S100A8 Gene Overexpressed In HL-60 Cell Line Promoting The Cells’ Invasion And Mediating Their Resistance To Etoposide

More and more evidence has showed that chronic inflammation may increase the risk of cancer(Inflammation will induce tumor). In recent years, the mechanisms of inflammatory factors involved in tumorigenesis become the new hot spot. S100 protein family has a close relationship with malignant tumor. S100A8 belongs to the S100 protein family. In recent years, it has been found high expressed in many tumors such as gastric cancer, colon cancer, pancreatic cancer, bladder cancer, ovarian cancer, thyroid cancer, breast cancer and skin cancer, which indicated that S100A8 plays an important role in the process of tumorigenesis. Our previous study had revealed that patients with the high transcriptional level of S100A8 had a shorter survival time in adult AML. High expression level of S100A8 indicated a poor response in early phase of pediatric,AML receiving induction therapy. In this study, we constructed S100A8 vector and transfected into AML cell line, HL-60, and explored the effects of S100A8 on the biological characteristics of leukemic cells and chemodrug response.Part I. Construction of S100A8 overexpressed lentiviral vector and package of lentivirusObjective: To explore the function of S100A8 in HL-60 cells, we construct a S100A8 overexpressed lentiviral vector and subsequently package the lenitvirusMethods: Full length of S100A8 c DNA was amplified and cloned from BMMC cells, and then inserted into p MD19-T vector. The bacterial colony was sequenced. S100A8 fragment ligated into the lentiviral vector p LVX-IRES-puro and then verified the restriction enzyme reaction, bacterial colony by PCR and sequencing. To produce lentivirus, 293 T cells were co-transfected with the S100A8 overexpression vector and package plasmids by the method of calcium phosphate. Supernatant containg lentiviral particle was collected.Results: The S100A8 gene was successfully cloned and ligated into the lentiviral vector p LVX-IRES-puro. 293 T cells were co-transfected with the S100A8 overexpression vector and package plasmids, and then collected lentiviral particle.Conclusion: We successfully constructed the S100A8 overexpressed lentiviral vector and packaged the lenivirus as well.Part II. S100A8 gene promoting HL-60 cells’ invasionObjective: To explore the effect of S100A8 gene on leukemia cell line HL-60, following experiments such as proliferation, apoptosis, cell cycle, migration and invasion were performed.Methods. 1. The experimental groups: the empty vector control(p LVX- control) and target gene vector(p LVX- S100A8); 2. Proliferation was determined by CCK-8; 3. Cell cycles and apoptosis were analyzed by Flow cytometry; 4.The transwell assay were used to detect the effect of S100A8 on cell invasion and migration ability in vitro.Results: 1. Western blot and real time RT-PCR showed the expression levels of S100A8 is high in the p LVX-S100A8 which indicated that S100A8 has been transfected into HL-60 cells; 2. The invasive ability in S100A8 overexpressed HL-60 cells significantly increased compared with control group(0.701±0.058 vs 0.289±0.032, p<0.0001), but the migration, proliferation, cell cycle, and apoptosis didn’t show any differences between the two groups(P>0.05).Conclusion: Exogenous S100A8 promotes the HL- 60 invasion, but had no effect on cell proliferation, apoptosis and migration in vitro.Part III. S100A8 gene mediating HL-60 cells’ resistance to Etoposide Objective: To explore the potential effect of S100A8 on chemodrug resistance, we did the experiments of cytotoxicity and related mechanisms.Methods: 1.Chemodrugs like Etoposide(VP-16), Doxorubicin, Cytarabine, and Homo-harringtonine(HHT) were used at different doses to treat HL-60 experimental cells for 72 hrs. CCK8 was performed to detect the inhibition of proliferation; 2.Cell cycle arrest and apoptosis were measured by FCM after the cells exposed to VP-16 at the dose of 300 ng/ml for 72 hrs. Meanwhile, PCR Aarry analysis and Western Blot were employed to detect the apoptosis related genes.Results: 1. S100A8 overexpressed HL-60 cells showed resistance to VP16 compared with control(p<0.0001). However, No significant differences were observed among Doxorubicin, Cytarabine and HHT(P>0.05). 2. Cell cycle didn’t show any difference. S100A8 overexpressed HL-60 cells showed anti-apoptosis to VP16. Apoptosis PCR Array revealed 36 genes were significantly downregulated and 12 genes upregulated in HL-60 overexpressed S100A8 compared with control. Western blot analysis revealed that Caspase-3 and Bax expression levels decreased in HL-60 overexpressing S100A8.Conclusion: Overexpression of S100A8 mediated HL-60 cells’ resistance to VP16 which mediated by decreasing the expression of Bax and Casapse-3.