SPOP overexpression accelerates AML cell proliferation and inhibits apoptosis
Analysis of GEPIA database revealed that SPOP was up-regulated in AML (p < 0.05, Fig. 1A). Furthermore, the expression of SPOP in the peripheral blood of AML group was higher than that in the normal group (p < 0.05, Fig. 1B, C). Therefore, we speculate that SPOP may mediate cell activities in AML. To verify this hypothesis, we first examined the expressions of SPOP in four AML cell lines (HL60, K562, U937, and KG-1). The expression of SPOP in K562, U937, and KG-1 cells was higher than that in PBMC cells (p < 0.05, Fig. 1D, E). We selected K562 and KG-1 cells for subsequent experiments. The efficiency of overexpressing or silencing SPOP in K562 and KG-1 cells was validated. The results manifested that the expression of SPOP in the oe-SPOP group was higher than that in the oe-NC group, and that in the sh-SPOP-1 and sh-SPOP-2 groups was lower than that of the sh-NC group (p < 0.05, Fig. 1F, G). The sh-SPOP-1 group (sh-SPOP group) was selected for subsequent experiments, due to its stronger silencing efficiency than the sh-SPOP-2 group.
CCK-8 assay identified increased cell viability in response to oe-SPOP treatment, and lowered cell viability to sh-SPOP treatment (Fig. 1H). EdU assay manifested that EdU-positive cells was increased in response to oe-SPOP treatment, along with lowered cell proliferation in response to sh-SPOP treatment (Fig. 1I). Flow cytometric data revealed attenuated AML cell apoptosis in response to oe-SPOP treatment, and that in response to sh-SPOP treatment was facilitated (Fig. 1J). Together, SPOP overexpression accelerates the proliferation of AML cells and inhibits their apoptosis.
SPOP enhances β-catenin protein expression and nuclear translocation leading to elevated miR-183 expression
Prior evidence proposed that β-catenin can facilitate the occurrence of AML, and SPOP can indirectly enhance the stability and nuclear translocation of β-catenin in the cytoplasm in renal cancer cells [21, 22]. Therefore, we speculate that in AML cells, SPOP may also stabilize β-catenin expression and nuclear translocation in the cytoplasm. To verify this hypothesis, we first tested β-catenin expression in K562 and KG-1 cells. The treatment of oe-SPOP resulted in elevated total β-catenin protein level, and cytoplasmic and nuclear levels, while β-catenin protein level was diminished in the presence of sh-SPOP transduction (Fig. 2A). Immunofluorescence assay found increased β-catenin distribution in the nucleus and cytoplasm following treatment of oe-SPOP (Fig. 2B). Thus, in AML cells, SPOP can augment the stability of cytoplasm and nuclear translocation of β-catenin.
MiR-183 has been reportedly suggested to highly express in AML and stimulate the occurrence of this disease [18]. Besides, β-catenin and TCF bind to the miR-183 promoter region to enhance the miR-183 expression in liver cancer cells [23]). Therefore, we speculate that the high expression of miR-183 in AML cells may also be regulated by β-catenin. First, we found that the expression of miR-183 in peripheral blood of the AML group was higher than that in the normal group (p < 0.05, Fig. 2C). Next, we assessed the efficiency of overexpressing and silencing β-catenin. The expression of β-catenin was increased upon treatment of oe-β-catenin, and reduced upon treatment of sh-β-catenin-1 and sh-β-catenin-2 (Fig. 2D, E). The sh-β-catenin-1 (sh-β-catenin group) with stronger silencing efficiency than sh-β-catenin-2 was selected for subsequent experiments. oe-β-catenin was co-transfected into HEK293T cells with miR-183 Wt and miR-183 Mut, respectively. Western blot assay revealed in the presence of miR-183 Wt and miR-183 Mut, the β-catenin expression was enhanced upon oe-β-catenin transduction versus oe-NC treatment (p < 0.05). The dual luciferase report assay suggested that the luciferase activity in the miR-183 Wt group increased following addition of oe-β-catenin, while the luciferase activity in the miR-183 Mut group did not change (Fig. 2F). The CHIP assay manifested that relative to IgG, β-catenin enrichment in the miR-183 promoter region was elevated, and the enrichment was facilitated after overexpressing β-catenin (Fig. 2G). In addition, we unraveled that miR-183 expression was elevated by oe-SPOP and oe-β-catenin, and reduced by sh-SPOP and sh-β-catenin. Whereas, combined treatment of oe-SPOP + sh-β-catenin showed lower miR-183 expression than oe-β-catenin treatment, and combined treatment of sh-SPOP + oe-β-catenin showed higher miR-183 expression than sh-SPOP treatment (Fig. 2H). These results indicate that SPOP accelerates β-catenin protein expression and nuclear translocation, thus elevating miR-183 expression.
MiR-183 overexpression enhances proliferation and inhibits apoptosis of AML cells by targeting METAP2
In order to further clarify the downstream mechanism of miR-183, we, through bioinformatic analysis, we obtained 8890 up-regulated and 7791 down-regulated mRNAs (Fig. 3A), and intersected the mRNAs from different databases (Fig. 3B). In these four databases, 12 genes (GCLM, GNG12, METAP2, EXO1, TET1, RHOBTB1, YOD1, SMCO4, XPOT, GTF2H1, SPATS2, LRP6) were found in the intersection. No previous studies could be found on target relationship between miR-183 and METAP2. The GEPIA database analysis showed that METAP2 was down-regulated in AML samples (p < 0.05, Fig. 3C). Moreover, our experimental data manifested that the expression of METAP2 in the AML group was lower than that in the Normal group (p < 0.05, Fig. 3D, E). The binding sites between miR-183 and METAP2 were predicted by Starbase database (Fig. 3F).
In KG-1 and K562 cells, miR-183 mimics treatment resulted in increased miR-183 expression, and miR-183 inhibitor treatment resulted in diminished miR-183 expression (Fig. 3G). In the presence of METAP2 WT and METAP2 Mut, the expression of miR-183 was elevated in response to miR-183 mimics (p < 0.05). The dual luciferase report assay manifested that the luciferase activity in the METAP2 WT group was reduced upon miR-183 mimics treatment, and the luciferase activity didn’t differ appreciably in the METAP2 Mut group (Fig. 3H). Moreover, we found that, in KG-1 and K562 cells, the expression of METAP2 was lowered following miR-183 mimics treatment, and which was elevated in response to miR-183 inhibitor treatment (Fig. 3I, J). The above data indicate that miR-183 targets and inhibits METAP2 expression.
Next, we further investigated whether miR-183 can expedite the proliferation and halt apoptosis of AML cells by inhibiting METAP2 expression. First, the experimental data suggested that miR-183 mimics led to miR-183 upregulation, and combined treatment of miR-183 mimics + oe-METAP2 resulted in higher miR-183 expression than oe-METAP2 alone did (Fig. 3K). Besides, miR-183 mimics led to reduced METAP2 expression, and combined treatment of miR-183 mimics + oe-METAP2 resulted in higher METAP2 expression than miR-183 mimics alone did (Fig. 3K, L). CCK-8 assay found that oe-METAP2 suppressed cell viability, and miR-183 mimics enhanced cell viability, whereas, combined treatment of miR-183 mimics + oe-METAP2 resulted in impeded cell viability induced by miR-183 mimics (Fig. 3M). EdU assay revealed that oe-METAP2 diminished EdU-positive cells, and miR-183 mimics elevated EdU-positive cells, whereas, combined treatment of miR-183 mimics + oe-METAP2 resulted in reduced EdU-positive cells that were increased by miR-183 mimics (Fig. 3N). Flow cytometric data manifested that oe-METAP2 accelerated cell apoptosis, and miR-183 mimics repressed cell apoptosis, whereas, combined treatment of miR-183 mimics + oe-METAP2 resulted in enhanced cell apoptosis that was suppressed by miR-183 mimics (Fig. 3O).
Together, miR-183 targets and negatively regulates METAP2 expression, thereby promoting the proliferation and inhibiting apoptosis of AML cells.
MiR-183 inhibition represses SPOP-induced proliferation of AML cells
Next, we further investigated whether miR-183 can restrict the SPOP overexpression-induced proliferation of AML cells. First, we found that the expression of SPOP and β-catenin was elevated in response to treatment of oe-SPOP + inhibitor-NC or oe-SPOP + miR-183 inhibitor, relative to treatment of oe-NC + inhibitor-NC or oe-NC + miR-183 inhibitor (p < 0.05). Besides, miR-183 expression was lower following oe-NC + miR-183 inhibitor treatment, relative to oe-NC + inhibitor-NC treatment (p < 0.05). The treatment of oe-SPOP + inhibitor-NC stimulated miR-183 expression versus oe-NC + inhibitor-NC treatment (p < 0.05). Moreover, combined treatment of oe-SPOP + miR-183 inhibitor reduced miR-183 expression versus oe-SPOP + inhibitor-NC treatment (p < 0.05) (Fig. 4A, B).
CCK-8 assay manifested that miR-183 inhibitor attenuated cell viability and oe-SPOP enhanced cell viability, but the combined treatment of oe-SPOP and miR-183 inhibitor appreciably repressed the promotion of cell viability induced by oe-SPOP (Fig. 4C). Additionally, EdU assay demonstrated that miR-183 inhibitor diminished EdU-positive cells and oe-SPOP elevated EdU-positive cells, but the combined treatment of oe-SPOP and miR-183 inhibitor appreciably diminished the increase in EdU-positive cells induced by oe-SPOP (Fig. 4D). Besides, flow cytometric results manifested that miR-183 inhibitor facilitated cell apoptosis and oe-SPOP restricted cell apoptosis, but the combined treatment of oe-SPOP and miR-183 inhibitor appreciably augmented the cell apoptosis that was repressed by oe-SPOP (Fig. 4E).
Therefore, inhibiting miR-183 can reverse SPOP-induced AML cell proliferation enhancement and apoptosis suppression.
METAP2 overexpression restricts SPOP-induced proliferation of AML cells
Next, we sought to assess whether METAP2 can restrict the SPOP overexpression-induced proliferation of AML cells. The oe-SPOP + oe-NC treatment and oe-SPOP + oe-METAP2 treatment showed higher expression of SPOP, β-catenin and miR-183 than the oe-NC treatment and oe-NC + oe-METAP2 treatment (p < 0.05). The oe-NC + oe-METAP2 treatment resulted in higher METAP2 expressions, relative to oe-NC treatment (p < 0.05). The expression of METAP2 was reduced upon the oe-SPOP + oe-NC treatment, relative to oe-NC treatment (p < 0.05), and the expression of METAP2 following the oe-SPOP + oe-METAP2 treatment was higher than that in response to oe-SPOP + oe-NC (p < 0.05, Fig. 5A, B).
CCK-8 assay suggested that oe-METAP2 led to lowered cell viability, and oe-SPOP enhanced cell viability, but the combined treatment of oe-METAP2 and oe-SPOP appreciably repressed the promotion of cell viability induced by oe-SPOP (Fig. 5C). Additionally, EdU assay demonstrated that oe-METAP2 diminished EdU-positive cells and oe-SPOP elevated EdU-positive cells, but the combined treatment of oe-SPOP and oe-METAP2 appreciably diminished the increase in EdU-positive cells induced by oe-SPOP (Fig. 5D). Besides, flow cytometric results manifested that oe-METAP2 facilitated cell apoptosis and oe-SPOP restricted cell apoptosis, but the combined treatment of oe-SPOP and oe-METAP2 appreciably augmented the cell apoptosis that was repressed by oe-SPOP (Fig. 5E).
Together, overexpression of METAP2 can reverse SPOP-induced AML cell proliferation enhancement and apoptosis suppression.
SPOP facilitates AML initiation by mediating β-catenin expression and the miR-183/METAP2 axis in vivo
Next, we further measured xenograft tumors in nude mice to verify that SPOP affects the growth of AML cells by mediating β-catenin expression and the miR-183/METAP2 axis in vivo. The tumor volume and weight were reduced in response to oe-METAP2 treatment, and they were elevated in response to oe-SPOP. Whereas, the combined treatment of oe-SPOP and oe-METAP2 appreciably repressed the tumor growth induced by oe-SPOP (Fig. 6A). Moreover, the expression of SPOP, β-catenin and METAP2 in the xenograft tumors was higher following treatment of oe-SPOP + oe-NC and oe -SPOP + oe-METAP2, relative to treatment of oe-NC and oe-NC + oe-METAP2 (p < 0.05). The oe-METAP2 treatment resulted in increased expression of METAP2, and the oe-SPOP treatment diminished expression of METAP2. Whereas, the combined treatment of oe-SPOP and oe-METAP2 appreciably enhanced the expression of METAP2 that was suppressed by oe-SPOP (Fig. 6B, C).
Ki67 immunohistochemical analysis manifested that oe-METAP2 diminished Ki67-positive cells and oe-SPOP elevated Ki67-positive cells, but the combined treatment of oe-SPOP and oe-METAP2 appreciably diminished the increase in Ki67-positive cells induced by oe-SPOP (Fig. 6D). Besides, TUNEL staining manifested that oe-METAP2 facilitated cell apoptosis and oe-SPOP restricted cell apoptosis, but the combined treatment of oe-SPOP and oe-METAP2 appreciably augmented the cell apoptosis that was repressed by oe-SPOP (Fig. 6E).
Together, SPOP affects the occurrence and growth of AML by mediating the expression of β-catenin and regulating the activation of miR-183/METAP2 axis.