Study On The Role Of MTHFR And NQO1 Gene Mutations In Myeloproliferative Neoplasms

Objective: JAK2V617 F mutation is one of the main driver gene mutations of myeloproliferative neoplasms(MPN).Next-generation sequencing(NGS)detected non-driver gene mutations in MPN.Our group performed second-generation sequencing mutations on 23 patients with triple-negative MPN in the early stage.It is found that high-frequency single-nucleotide mutations in the MTHFR and NQO1genes: NQO1 C559 T,MTHFR C665 T,whether they are related to the occurrence and development of MPN is still unclear.This study aims to study the effects of MTHFR and NQO1 gene mutations in MPN and their effects on the proliferation,apoptosis,cycle and enzyme activity of JAK2V617 F positive HEL cells.Methods:1.Lentiviral infection technology was used to overexpress wild-type MTHFR gene and mutant MTHFR,wild-type NQO1 gene and mutant NQO1 gene on HEL cell lines,respectively,and divided into HEL/NC group,HEL/WT-MTHFR group and HEL/MUT-MTHFR Group and HEL/NC group,HEL/WT-NQO1 group and HEL/MUT-NQO1 group;2.The CCK-8 method was used to detect the proliferation changes of overexpressing wild-type and mutant MTHFR and wild-type and mutant NQO1 on HEL cells;3.Flow cytometry was used to detect the apoptosis and cycle of HEL cells overexpressing wild-type and mutant MTHFR and wild-type and mutant NQO14.ELISA method was used to detect the activity of MTHFR and NQO1 in HEL cells by overexpression of wild-type and mutant MTHFR and wild-type and mutant NQO1;5.RT-PCR detected the effect of overexpression of wild-type and mutant MTHFR and wild-type and mutant NQO1 on the expression of JAK2 in HEL cells.Results:1.Compared with the control group,the HEL/WT-MTHFR group promoted the proliferation of HEL cells,and the HEL/MUT-MTHFR group inhibited the proliferation of HEL cells,and the difference were statistically significant(P<0.05);compared with the control group,the HEL/WT-NQO1 group inhibited The proliferation of HEL cells,the HEL/MUT-NQO1 group promoted the proliferation of HEL cells,and the difference were statistically significant(P<0.05).2.Compared with the control group,the HEL/MUT-MTHFR group promoted HEL cell apoptosis,and the difference were statistically significant(P<0.05),and the HEL/WT-MTHFR group did not promote HEL cell apoptosis,and the difference were not statistically significant(P>0.05));Compared with the control group,neither HEL/WT-NQO1 nor HEL/MUT-MTHFR promoted HEL cell apoptosis.3.Compared with the control group,the MTHFR enzyme activity in the HEL cells of HEL/WT-MTHFR and HEL/MUT-MTHFR increased;compared with the control group,the NQO1 enzyme activity in the HEL cells of the HEL/WT-NQO1 group did not change significantly.The NQO1 enzyme activity in HEL cells in the HEL/MUT-NQO1 group decreased,and the difference were statistically significant(P<0.05).4.Compared with the control group,there was no significant difference in JAK2 expression in HEL cells between the HEL/WT-MTHFR group and the HEL/MUT-MTHFR group;compared with the control group,the JAK2 expression in the HEL/WT-NQO1 group and the HEL/MUT-NQO1 group There is no significant difference.Conclusion:1.Compared with control cells,HEL/MUT-MTHFR can inhibit the proliferation of HEL cells and increase the activity of MTHFR enzyme;compared with control cells,HEL/WT-MTHFR can promote the proliferation of HEL cells.2.Compared with control cells,HEL/MUT-NQO1 can promote the proliferation of HEL cells and decrease the enzyme activity of NQO1;compared with control cells,HEL/WT-NQO1 can inhibit the proliferation of HEL cells.