| Objective Leukemia is a malignant disease of abnormal cloned hematopoietic stem cell, the incidence rate accounted for sixth of all malignant tumors in China. LukS-PV, a component of Panton-Valentine leukocidin (PVL), is a pore-forming cytotoxin secreted by Staphylococcus aureus. In previous studies, we reported that LukS-PV induced acute myeloid leukemia cell lines HL-60 and THP-1 differentiation in vitro, but the molecular mechanism remains unclear. This paper discovers the expression changes of microRNA (miRNA) in LukS-PV stimulating THP-1. Then we further explores the influences of differentiation effect induced by LukS-PV when over-expression and inhibit-expression of miR-125a-3p in THP-1 cells, which may provide theoretical support for the anticancer approach of LukS-PV.Methods (1) THP-1 cells were treated with 1.00 μM of LukS-PV for 48h. The miRNAs involved in the anti-leukemia effect of LukS-PV were screened by miRNA PCR gene chip. Results of miRNA gene chip were verificated using real-time fluorescence quantitative polymerase chain reaction (quantitative real-time polymerase chain reaction, qRT-PCR).(2) Lentiviral vector of over-expression, inhibit-expression and non-carrier of miR-125a-3p were transfected into THP-1 cells. Then THP-1 cells screened by puromycin and divided into following four groups for further trials. ① miR-125a-3p NC ② over-expression miR-125a-3p ③ 1.00 μM LukS-PV+miR-125a-3p NC ④ 1.00 μM LukS-PV+inhibit-expression miR-125a-3pCell surface antigens CD 11b and CD 14 were detected by flow cytometry. mRNA levels of differentiation related genes were detected by qRT-PCR and the changes in protein expression were using Western blot. By the above experiments, we discovered the effects of miR-125a-3p in LukS-PV stimulated THP-1 process.(2) The bioinformatics technology was used to predicted and screened the target gene of miR-125a-3p. The plasmid of miR-125a-3p, wild type of target gene mRNA 3’ UTR and mutation type of target gene mRNA 3’UTR were constracted and tansfected into cells. After 48h, cells were lysis and determinated the ration of Firefly Luciferase and Renilla Luciferase to confirmed that miR-125a-3p combined with target gene’s mRNA 3’UTR.Results (1) After preliminary screening of the miRNA gene chip, we found that there are 161 miRNAs expression increased,35 miRNAs expression decreased, one of the most significant miR-125a-3p (102.5 up-fold) compared with the control group when 1.00 μM LukS-PV given stimulated THP-1 cells for 48 hours. In order to validated miRNA gene chip results, miR-125a-3p, miR-181a-3p, miR-761 expression changes were detected using qRT-PCR after 1.00 μM LukS-PV given stimulated cells 48 h. The results show miR-125a-3p (56.2+7.2) up-fold, miR-181a-3p (22.3+3.4) up-fold, miR-761 (9.2+1.3) down-fold (P< 0.05), the change trend was consistent with the microarray results.(2) over-expression vector and inhibit-expression of miR-125a-3p were transfected into THP-1, the expression level of miR-125a-3p were determined using qRT-PCR method. Compared with the control group, over-expression miR-125a-3p increased (5.94+1.4) fold (P< 0.05), inhibit-expression miR-125a-3p decreased (2.61+0.5) fold (P< 0.05). There were no significant differences between the control group and the empty vector miR-125a-3p. The above results showed that miR-125a-3p lentivirus vectors have successfully transfected into THP-1 cells.Flow cytometry results showed that compared with miR-125a-3p NC group, The expression of CD11b and CD 14 were significant increased in the group of over-expression miR-125a-3p (P< 0.05); Compared with 1.00 μM LukS-PV+ miR-125a-3p NC, the expression of CD11b and CD14 were significant decreased in the group of 1.00 μM LukS-PV+inhibit-expression miR-125a-3p (P< 0.05). qRT-PCR and Western blot were used to detect the expression of differentiation related genes, including chemokine chemokine receptor 1 (CCR1), chemokine chemokine receptor 2 (CCR2), nuclear transcription factor JUN and FOS. Compared with miR-125a-3p NC, CCR1, CCR2, JUN and FOS mRNA and protein level increased significantly in the group of over-expression miR-125a-3p (P< 0.05). Compared with 1.00 μM LukS-PV+miR-125a-3p NC, CCR1, CCR2, JUN and FOS mRNA and protein level increased significantly in the group of 1.00 μM LukS-PV+ inhibit-expression miR-125a-3p (P< 0.05).(3) There were 42 target genes regulated by miR-125a-3p in bioinformatics prediction. Neurofibromin 1 (NF1) is closely related with tumour progression. Using dual luciferase reporter assay demonstrated that miR-125a-3p can combined with NF1 3’UTR.Conclusion (1) The expression of miR-125a-3p significant increased after treatment with LukS-PV in THP-1 cells. (2) In THP-1 cells, over-expression miR-125a-3p can induce cell differentiation, on the contrary, the inhibit-expression miR-125a-3p can block cell differentiation induced by LukS-PV. (3) miR-125a-3p combined with NF13’ UTR, which provided foundation for further study of mechanism in miR-125a-3p in LukS-PV-induced cell differentiation in THP-1 cells. |
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