Cell Differentiation Agent-2 (CDA-2) Inhibits the Growth and Migration of Saos-2 Cells via miR-124/MAPK1

Background CDA-2 (cell differentiation agent 2), isolated from healthy human urine, exerts antitumor effects in multiple types of cancer cells. However, its role in osteosarcoma has not been studied. Methods The MTT assay was used to examine the cell proliferation rate. A colony formation assay was used to examine cell growth. The Transwell assay was used to examine cell migration ability. A real-time PCR assay was used to examine the expression levels of miR-124 and MAPK1. A Western blot assay was used to examine protein expression levels. MAPK1 was selected as a possible target of miR-124, and the targeting relationship was examined by a luciferase reporter assay. Results We revealed that CDA-2 decreased the growth, migration and invasion ability of the osteosarcoma cell line Saos-2. Further study revealed that CDA-2 elevated the expression level of miR-124. MAPK1 was identified as a downstream target of miR-124. Knockdown of miR-124 or overexpression of MAPK1 counteracted CDA-2’s effects on cell growth and invasion. Conclusion Our data revealed that the miR-124/MAPK1 axis mediated CDA-2’s function in Saos-2 cells. CDA-2 can be used as a new treatment strategy for osteosarcoma.


Introduction
CDA-2 (cell differentiation agent 2) was first extracted from healthy human urine by Chinese researchers. CDA-2 has multiple functions, including inhibiting cell growth and invasion and promoting cell apoptosis. 1 It is also involved in preclinical studies, including those for breast cancer, leukemia and so on. The underlying mechanism by which CDA-2 kills cancer cells is complicated. For instance, CDA-2 can inhibit the PI3K/Akt signaling pathway and NF-kappaB signaling pathway. 2 Thus, CDA-2 exerts its antitumor function through these pathways. Recently, it was also reported that CDA-2 modulates microRNA (miRNA) expression in cancer. 3 MicroRNAs (miRNAs) are a new class of noncoding RNAs that can effectively silence their target genes at the posttranscriptional level. 4 miRNAs are involved in many biological processes (eg, cell proliferation, apoptosis and invasion). 5 The role of miRNAs in osteosarcoma has been fully investigated. For instance, microRNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy. 6 MicroRNA-381 suppresses the proliferation of osteosarcoma cells through the LRH-1/Wnt/β-catenin signaling pathway. 7 MicroRNA-520d-3p inhibits osteosarcoma progression by degrading Akt1. 8 These studies suggest that miRNAs could be a new treatment target for osteosarcoma.
The mitogen-activated protein kinase (MAPK) signaling cascade, which belongs to the membrane-nuclear signaling pathway, plays an important role in a variety of physiological processes, including cancer. MAPK1 belongs to one of the downstream oncogenes of the MAPK signaling pathway. Previous studies have found that MAPK1 is significantly upregulated in different types of human cancer, including osteosarcoma. 9,10 Osteosarcoma, one of the most common solid bone tumors in children and adolescents, often occurs in the metaphyseal region of the proximal humerus and proximal tibia. Though a combination of surgery, postoperative radiotherapy and chemotherapy is often used to treat osteosarcoma, osteosarcoma often has high morbidity and mortality. 11 New methods for osteosarcoma treatment are urgently needed.
Here, we report that CDA-2 has an antitumor effect in Saos-2osteosarcoma cells. The mechanism of CDA-2 in Saos-2 cells is modulated by the miR-124/MAPK1 axis. Our data suggest that CDA-2 is a promising drug for osteosarcoma treatment.

Cell Line Culture and Cell Transfection
The Saos-2 cells were maintained in our lab. The use of this cell line had been approved by the Institutional Committee on Animal Care of the Third Affiliated Hospital of Shenzhen University. Saos-2 cells were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS (Gibco). Cells were maintained at 37°C in a 5% CO2 incubator.
For cell transfection, lipofectamine 2000 reagent (Invitrogen) was used according to the manufacturer's instructions. miR-124 and miR-control were purchased from Ruibo (Guangzhou, China). pcDNA3.1 and pcDNA3.1-MAPK1 were purchased from Santa cruz. Cell transfection was carried out as previously described. 12

Cell Growth Assay
To carry out the MTT assay, 2000 cells were seeded in 96well plates per well. Different concentrations of CDA-2 were added to each well. After culture for different times, MTT solution (sigma) was added to each well. Two hours later, DMSO (sigma) was added to each well after the suspension was removed. The absorbance was measured at 490 nm with a microplate reader.

Cell Migration and Invasion Assays
Transwell assays were used to measure cell migration. Saos-2 cells were inoculated into the upper chamber of the Transwell in serum-free RPMI-1640. Later, we added 500 µlRPMI-1640 containing 10% FBS to the lower chamber. Twenty-four hours later, unmigrated cells remaining in the upper chamber were wiped. Migrated cells were then fixed with 90% ethanol (Beyotime Biotechnology, China) and stained with 0.1% crystal violet (Beyotime Biotechnology, China).An inverted microscope was used to count cells.
The boyden assay was used to examine cell invasion ability. To carry out boyden assay, the upper chamber of the transwell was coated with Matrigel, the experimental procedure is similar to transwell assay.

Western Blot Assay
Total protein was extracted from RIPA lysis buffer (Beyotime Biotechnology, China) and identified according to the manufacturer's protocol using a BCA detection kit (Beyotime Biotechnology, China).An equivalent amount of protein was then loaded onto 10% SDS-PAGE and transferred to PVDF membranes. After blocking the membranes with 5% nonfat milk, cyclinD1 (Santa Cruz biotechnology), MMP-3 (Santa Cruz biotechnology), MMP-9 (Santa Cruz biotechnology), MAPK1 (Santa Cruz biotechnology) and GAPDH (Santa Cruz biotechnology) primary antibodies were added to the membranes and incubated overnight at 4°C; then, the membranes were incubated with secondary antibody HRP (Santa Cruz biotechnology) binding antibody. Finally, the signal was detected using the enhanced chemiluminescence imaging system.

Luciferase Reporter Assay
The wild-type or mutant 3-'UTR of MAPK1was cloned into the pGL3-controlled luciferase reporter vector (Promega). We named the resulting vectorsMAPK1-wt or -mut luciferase reporter vector. Saos-2 cells were cotransfected with the corresponding luciferase reporter vector and miR-124 mimic or miR-ctrl. After 48 h of transfection, the cells were lysed, and luciferase activity was determined with a dual-luciferase assay kit (Promega).

Quantitation of miRNA-124 Levels
The extraction and examination of miR-124 was carried out as previously described. 3 In vivo Study CDA-2-treated or 0.9% NaCl-treated Saos-2 cells were injected into the posterior flanks of the mice. The tumor volumes were measured by using the formula (volume = length × width 2 /2). Five weeks after the implantation, the xenografts were removed from the mice and weighed. All procedures involving animals were approved by the Institutional Committee on Animal Care of the Third Affiliated Hospital of Shenzhen University. The female BALB/c nude mice (five-week-old) were fed under standard conditions and cared according to the Institutional Committee on Animal Care of the Third Affiliated Hospital of Shenzhen University for animal care.

Statistical Analysis
The statistical analysis was carried out by using Graph Pad Prism 5.0 software. Values are presented as the mean ± the standard error of the mean. The two-tailed Student's t-test was used to analyze difference among groups. P value < 0.05 was considered statistically significant.

CDA-2 Treatment Inhibited Saos-2 Cell Growth and Migration in vitro
First, we determined the IC50 of CDA-2 on Saos-2 cells. An MTT assay was performed to examine cell growth ability. Saos-2 cells were treated with different concentrations of CDA-2 (indicated in Figure 1A, P<0.05). We measured cell viability 72 hours after treatment. CDA-2 treatment decreased cell viability. The IC50 of CDA-2 for Saos-2 cells was 4.2 mg/L ( Figure 1A). We then carried out a colony formation assay. CDA-2 treatment decreased Saos-2 cell colony formation ability ( Figure 1B, P<0.05). We then asked whether CDA-2 treatment affected cell cycle distribution. After treatment with CDA-2, Saos-2 cells presented a significantly higher frequency of cells in G1 phase and a lower frequency of cells in S phase ( Figure 1C, P<0.05). We then examined the expression level of cyclin D1, which controls cell cycle progression from G1 phase to S phase.CDA-2 treatment decreased cyclinD1 expression ( Figure 1D).
Taken together, our data suggest that CDA-2 increased miR-124 expression via DNMT1.

MAPK1 Was Identified as a Downstream
Target of miR-124 miRNAs exert their function via multiple pathways and targets. The online microRNA prediction tool TargetScan was used to predict the target of miR-124. There was a list of mRNAs that predicted to be the targets of miR-124. Among these targets, we focused on MAPK1. Because MAPK1 was an oncogene that promoted origin and progression of cancer. The binding sites of miR-124 in the MAPK1 3ʹ-UTR are indicated in Figure 3A. We generated a mutant type of the MAPK1 3ʹ-UTR (MAPK1-mut) that did not have miR-124 binding sites ( Figure 3A).
Subsequently, we performed a dual luciferase reporter gene assay. The transfection efficiency of miR-124 was indicated in Figure 3B. Saos-2 cells were cotransfected with miR-124 mimic/miR-ctrl and MAPK1-wt/mut plasmids. The relative luciferase activity was decreased in cells cotransfected with pGL3-MAPK1-Wt and miR-124 mimic but not in cells cotransfected with pGL3-vector. In addition, no significant difference was observed in cells cotransfected with pGL3-MAPK1-Mut and miR-124mimic ( Figure 3C, P<0.05). RT-PCR assays revealed that miR-124 mimic treatment decreased MAPK1 mRNA expression levels ( Figure 3D, P<0.05). Western blot assays revealed that miR-124 mimic treatment decreased MAPK1 protein expression levels ( Figure 3E). Overall, these data revealed that MAPK1 was a direct target of miR-124.

CDA-2 Decreased Cell Growth in vivo
We finally examined the effect of CDA-2 on cell growth in vivo. CDA-2-treated cells or 0.9% NaCl-treated cells were inoculated into the backs of the nude mice. Compared with 0.9% NaCl-treated cell-derived xenograft tumors, CDA-2-treated cell-derived xenograft tumors grew more slowly ( Figure 5A, P<0.05). Twenty-five days later, the mice were sacrificed, and xenograft tumors were removed from the mice. The mean weight of xenograft tumors in the CDA-2-treated group was less than that in the 0.9% NaCl-treated group ( Figure 5B, P<0.05). Taken together, these results suggested thatCDA-2 decreased cell growth in vivo.

Discussion
Previous studies have documented that CDA-2 exerts anticancer effects in multiple cancers. CDA-2 has an effect on glioma, breast cancer, lung cancer and human leukemia cells. 1,15 The wide spectrum of the antitumor effects of CDA-2 has attracted researchers' attention. Further studies have investigated the underlying mechanism of how CDA-2 exerts its function. In myeloid cells, CDA-2 leads to suppression of NF-kappaB activation. 2 In myelodysplastic syndrome (MDS)-derived MUTZ-1 cells, CDA-2 induces apoptosis through the PI3K/Akt signaling pathway. 16 Interestingly, CDA-2 also affects miRNA expression and therefore may exert its function through  an miRNA-mediated mechanism. 12 These studies suggest that the function of CDA-2 is complex. However, whether CDA-2 exerts its functions via different mechanisms in different cell contexts needs further investigation.
The biological role of CDA-2 in osteosarcoma has not been reported before. We treated osteosarcoma cells with CDA-2 and found that CDA-2 has an antitumor effect in these cells. Functional studies (eg, MTT and colony formation assays) revealed that CDA-2 decreased osteosarcoma cell growth ability. In parallel, the in vivo study found that CDA-2 had an antitumor effect in nude mice. CDA-2 treatment led to cell cycle arrest in G1 phase and decreased cyclin D1 expression. This is the main reason why CDA-2 decreased cell growth. However, whether CDA-2 treatment also contributed to apoptosis in osteosarcoma cells needs further study. The Transwell assay showed that CDA-2 treatment inhibited osteosarcoma cell invasion. The EMT phenotype is considered a key factor leading to cancer cell migration. We also examined the effect of CDA-2 treatment on EMT markers. The E-cadherin expression level was increased, while the N-cadherin and vimentin expression levels were decreased after CDA-2 treatment. These data suggest that CDA-2 treatment reverses the EMT phenotype. A previous study found that CDA-2 treatment decreased twist/slug expression levels. 3 Twist/slug are key masters of EMT, and we speculate that CDA-2 reverses the EMT phenotype via the twist/slug pathway. Evasion of growth suppression and invasion are two important hallmarks of cancer. 17 Our data provided evidence that CDA-2 inhibited osteosarcoma cell growth and invasion ability, which implicated that CDA-2 may be a useful anti-tumor drug for osteosarcoma.
miR-124 is a tumor suppressor, including in osteosarcoma. 18,19 Hypermethylation-mediated inactivation of miR-124 is often observed in cancer cells. 20,21 We observed that a DNA methylation inhibitor (5-Aza-dC) elevated miR-124 expression in Saos-2 cells, which suggests that methylation contributed to miR-124 downregulation in osteosarcoma cells. Interestingly, CDA-2 treatment also elevated miR-124 expression levels. However, knockdown of DNMT1 counteracted CDA-2's effect on miR-124 expression. Epigenetic modification is a powerful mechanism that modify gene function, including miRNAs. DNA methyltransferases usually modulate miRNAs expression levels. For instance, DNMT1 mediated methylation silences the microRNA-200b/a/429 gene and promotes tumor progression. 22 Overexpression of DNMT1 blocked the function of miR-200a on repressing proliferation. 23 These literatures provided the evidence that DNMT1 played significant role in regulating miRNAs expression. In parallel, our data found that DNMT1 also repressed miR-124 expression via epigenetic modification. We speculated that CDA-2 elevated miR-124 expression level via the DNA methyltransferase DNMT1.
Among the potential targets of miR-124, we focused on a new target, MAPK1. MAPK1 is a well-known oncogene that is widely overexpressed in multiple human cancers. 24,25 In addition, MAPK1 is highly expressed in osteosarcoma cells and promoted proliferation and invasion in osteosarcoma cell lines. 9 These literatures suggest that MAPK1 may be a useful target in treating osteosarcoma. We used a dual luciferase reporter gene assay, which showed that that miR-124 directly bound to the MAPK1 3ʹ-UTR. RT-PCR and Western blot assays revealed that miR-124 decreased MAPK1 expression levels. CDA-2 treatment elevated miR-124 expression and decreased MAPK1 expression levels. Downregulation of miR-124 or overexpression of MAPK1 counteracted CDA-2's effect on Saos-2 cells. Our data suggest that CDA-2 may exert its function via the miR-124/MAPK1 axis.
Overall, our data suggest that CDA-2 has an antitumor effect on Saos-2 cells and that the miR-124/MAPK1 axis mediates CDA-2's effect. CDA-2 may be a new useful treatment for osteosarcoma.