Fig 1.
The proliferation and cell cycle analysis of leukemia cells after exposure to PMA.
K562, U937 and THP-1 leukemia cells were treated with PMA 100 ng/mL for 48 hours, the cell density were directly observed under a microscope, and proliferation of these leukemia cells were detected by CCK8 cell counting assay. Cell cycle was detected by Flow cytometry assay. A: Proliferation of K562, U937 and THP-1 leukemic cells before and after exposure to PMA; B. Cell density of THP-1 cells under microscopy; C: CCK8 assay to detect the proliferation of leukemia THP-1 cells; D. Cell cycle changes of control group THP-1 cells by flow cytometry assay; E: Cell cycle changes of PMA group cells by flow cytometry assay; F: The average percentage of cell cycle distribution. Each experiment was repeated three times. (* P < 0.05, ** P < 0.01, **** P < 0.0001).
Fig 2.
Effects in differentiation of THP-1 leukemia cells after PMA treatment.
Induced by PMA for 48 hours, the THP-1 cells were collected and stained with Wright-Giemsa staining, and expression of CD11b and CD14 antigens on the cell surface was detected by flow cytometry. A. Wright-Giemsa staining of THP-1 cells control group; B. Wright-Giemsa staining of THP-1 cells induced by PMA; C. The mean rate of differentiated THP-1 cells; D: Expression changes of CD11b expresson percent in THP-1 cells in control group and PMA-induced group by flow cytometry assay; E. Mean expression of CD11b in control group and PMA-induced group THP-1 cells; F. Expression changes of CD14 expresson percent in control group and PMA-induced group by flow cytometry assay; G: Mean CD14 expression percent in control and PMA-induced group. Each experiment was repeated three times. (**** P < 0.0001).
Fig 3.
Effect of WT1 gene overexpression on THP-1 cell differentiation induced by PMA.
Induced by PMA for 48 hours, the THP-1 cells were collected, the expression of WT1 cells was detected by western blotting assay, and the CD11b and CD14 of THP-1 cells was detected by flow cytometry. A. WT1 expression of THP-1 cells induced by PMA; B: Mean WT1 protein expression of THP-1 cells induced by PMA. C: WT1 expression of THP-1 cells after exposure to PMA or PMA+empty vector; D:Mean WT1 expression of THP-1 cells after exposure to PMA or PMA+empty vector; E: WT1 expression of THP-1 cells transfected with WT1 vector as compared with control group or PMA group; F: Mean relative expression of WT1 in THP-1 cells transfected with WT1 vector as compared with control group or PMA group; G. Inhibiting percent of WT1 expression by PMA and the restoration percent of WT1 expression by PMA + WT1 vector to that of control group, which were based on the results of Fig 3E and 3F; H. The WT1 expression after exposure to PMA or PMA + WT1 vector by Western blot assay; I. Mean relative expression of WT1 in THP-1 cells after exposure to PMA or PMA + WT1 vector; J. The CD11b expression by flow cytometry assay; K. Expression of CD11b antigen after exposure to PMA or PMA + WT1 vector; L. The CD14 expression after exposure to PMA or PMA + WT1 vector by flow cytometry assay; M: Expression of CD14 antigens after exposure to PMA or PMA + WT1 vector. Each experiment was repeated three times. (* P < 0.05, ** P < 0.01, *** P < 0.001).
Fig 4.
Potential miRNA targeting WT1 and their relationship during committed differentiation towards macrophages.
TargetScan and miRTarBase were used to predict potential upstream associated miRNAs of WT1. RNA seq technology to identify differentially altered genes (greater than 2 and a P < 0.05) as the boundary. TargetScan and MirDB databases were used to predict the miRNAs associated with monocyte/macrophage differentiation, and the intersection of the two databases was tried. Further validation of positively related miRNAs was demonstrated by qRT-PCR assay. In order to further verify whether miRNA-132-3p could bind WT1 3’UTR and modulate its promoter activity, a dual luciferase reporter assay was used. The relative expression of miRNAs was detected by qRT-PCR assay, and the correlation analysis between expression of miRNA-132-3p and WT1was indicated by Pearson analysis. A. TargetScan database predicts the possible miRNAs that target and bind to WT1; B. The number of differential miRNA species before and after differentiation and the number of macrophage differentiation-associated miRNA species analyzed by database; C. Target gene prediction, differential detection before and after differentiation, and database analysis macrophage-associated miRNA intersection analysis; D. miRNA-132-3p and miR-212-3p related to WT1 expression by intersection analysis; E: related expression of miRNA-132-3p before and after differentiation. F: miR-212-3p expression before and after differentiation; G: miRNA-132-3p targets WT1 3̛ UTR sequence prediction; H. Luciferase activity assay of miRNA-132-3p targeting WT1; I. The relative expression level of miRNA-132-3p in THP-1 cells before and after PMA induction; J. Relative expression levels of WT1 in THP-1 cells before and after PMA induction; K. Correlation analysis between miRNA-132-3p and WT1 expression. (ns: no significant, * P < 0.05, **** P < 0.0001).
Fig 5.
Results of rescue experiments of miRNA-132-3p targeting WT1 to regulate the differentiation of THP-1 cells.
Rescue experiment about miRNA-132-3p to regulate differentiation of THP-1 cells by targeting WT1 was tried. WT1 protein expression was detected by Western blotting assay, the expression level of CD11b and CD14 differentiation antigen were detected by flow cytometry assay. A. The transfection efficiency of miRNA-132-3p mimics was detected by qRT-PCR; B. WT1 expression after exposure to mimics NC, miR-132-3p mimics or miR-132-3p mimics+empty vector; C.mean WT1 expression after exposure to mimics NC, miR-132-3p mimics or miR-132-3p mimics+empty vector; D. WT1 protein expression was detected by Western blot; E. Mean WT1 expression level; F. CD11b expression of mimics NC group by flow cytometry; G.CD11b expression of miR-132-3p mimics group by flow cytometry; H. CD11b expression of miR-132-3p mimics+WT1 vector group by flow cytometry; I.Mean expression level of CD11b differentiation antigen; J. CD14 expression of mimics NC group by flow cytometry; K.CD14 expression of miR-132-3p mimics group by flow cytometry; L.CD14 expression of miR-132-3p mimics+WT1 vector group by flow cytometry M. Mean expression levels of CD14 differentiation antigens. Each experiment was repeated three times.(** P < 0.01, *** P < 0.001).
Fig 6.
β1 expression by WT1 on committed differentiation of monocytes/macrophages. WT1 protein expression was detected by Western blotting assay, and the expression level of CD11b and CD14 differentiation antigen were detected by flow cytometry assay. A. Western blot was used to detect the protein level of TGF-β1 in THP-1 cells after PMA treatment; B. Changes in the mean level of TGF-β1 protein in THP-1 cells after PMA induction; C. The expression of WT1 or TGF-β1 protein in THP-1 cells transfected with si-WT1 scramble NC or si-WT1; D. Mean WT1 protein expression level in THP-1 cells transfected with si-WT1 scramble NC or si-WT1; E. Mean TGF-β1 protein expression in THP-1 cells transfected with si-WT1 scramble NC or si-WT1; F. Expression of CD11b antigens of control group by flow cytometry; G. Expression of CD11b antigens of si-WT1 scramble NC group by flow cytometry; H. Expression of CD11b antigens of si-WT1 group by flow cytometry; I. Mean CD11b antigens of above-mentioned indicated group; J. Expression of CD14 antigens of control group by flow cytometry; K. Expression of CD14 antigens of si-WT1 scramble NC group by flow cytometry; L. Expression of CD14 antigens of si-WT1 group by flow cytometry; M. Mean CD14 antigens of above-mentioned indicated group;. Each experiment was repeated three times, (* P < 0.05, *** P < 0.001, **** P < 0.0001).
Fig 7.
Chromatin immunoprecipitation (ChIP) assay for WT1 targeting and regulation of TGF-
β1 expression.To further verify whether WT1 targets binding and regulates expression of TGF-β1, Chromosome immunoprecipitation (ChIP) assay for targeting regulation of TGF-β1 by WT1. A. Prediction of the binding region of WT1-targeted TGF-β1 promoter; B. Primer sequence design for ChIP assay of TGF-β1; C. ChIP assay results of WT1 targeting TGF-β1.
Fig 8.
Rescue assay results of miRNA-132-3p targeting TGF-
β1 to induce differentiation of THP-1 cells. miRNA-132-3p mimics-NC group, miRNA-132-3p mimics group, and miRNA-132-3p mimics+si-TGF-β1 group three groups were used to try rescue assay about miRNA-132-3p modulating expression of TGF-β1. Western blotting assay was used for detecting expression of TGF-β1 protein. A. TGF-β1 expression in the indicated mimics NC, miR-132-3p mimics or miR-132-3p mimics+si-TGF-β1 scramble NC groups by Western blot assay; B.Mean expression of TGF-β1 protein in the above indicated groups; C. TGF-β1 expression in the indicated mimics NC, miR-132-3p mimics or miR-132-3p mimics+si-TGF-β1 groups by Western blot assay; D. Mean expression of TGF-β1 protein in the above indicated groups; E. Expression of CD11b antigens of mimics NC group by flow cytometry;.F. Expression of CD11b antigens of miR-132-3p mimics group by flow cytometry; G. Expression of CD11b antigens of miR-132-3p mimics+WT1 vector group by flow cytometry; H.Mean expression of CD11b in the indicated groups; I. Expression of CD14 antigens of mimics NC group by flow cytometry;.J. Expression of CD14 antigens of miR-132-3p mimics group by flow cytometry; K. Expression of CD14 antigens of miR-132-3p mimics+WT1 vector group by flow cytometry; L.Mean expression of CD14 antigens in the indicated groups;.Each experiment was repeated three times, (* P < 0.05, ** P < 0.01).
Fig 9.
Schematic of the signaling pathway of the miRNA-132-3p/WT1/TGF-
β1 axis contributed to inducing differentiation of THP-1 cells into macrophages. In THP-1 cells, PMA could induce the upregulation of miRNA-132-3p, the upregulation of miRNA-132-3p results in the downregulation of WT1 by sponge action with binding on its 3’UTR, the down regulation of WT1 contributes to the upregulation of TGF-β1, then the polarization of macrophages is induced, which is indicated by the upregulation of CD11b and CD14 expression. Therefore, the miRNA-132-3p/WT1/TGF-β1 axis is involved in the committed differentiation of THP-1 leukemia cells into macrophages.