The Biological Function And Mechanism Of Tumor Necrosis Factor ? In K562 Cells

Objective Despite the success of tyrosine kinase inhibitors(TKIs) targeting the oncokinase of chronic myelogenous leukemia(CML), there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response, TKI resistant in some patients and so on. Thus highlighting the need to identify novel therapeutic targets. Tumor necrosis factor alpha(TNF?) plays an important role in the occurrence and development of tumor, which has been shown to support CML stem and progenitor cell survival and enhance their proliferation. We establish the TNF?knock-out CML cell model with completely controllable genetic background using genome editing tool CRISPR-cas9. We also assess the biological effect caused by targeted knock-out of TNF? and explore the underlying mechanism.Methods In this study, appropriate target site and multiple CRISPRs were designed against human TNF? gene.The CRISPR/Cas9 plasmid was transfected into K562. The mutant clones were selected and defined by isogenic flow sorting and sanger sequencing analysis.Protein level and m RNA level of the initial mutated clones were detected by RT-PCR and Western-blot. The cell survival,proliferation in vitro and in vivo and microvesicles were compared between the initial mutated clones and the wild-type cells. We explore the underlying mechanism using RNA sequence.Results We designed multiple CRISPR/Cas9 genome editing tools and obtained more than 30 TNF? knock-out K562 clones. No off-target effect was detected by extensive PCR and sequencing results. Protein level and m RNA level of TNF? of the initial mutated clones have an significant decrease compared to the wild-type cells. RT-PCR analysis assay give a negative result that this assay has an effect on BCR/ABL gene.Mutated clones showed higher apoptosis rate than wild-type cells when cells incubated with Imatinib. The proliferation capacity and colony-forming capacity of the mutant clones was consistently reduced than those of the corresponding wild-type cells. There were no difference in the quantity and quality of the microvesicles between the mutant clones and the wild-type cells. The STAT5 A and Cancer promoting miRNA down regulated of the mutant clones.Conclusions We found that the CRISPR/Cas9 knock-out model is more simple than models using other gene editing tools. The results are more reliable, stable and comprehensive. TNF? supports CML cells survival and enhances their proliferation.The above effects may be achieved by changing the STAT5 A expression and related miRNA profiles.