MiR-20a-5p functions as a potent tumor suppressor by targeting PPP6C in acute myeloid leukemia

Acute myeloid leukemia (AML) is as a highly aggressive and heterogeneous hematological malignancy. MiR-20a-5p has been reported to function as an oncogene or tumor suppressor in several tumors, but the clinical significance and regulatory mechanisms of miR-20a-5p in AML cells have not been fully understood. In this study, we found miR-20a-5p was significantly decreased in bone marrow from AML patients, compared with that in healthy controls. Moreover, decreased miR-20a-5p expression was correlated with risk status and poor survival prognosis in AML patients. Overexpression of miR-20a-5p suppressed cell proliferation, induced cell cycle G0/G1 phase arrest and apoptosis in two AML cell lines (THP-1 and U937) using CCK-8 assay and flow cytometry analysis. Moreover, miR-20a-5p overexpression attenuated tumor growth in vivo by performing tumor xenograft experiments. Luciferase reporter assay and western blot demonstrated that protein phosphatase 6 catalytic subunit (PPP6C) as a target gene of miR-20a-5p was negatively regulated by miR-20a-5p in AML cells. Furthermore, PPP6C knockdown imitated, while overexpression reversed the effects of miR-20a-5p overexpression on AML cell proliferation, cell cycle G1/S transition and apoptosis. Taken together, our findings demonstrate that miR-20a-5p/PPP6C represent a new therapeutic target for AML and a potential diagnostic marker for AML therapy.


Introduction
Acute myeloid leukemia (AML), as a highly aggressive and heterogeneous hematological malignancy, is characterized by abnormal growth of bone marrow stromal cells and undifferentiated myeloid progenitor cells in the bone marrow and peripheral blood [1,2]. At present, the principal therapeutic approaches, including chemotherapy, targeted therapy, and hematopoietic stem cell transplantation, have greatly improved the therapeutic outcomes of patients with AML [3,4]. Unfortunately, the prognosis and outcomes remain still poor in AML patients at advanced stage, which might be correlated with major problems, such as serious infection and bleeding [5][6][7]. Therefore, it is of great importance to elucidate the molecular mechanisms responsible for the progression of AML. MicroRNAs (mRNAs/miRs) are small noncoding RNA molecules consisting of 18-24 nucleotides that function as post-transcriptional regulator on gene expression by targeting mRNAs via binding to their 3 0 -untranslated region (3 0 -UTR) [8]. Increasing evidence suggests that miR-NAs are frequently aberrantly expressed in malignancies and function as tumor suppressors or oncogenes by participating in diverse biological functions, including cell proliferation, apoptosis and differentiation [9,10]. In recent years, miR-20a-5p, a 23-nucleotides-length non-coding RNA, has been reported to be involved in carcinogenesis. For instance, Bai et al [11] showed that miR-20a-5p was highly expressed in triple-negative breast cancer and promoted the cell proliferation through targeting runt-related transcription factor 3 (RUNX3). MiR-20a-5p overexpression contributed to hepatocellular carcinoma (HCC) cell proliferation and migration through reducing the translation of RUNX3 [12]. Similarly, Huang et al [13] demonstrated that miR-20a-5p promoted radio-resistance in nasopharyngeal cancer cells via repression of Rab27B. In addition to act as an oncogene, the suppressive role of miR-20a-5p was also manifested in endometrial cancer cells by targeting STAT3 [14], neuroblastoma cells by targeting ATG7 [15], as well as multi-drug resistance in osteosarcoma by cells targeting KIF26B [16] and SDC2 [17]. What interests us most is that a recent study by Ping et al [18] reported that knockdown of circ_0009910 inhibited AML cell proliferation and induced apoptosis through sponging miR-20a-5p. Nevertheless, the clinical significance and regulatory mechanisms of miR-20a-5p in AML cells have not been fully understood and needs to be further explored.
Protein phosphatase 6 catalytic subunit (PPP6C/PP6C) is a human ortholog of the yeast protein Sit4 and conserved phosphatase among eukaryotes from yeast to humans, which has been shown to regulate mitotic spindle formation by dephosphorylating Aurora A bound to its activator TPX2 and be required for normal cell cycle G1 to S transition [19]. PPP6C protein expression was significantly increased in glioma tissues [20]. Similarly, Zhu et al [21] reported that PPP6C expression was increased and directly regulated by miR-335 in cervical cancer. Functionally, loss of PPP6C in mouse keratinocytes increases susceptibility to ultraviolet-Binduced carcinogenesis [22]. NF-κB activation enhances keratinocyte proliferation by inhibiting PPP6C expression directly through the induction of miR-31 [23]. Our online bioinformatic prediction identified that PPP6C as a target gene of miR-20a-5p. However, the functional role of PPP6C and miR-20a-5p/PPP6C axis in AML cell proliferation have not been investigated.
In the present study, we first determined the expression of miR-20a-5p in bone marrow from AML patients and healthy controls. The correlation between miR-20a-5p and clinicopathological variables and survival prognosis was analyzed in AML patients. The in vitro experiments, including CCK-8 assay and flow cytometry and in vivo tumor xenograft experiments were performed to investigate the role of miR-20a-5p on AML tumorigenesis. Moreover, the association between miR-20a-5p and PPP6C was validated in AML cells.

Patients and specimen collection
All specimens were collected through bone marrow aspiration from patients diagnosed as AML (n = 61) and age matched healthy volunteers (n = 61) in the Henan Provincial People's Hospital (Henan, China) between September 2017 and October 2019. None of the patients received anti-tumor therapies, including chemotherapy or radiotherapy before sample collection. All obtained specimens were immediately kept at −80˚C until further analysis. Some basic clinicopathological variables of AML patients with survival information were recorded. All patients were followed-up every 2-4 months for at least 5 years and the overall survival was defined as the period of time from diagnosis to death. This study was performed in accordance with the declaration of Helsinki and Ethic Committee of Henan Provincial People's Hospital.

Cell proliferation analysis
Cell proliferation was assessed using Cell Counting Kit-8 assay (Dojindo Laboratories). In brief, approximately 0.5 × 10 4 transfected cells were seeded in each well of 96-well plates and cultured overnight. After incubation for 24, 48, 72 and 96 h, respectively, cells in each well were incubated with 10 μl CCK-8 reagents for another 1 h before the optical density (OD) value was measured at 450 nm by a microplate reader.

Cell cycle analysis
For cell cycle analysis, approximately 2 × 10 6 transfected cells were harvested by trypsin treatment and centrifugation at 300 g for 5 min. After washed with PBS twice, cells were fixed with 75% ethanol overnight at 4˚C, rehydrated with PBS at room temperature and stained with 500 μL propidium iodide (PI) solution for 15 min in darkness. Finally, cell cycle distribution was analyzed by a BD FACSCanto II (BD Biosciences, San Jose, CA).

Apoptosis analysis
After 48 h transfection, cells were collected and seeded into 24-well plates at a density of 2 × 10 5 cells per well. After washed twice with PBS, cells were resuspended in 500 μl binding buffer containing 10 μl fluorescein isothiocyanate (FITC) Annexin V and 10 μl PI solution (Sigma-Aldrich) for 10 min, followed by apoptotic analysis through a BD FACSCanto II.

Western blot analysis
Total protein sample was extracted using a RIPA lysis kit (Beyotime, Shanghai, China) and protein concentration was determined by BCA protein assay (Beyotime). Approximately 30 μg of protein sample was separated on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Block of membranes was performed with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h, the membranes were incubated with primary antibodies against PPP6C and GAPDH (Abcam, Cambridge, MA, USA) overnight at 4˚C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized with an enhanced chemiluminescence kit (Millipore).

Tumor xenograft experiments
Four-week-old male BALB/c nude mice were purchased from the Animal Center of Nanjing Medical University and kept in specific pathogen-free cages with standard rodent chow. A total of 3 × 10 6 miR-20a-5p mimics or miR-NC transfected THP-1 cells in 200 μl PBS were subcutaneously injected into the right posterior flank of equal nude mice. The tumor size was measured at 3, 7, 14 and 28 day, respectively by a caliper after cell implantation. Accordingly, tumor volume was calculated by the formula: volume (cm 3 ) = (length × width 2 )/2. On 28 day, the mice were sacrificed and tumor tissues were removed for weighing and miR-20a-5p expression analysis. All procedures of animal experiments were conducted in accordance with the Declaration of Helsinki and approved by the Animal Care Committee of the Xi'an Jiaotong University.

Statistical analysis
After statistical analysis by GraphPad Prism (Version 6.0), the quantitative data were expressed as mean ± SD from three independent experiments. The association of miR-20a-5p expression with clinicopathological variables of patients with AML was analyzed by Chi-square test. The overall survival rate estimates survival time were calculated using the Kaplan-Meier method with log-rank test. The differences among two groups were estimated by Student's t-test or one-way ANOVA with Tukey post hoc test for multiple groups. The values of p less than 0.05 were considered to be statistically significant.

MiR-20a-5p was downregulated and correlated with worse prognosis in AML patients
To explore the potential role of miR-20a-5p in AML development, the expression of miR-20a-5p was first analyzed by quantitative real time PCR in bone marrow from 61 AML patients and healthy controls. As shown in Fig 1A, miR-20a-5p expression was significantly downregulated in AML patients compared with matched healthy volunteers. Next, all AML patients were allocated to high and low miR-20a-5p expression group based on the median value, which were applied to research the correlation between miR-20a-5p expression and clinical features. As illustrated in Table 1, miR-20a-5p expression was associated with risk status, but not correlated with age, gender, FAB subtypes and relapse. Moreover, we further analyzed the relationship between miR-20a-5p levels and survival time in AML patients. The Kaplan-Meier survival cure showed that the patients with low miR-20a-5p expression had shorter overall survival rate than those with high miR-20a-5p expression (Fig 1B). These data indicated that decreased miR-20a-5p expression might predict worse prognosis in AML patients.

MiR-20a-5p overexpression suppressed cell proliferation, induced G0/G1 phase arrest and apoptosis in AML cells
Consistently, we found that miR-20a-5p was significantly decreased in four AML cell lines (Kasumi-1, THP-1, U937 and HL-60) compared with a normal human bone marrow stromal cell line HS-5 (Fig 2A). Then, two AML cell lines (THP-1 and U937) were selected for gain-offunction assays, which owned lowest miR-20a-5p expression level among four AML cell lines, demonstrating better typicality and representation. Quantitative real time PCR analysis confirmed that the expression of miR-20a-5p was significantly elevated in THP-1 and U937 cells after miR-20a-5p mimics transfection compared with miR-NC transfection (Fig 2B). Results of CCK-8 showed that upregulated miR-20a-5p inhibited the proliferation of THP-1 and U937 cells at 48, 72 and 96 h, respectively (Fig 2C). Flow cytometry was utilized to further analyze the effects of upregulated miR-20a-5p on cell cycle progression and apoptosis status. Cell cycle analysis showed that overexpression of miR-20a-5p presented a significant increase in the percentage of cells at G0/G1 phase while a significant decrease in the percentage of cells at G2/M phase in THP-1 (Fig 2D) and U937 (Fig 2E) cells, which indicated that cell cycle G0/G1 phase arrest was induced by miR-20a-5p overexpression. In addition, apoptosis analysis showed that miR-20a-5p mimics transfection remarkably promoted cell apoptosis in both THP-1 (Fig 2F) and U937 (Fig 2G) cells.

MiR-20a-5p negatively regulated PPP6C expression by targeting its 3'UTR
The online public bioinformatics tool TargetScan was used to predict the potential target of miR-20a-5p. Among these predicted target genes, PPP6C, associated with cell cycle and tumor formation/progression, was selected as a potential target of miR-20a-5p for further analysis. To test the effect of miR-20a-5p on PPP6C, WT and MUT PPP6C-3'UTR sequences were cloned into reporter plasmids (Fig 3A). The results of luciferase reporter assay showed that ectopic miR-20a-5p expression significantly decreased the luciferase activities of PPP6C-WT in THP-1 (Fig 3B) and U937 (Fig 3C) cells. Western blot analysis further demonstrated that miR-20a-5p mimics transfection obviously downregulated the expression of PPP6C protein in THP-1 and U937 cells compared with miR-NC transfection (Fig 3D). These data suggested that miR-20a-5p mediated PPP6C expression by directing targeting its 3'UTR, which may be involved in the suppressive role in AML.

Knockdown of PPP6C suppressed cell proliferation, induced G0/G1 phase arrest and apoptosis in AML cells
Since PPP6C was negatively regulated by miR-20a-5p in AML cells, we then performed lossof-function assay in AML cells by transfection with si-PPP6C or si-NC into THP-1 cells. As shown in Fig 4A, the protein level of PPP6C was obviously downregulated in THP-1 cells after si-PPP6C transfection. Consistent with the effects of miR-20a-5p in AML cells, we observed that knockdown of PPP6C markedly suppressed cell proliferation (Fig 4B), induced cell G0/ G1 phase arrest (Fig 4C) and promoted apoptosis (Fig 4D) in THP-1 cells. These data indicated that PPP6C played a positive role in AML cell growth and proliferation.

PPP6C overexpression reversed the effects of miR-20a-5p on AML cell proliferation, G1/S transition and apoptosis
Next, we performed rescue experiments to confirm whether PPP6C was the downstream regulator involved in miR-20a-5p regulating cell functions in AML cells. THP-1 cells were co-transfected with miR-20a-5p mimics and PPP6C. As shown in Fig 5A, the expression of PPP6C protein was decreased after miR-20a-5p + Vector, compared with miR-NC + Vector transfection, which was recovered after co-transfection with miR-20a-5p mimics and PPP6C. CCK-8 assay showed that increased PPP6C expression partially restored the impaired proliferation induced by miR-20a-5p overexpression in THP-1 cells (Fig 5B). Moreover, PPP6C

MiR-20a-5p inhibited tumor growth in mice xenograft models
To reveal the in vivo tumor suppressive effects of miR-20a-5p, THP-1 cells transfected with miR-20a-5p mimics or miR-NC were injected into the flanks of nude mice to generate tumors ectopically. As depicted in Fig 6A, mice injected with miR-20a-5p mimics transfected-THP-1 cells generated smaller tumor size compared with those with miR-NC transfected cells. After injections of miR-20a-5p mimics at consecutive 28 days, tumor growth was significantly lower than that following injection of miR-NC (Fig 6B). Moreover, the formed tumor weight was significantly decreased in miR-20a-5p mimics group, in comparison with miR-NC group (Fig  6C). In addition, quantitative real time analysis confirmed that miR-20a-5p expression was upregulated in miR-20a-5p mimics-transfected tumors when compared with miR-NC-transfected tumors (Fig 6D). Taken together, these results clearly demonstrate that miR-20a-5p functioned as a tumor suppressor in vivo.

Discussion
In this study, we found that miR-20a-5p was significantly decreased in bone marrow from AML patients, compared with that in healthy controls. Moreover, decreased miR-20a-5p expression was correlated with risk status and poor survival prognosis in AML patients. Consistently, the expression level of miR-20a-5p was reported to be downregulated along with clinical staging of neuroblastoma progression [15], in drug-resistant pancreatic cancer cells [24], breast tumors [25,26] and endometrial cancer tissues [14]. Downregulation of miR-20a-5p was correlated with the malignancy and poor prognosis of glioma patients [27]. HCC patients  with lower level of miR-20a-5p had significantly shorter OS and PFS survivals after surgical resection [28]. Contrary to our results, miR-20a-5p was upregulated in lung squamous cell carcinoma (SCC) tissues and serum exosomes with considerable clinical value in the diagnosis of male lung SCC patients [29]. The upregulation of miR-20a-5p was associated with advanced clinical stages of astrocytoma [30]. Notably, the downregulation of miR-20a-5p identified by Ping et al [18] confirmed our data on the decreased expression of miR-20a-5p and its correlation with worse prognosis in AML patients. Functionally, we further demonstrated that overexpression of miR-20a-5p significantly suppressed cell proliferation, induced cell cycle G0/G1 phase arrest and apoptosis in two AML cell lines (THP-1 and U937), as well as inhibited tumor growth in mice xenograft models. These findings indicated the tumor suppressive role of miR-20a-5p in AML progression. In line with our data, miR-20a-5p inhibited cell proliferation and promoted apoptosis in SH-SY5Y cells [15]. Forced expression of miR-20a-5p counteracted osteosarcoma cell chemoresistance in both cell culture and tumor xenografts in nude mice [16]. Ping et al [18] further confirmed that miR-20a-5p inhibitors reversed circ_0009910 siRNA-mediated cell proliferation inhibition, cell cycle arrest and cell apoptosis in AML5 cells. Different from the study from Ping et al, our study used two AML cell lines (THP-1 and U937) and performed gain-of-function assay to consistently demonstrated the suppressive effects of miR-20a-5p in AML cell proliferation and tumor growth. On the contrary, some studies described the oncogenic role of miR-20a-5p in other tumor cells, including colorectal cancer [31], HCC [12] and radio-resistance in nasopharyngeal cancer [13]. These differences in the regulatory role of miR-20a-5p might be ascribed to different tissue resources, cell types and different culture conditions. As our best knowledge, miRNAs bind to the 3'-UTR of their target mRNAs, leading to degradation of mRNA or inhibiting mRNA translation. Herein, PPP6C was a bioinformatics target of miR-20a-5p, and we further validated miR-20a-5p directly targeted PPP6C and reduced the expression of PPP6C protein. And indeed, PPP6C has been demonstrated to be a target gene of several miRNAs, such as miR-31 [32], miR-373 [33] and miR-335 [21]. Our data additionally indicated that PPP6C knockdown imitated, while overexpression reversed the effects of miR-20a-5p overexpression on AML cell proliferation, cell cycle G1/S transition and apoptosis, which suggested that miR-20a-5p regulated AML cell proliferation, G1/S transition and apoptosis via PPP6C repression. In fact, PPP6C protein expression was significantly upregulated in glioma [20] and cervical cancer [21]. The pro-survival effects of PPP6C have been manifested in ultraviolet-B-induced carcinogenesis [22] and keratinocyte proliferation [23]. By searching the members of the protein serine/threonine phosphatase family, we found PPP5C could significantly promote the tumor cell proliferation and survival, including HCC [34], ovarian cancer [35] and prostate cancer [36]. Interestingly, knockdown of PPP5C suppressed the proliferation ability, and led to G0/G1 phase arrest, induced cell apoptosis in leukemic cell line U937 cells [37], which is line with our data showed that PPP6C promoting AML cell proliferation.

Conclusions
In summary, we demonstrated that miR-20a-5p was significantly downregulated and correlated with worse prognosis in AML patients. MiR-20a-5p overexpression impaired AML cell proliferation, induced cell cycle G0/G1 phase arrest, apoptosis in vitro and tumor growth in vitro. Moreover, we confirmed that miR-20a-5p negatively regulated PPP6C expression by binding to its 3'-UTR and further confirmed that PPP6C was the downstream regulator involved in miR-20a-5p regulating AML cell functions. The results of this study indicate that the miR-20a-5p/PPP6C axis closely correlates with the malignant progression of AML, which might be of potential value as novel therapeutic targets for AML treatment.