NC groups(P < 0.05).The inhibitor group was significantly higher than the control and NC groups(P < 0.05).Double luciferase reporter gene showed that mi R-210 inhibitor could up-regulate the activity of luciferase in the wild-type reporter gene vector compared with mi R-210 inhibitor NC(P < 0.05),while there was no significant change in no-load and mutant vector.ConclusionThe low-dose long-term exposure to Nano-Co can induce DSBs and the genomic instability.Long-term stimulation of Nano-Co can induce hypoxic injury and down-regulate the expression of Rad52.Long-term treatment of Nano-Co promotes the activation of HIF-1?/ mi R-210 pathway and the inhibition of Rad52 expression that is an important influencing factor of NanoCo-induced cell genomic instability.Our study further confirmed that long-term exposure to Nano-Co has potential carcinogenic effects.