Aged ZnO NPS Enhance Malignant Transformation And Tumorigenicity Of Mefs With Mutant SHP-2 And Its Molecular Mechanism

[Background]Tumor is one of the most serious diseases that threaten human health in the world.In recent years,not only morbidity and mortality of tumor are increasing year by year,but it also shows a trend of early onset.As we all know,the occurrence of cancer is a complex multi-factor process,influenced by environment and genetic factors.On one hand,long-term exposure to environmental pollutants may lead to genetic mutations that trigger tumors.On the other hand,individuals who have already had genetic mutations are more likely to cause tumors after they are stimulated by environmental pollutants.Our laboratory has long been committed to the mechanism of tumorigenesis and development of SHP-2 mutation and environmental pollutants.SHP-2?Src homology 2-containing protein tyrosine phosphatase?is a non-receptor type tyrosine phosphatase encoded by the PTPN11 gene in various tissues of mammalian cells,which is closely related to the proliferation,invasion and metastasis of tumor.SHP-2 mutation was found in Noonan syndrome,various blood diseases and many solid tumors,and it plays a very important role in tumorigenesis.In the present study,the most common mutant of SHP-2 is D61G/D61Y,which is also an activating mutant in this study.Previous studies in the laboratory found that heavy metal pollutants can synergize the SHP-2 mutant to promote the malignant transformation of mouse embryonic fibroblast?MEF?cells and increase the tumorigenic ability of MEF cells in vivo,mutant SHP-2plays a positive role in this process.ZnO NPs?Zinc oxide nanoparticles?is a type of nano-metal oxide widely used in life.It is precisely because of its wide application,resulting in its release in the environment increased year by year.Studies have found that with the passage of time,nano-zinc oxide will be subject to various factors in the environment changes in the physical and chemical properties,from the original state into the aging state,and the aging state of zinc oxide has a higher genotoxicity and lower cytotoxicity.Therefore,we speculate whether ZnO NPs with special physicochemical properties can synergize the SHP-2 mutation to promote the malignant transformation of MEF cells.According to the assumption,we use the original state and the aging state of ZnO NPs to treat MEF(SHP-2^+/+)and MEF(SHP-2^D61G/+)cells?hereinafter,WT and D⁺?,and observe cells biological behavior of MEFs and the tumorigenesis in nude mice,and study the role of aging zinc oxide and SHP-2 mutation in this process.[Methods]1 Long-term treatment of WT and D⁺cells with 0.75?g/ml and 1.5?g/ml original and aged ZnO NPs to build the transformed cell models.2 The proliferation ability of the cells was detected by the clone formation experiment;the non-anchored growth ability was detected by soft agar colony formation assay;the invasion and migration ability were detected by Transwell assay;the tumorigenicity was detected by nude mouse tumorigenesis assay in vivo.3 Flow cytometry was used to detect the change of intracellular ROS levels in MEFs after exposure of ZnO NPs.4 ICP-MS?Inductively coupled plasma mass spectrometry?experiment to detect the level of intracellular zinc element after treatment with NPs.5 The expression of?-H2AX,p-erk and erk were detected by Western blotting after treatment with 10?g/ml ZnO NPs.6 WT and D⁺cells were treated with MEK inhibitor U0126.Western blotting was used to detect whether p-erk expression was inhibited.Then it was tested whether the malignant transformation of cells induced by ZnO NPs was inhibited after p-erk inhibition.[Result]1 Obtained the cell model with original and aged ZnO NPs induced.2 Experimental results showed that the biological behavior of cells after induction such as proliferation ability,non-anchored growth ability,invasion and migration ability,nude mice tumorigenicity were significantly higher than the cells of control group,and the aging state of zinc oxide has a stronger effect,activated SHP-2 mutant play a positive regulatory role in this process.3 Flow cytometry results showed that intracellular ROS levels were significantly increased after ZnO NPs acute and chronic treatment.4 ICP-MS experiments found that the level of intracellular zinc element with longer induction was higher,and the level of zinc element in cells treated with aging zinc oxide was higher.5 After acute treatment of ZnO NPs,the expression levels of?-H2AX and p-erk were significantly increased.6 U0126 treatments significantly inhibited the expression of p-erk,and the malignant transformation of cells induced by ZnO NPs was significantly inhibited after U0126 treatment.[Conclusion]1 Long-term treatment of low dose ZnO NPs can promote the malignant transformation of MEFs and the tumorigenesis of nude mice.The aging zinc oxide is stronger than the original zinc oxide,and the SHP-2 mutation plays a positive regulatory role in this process.2 In the process of malignant transformation of MEF cells,there are ROS production and the activation of ERK signaling pathway.