circATP2A2 promotes osteosarcoma progression by upregulating MYH9

Abstract Osteosarcoma (OS) is a highly metastatic primary malignant tumor. CircRNA hsa_circ_0028173 (circATP2A2) has been uncovered to be related to the advancement of OS. However, the biological role of circATP2A2 in OS has not been validated. circATP2A2 and MYH9 were upregulated while miR-335-5p was downregulated in OS. OS patients with high circATP2A2 expression displayed a shorter overall survival and the area under curve of circATP2A2 was 0.77, manifesting that circATP2A2 might be a diagnostic and prognostic biomarker. circATP2A2 silencing repressed OS cell proliferation and glycolysis in vivo and constrained OS cell proliferation, glycolysis, migration, and invasion in vitro. circATP2A2 regulated MYH9 expression through sponging miR-335-5p. MiR-335-5p inhibitor reversed the repressive effect of circATP2A2 knockdown on OS cell malignancy and glycolysis. MYH9 overexpression overturned miR-335-5p upregulation-mediated OS cell malignancy and glycolysis. circATP2A2 accelerated OS cell malignancy and glycolysis through upregulating MYH9 via sponging miR-335-5p, offering a promising target for OS treatment.


Introduction
Osteosarcoma (OS) is a common primary malignant tumor that occurs mainly in children and adolescents near the shoulder joint and the long bone metaphysis [1]. Also, diffusion to the lungs is the leading cause of OS death [2].
With the advancement of medical technologies, the longoverall survival rates of OS patients have enhanced conspicuously to 65-70% [3]. So far, the exact molecular mechanism of the occurrence and progression of OS is still unclear. Therefore, exploring the molecular mechanism of OS progression is crucial to finding specific targets for OS.
Changes in energy metabolism are one of the hallmarks of cancer, including OS. The metabolism of cancer cells is characterized by preferential reliance on glycolysis, which produces low adenosine triphosphate (ATP), but cancer cells can compensate for this deficiency by increasing glucose uptake [4]. Studies have shown that inhibiting malignant cells with increased glycolytic capacity may be a key anti-cancer strategy [5]. Therefore, it is very important to explore the mechanism related to glycolysis in OS.
Circular RNAs (circRNAs), which have a covalent closed-loop structure, are important elements for regulating the genome [6]. CircRNAs can function as microRNA (miR) sponges and decrease miR activity by binding to miRs [7,8]. Recent research studies have proved that cir-cRNAs exert crucial roles in the initiation and advancement of human diseases, especially in cancer [9]. For instance, circRNA TADA2A upregulation promoted OS cell growth and metastasis by increasing CREB3 expression [10]. CircRNA FAT1 facilitated the tumorigenesis of OS by adsorbing miR-375 and increasing YAP1 expression [11]. CircRNA hsa_circ_0028173 (circATP2A2), located on chr12: 110764194-110780253, is derived from the ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2) gene. However, the biological role of circATP2A2 in OS has not been verified.
MiRs are short noncoding RNAs (20-22 nucleotides) that regulate gene expression [12] and participate in a battery of biological processes [13]. The dysregulation of miRs is related to the occurrence of many diseases [14]. MiR-335-5p has been revealed to play a suppressive role in various tumors, such as lung adenocarcinoma [15], thyroid cancer [16], and colorectal cancer [17]. MiR-335-5p also exerted an inhibitory effect in OS [18]. However, the mechanism of the abnormal expression of miR-335-5p in OS is unclear.
Non-muscle myosin (NM IIA), a cytoplasmic myosin, was involved in several processes that require actin cytoskeletal translocation and intracellular chemical mechanical forces [19]. The myosin heavy chain 9 (MYH9) gene, which encodes an NM IIA heavy chain, is associated with human diseases [19]. MYH9 has been uncovered to be connected with tumor progression, tumor differentiation, lymph node metastasis, and poor prognosis [20]. It was reported that high MYH9 expression was associated with OS cell metastasis and invasion [21]. Nevertheless, the mechanism that regulates MYH9 in OS has not been fully explained.
Herein, we validated circATP2A2 as an oncogene in OS. Also, circATP2A2 promoted OS cell malignancy and glycolysis by regulating miR-335-5p/MYH9. Our research provided a novel mechanism for understanding the progression of OS.

OS samples
This research was permitted by the Ethics Committee of Shengli Oilfield Central Hospital. Blood samples were from 51 OS patients and 47 healthy people. Fifty-one paired OS tissues and adjacent normal bone tissues were obtained from 51 primary OS patients who were diagnosed at Shengli Oilfield Central Hospital. All patients signed informed consent and did not receive chemotherapy or radiotherapy before surgery. The clinic-pathological information of the 51 OS patients is exhibited in Table 1.

Real-time reverse transcription polymerase chain reaction (RT-qPCR)
The TRIzol reagent (Invitrogen) was utilized to isolate total RNA from the serum, tissues, and cells. The high-capacity complementary DNA Reverse Transcription Kits (Invitrogen) or MicroRNA Reverse Transcription Kit (Invitrogen) was used for cDNA generation. The SYBR Green PCR Master Mix (Vazyme, Nanjing, China) was utilized for qPCR analysis. Primer sequences were synthesized by Ribobio ( Table 2). Expression levels of circATP2A2, miRs, or MYH9 mRNA were calculated using the 2 −ΔΔCt method, and β-actin or U6 was deemed as an internal control.

Transwell assay
Transwell chambers (Corning) were used for migration and invasion analysis. In short, the transfected OS cells (1 × 10 5 ) were resuspended in McCoy's 5A medium without FBS and then placed in the apical chamber. The McCoy's 5A medium containing 10% FBS (Procell) was added to the basolateral chamber. After 24 h, the migrating and invading cells were stained with 0.1% crystal violet (Sigma), followed by counting with a microscope (Nikon Instruments).

Metabolism analysis
The levels of glucose uptake and lactate product in OS cells were detected using the glucose uptake colorimetric assay kit (BioVision, Milpitas, CA, USA) or lactate assay kit II (BioVision) based on the manufacturer's instructions.

RNA pulldown assay
The circATP2A2 probe and oligo probe were designed and synthesized by RiboBio, followed by incubating with C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA). Then, the lysates of OS cells were incubated with C-1 magnetic beads. RNA complexes bound to the C-1 magnetic beads were eluted and then analyzed via RT-qPCR.

Bioinformatics analysis
The candidate miRs containing the putative binding sites of circATP2A2 were analyzed using circinteractome, starBase,

RIP (RNA immunoprecipitation) assay
RIP assay was conducted with the Magna RIP kit (Millipore, Bedford, MA, USA) in accordance with the manufacturer's instructions. The lysates of OS cells were incubated with immunoprecipitation buffer containing magnetic beads conjugated to IgG antibody (Abcam, Cambridge, MA, USA) or Ago2 antibody (Abcam). The enrichment of circATP2A2 or MYH9 and miR-335-5p was assessed by RT-qPCR.

Xenograft assay
The experiment was authorized by the Animal Ethics Committee of Shengli Oilfield Central Hospital. Ten BALB/c nude mice (4-6 weeks old) were obtained from Vital River Laboratory (Beijing, China) and divided into 2 groups (n = 5) by random number table. Briefly, Saos-2 cells (5 × 10 6 cells/100 μL) with stable knockdown of circATP2A2 were injected into nude mice (n = 5), and sh-NC was used as a control. The tumor volume was measured with a digital caliper every 7 days and calculated with the following equation: volume = (length × width 2 )/2. After 28 days, the mice were euthanized under anesthesia to obtain the tumor tissues for subsequent analysis.

Statistical analysis
SPSS 18.0 (SPSS, Chicago, IL, USA) was employed for statistical analysis. Data were produced from three independent experiments and presented as mean ± standard deviation. The Kaplan-Meier method with the log-rank (Mantel-Cox) test was utilized for analysis of the overall survival rate of OS patients. Receiver operating characteristic (ROC) curves were plotted using GraphPad Prism 5 (GraphPad Software 5.0, LaJolla, CA). The Student's t-test or one-way analysis of variance was used to compare the differences between two groups and more than two groups. When P＜0.05, the data were deemed statistically significant.

circATP2A2 might be a diagnostic and prognostic biomarker for OS patients
By analyzing the GSE96964 dataset downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE96964), we found that circATP2A2 was a differentially expressed circRNA in OS (Figure 1a). To survey the biological role of circATP2A2 in OS, we detected circATP2A2 expression in OS. Expression of circATP2A2 was apparently increased in OS cells (U2OS, HOS, and Saos-2) and tissues in contrast to their matched control groups (Figure 1b and c). Also, Kaplan-Meier curves and log-rank (Mantel-Cox) test were used for survival analysis, and OS patients with low circATP2A2 expression displayed a higher overall survival (Figure 1d). In addition, cir-cATP2A2 was drastically upregulated in the serum of OS patients (Figure 1e). The ROC curve analysis exhibited that the area under curve (AUC) of circATP2A2 was 0.77, manifesting that circATP2A2 might be a clinical diagnostic biomarker for OS (Figure 1f). circATP2A2 is derived from the reverse splicing of the ATP2A2 gene and forms a 1,774 bp circular transcript (Figure 1g). Moreover, circATP2A2 was resistant to RNase R treatment (Figure 1h and i). Additionally, circATP2A2 was more distributed in the cytoplasm of OS cells (Figure 1j and k). These results indicated that circATP2A2 might be related to OS progression.

circATP2A2 facilitated OS cell malignancy and glycolysis
Subsequently, the biological function of circATP2A2 in OS was further surveyed. The interference efficiency of sh-circATP2A2 in OS cells is presented in Figure 2a. Downregulation of circATP2A2 decreased the OS cell growth and colony formation in MTT and plate clone assays (Figure 2b-d). cir-cATP2A2F inhibition induced cell cycle arrest in OS cells in flow cytometry assay (Figure 2e and f). The migration and invasion of OS cells were also remarkably repressed in transwell assays (Figure 2g and h). In addition, the levels of glucose uptake and lactate product were lower in OS cells with low circATP2A2 expression (Figure 2i and j). HK2 and PKM2 are the key rate-limiting enzymes for glycolysis [23]. HK2 catalyzes the conversion of glucose to 6-phosphate glucose (G-6-P), while PKM2 catalyzes phosphoenolpyruvate (PEP) to produce ATP and pyruvate [24]. As expected, the levels of HK2 and PKM2 protein were downregulated in circATP2A2-repressed OS cells (Figure 2k). The results indicated that circATP2A2 promoted OS cell malignancy and glycolysis.

circATP2A2 adsorbed miR-335-5p to accelerate OS cell malignancy and glycolysis
To validate whether circATP2A2 regulated OS cell malignancy and glycolysis via serving as a miR-335-5p decoy, we co-transfected sh-circATP2A2 and anti-miR-335-5p into OS cells. The results exhibited that miR-335-5p silencing restored the suppressive impact of circATP2A2 knockdown on growth, colony formation, and cell cycle progression of OS cells (Figure 4a-e). Moreover, the miR-335-5p inhibitor abrogated the inhibition of migration and invasion of OS cells induced by circATP2A2 silencing (Figure 4f and g). Furthermore, the decreases of glucose uptake and lactate product in circATP2A2-suppressed OS cells were overturned after the anti-miR-335-5p introduction (Figure 4h and i). Furthermore, the downregulation of HK2 and PKM2 in OS cells with low circATP2A2 expression was restored by miR-335-5p silencing (Figure 4j). Therefore, these data disclosed that circATP2A2 promoted OS cell malignancy and glycolysis by adsorbing miR-335-5p.

MYH9 was a target of miR-335-5p
Previous research studies revealed that MYH9 acted as an oncogene in OS. To survey the relationship between  MYH9 and miR-335-5p, we first verified MYH9 expression in OS tissues. The results presented that the levels of MYH9 mRNA and protein were increased in OS tissues (Figure 5a-c). As expected, the level of MYH9 protein was also elevated in OS cells (Figure 5d). Through bioinformatics prediction, we discovered that MYH9 might be a target for miR-335-5p (Figure 5e). Furthermore, the luciferase activity of the MYH9 3′UTR-wt reporter was remarkably decreased in OS cells with overexpression of miR-335-5p (Figures 5f and g). Also, the abundance of miR-335-5p and MYH9 in the immunoprecipitates of the anti-Ago2 group was significantly increased (Figure 5h and i).
In addition, miR-335-5p overexpression repressed the protein level of MYH9 in OS cells, while miR-335-5p inhibition had an opposing impact (Figure 5j). There was also downregulation of MYH9 protein in OS cells with the inhibition of circATP2A2, whereas this downregulation was restored after miR-335-5p silencing (Figure 5k). Therefore, circATP2A2 could modulate MYH9 expression by sponging miR-335-5p.

MiR-335-5p repressed OS cell malignancy and glycolysis by targeting MYH9
To investigate whether miR-335-5p affected OS cell malignancy and glycolysis via MYH9, we performed rescue experiments. As exhibited in Figure 6a, the MYH9 protein level was increased in OS cells after MYH9 transfection. Furthermore, miR-335-5p elevation constrained growth, colony formation, and cell cycle progression of OS cells but this repression was reversed after MYH9 overexpression (Figure 6b-f). Moreover, MYH9 upregulation reversed the repression of cell migration and invasion in OS cells induced by miR-335-5p overexpression (Figure 6g and h). Also, the elevation of MYH9 overturned the decrease of glucose uptake and lactate production in miR-335-5p-elevated OS cells (Figure 6i and j). In addition, the downregulation of HK2 and PKM2 in miR-335-5p-increased OS cells was restored after MYH9 upregulation (Figure 6k). These results proved that miR-335-5p curbed OS cell malignancy and glycolysis via targeting MYH9.

circATP2A2 knockdown repressed OS cell proliferation and glycolysis in vivo
The biological function of circATP2A2 in OS was further verified through xenograft assay. The results displayed that the volume and weight of xenograft tumors in mice injected with OS cells carrying sh-circATP2A2 were smaller and lower (Figure 7a and b). Also, circATP2A2 expression was decreased in xenograft tumors in mice injected with OS cells carrying sh-circATP2A2 (Figure 7c). IHC revealed that the expression of Ki67 was also reduced in xenograft tumors in the sh-circATP2A2 group (Figure 7d). In addition, the protein levels of MYH9, HK2, and PKM2 were downregulated in xenograft tumors in the sh-circATP2A2 group (Figure 7e). These data suggested that circATP2A2 silencing constrained OS cell proliferation and glycolysis in vivo.

Discussion
A series of studies have proved that circRNAs are promising diagnostic and prognostic biomarkers and therapeutic A targets for diverse tumors [25][26][27]. Our results exhibited that circATP2A2 was overexpressed in OS tissues and cells, which was in line with the report of Chen et al. [28]. Also, OS patients with high circATP2A2 expression displayed a worse overall survival, indicating that circATP2A2 might be a prognostic biomarker. Also, the AUC of circATP2A2 was 0.77, manifesting that circATP2A2 might be a diagnostic biomarker. In addition, circATP2A2 silencing repressed OS cell proliferation, migration, and invasion in vitro. Furthermore, circATP2A2 knockdown decreased the xenograft tumor growth in vivo, and the protein level of Ki67 (a good marker of proliferation marker) was lower in cir-cATP2A2-knockdown xenograft tumors. These results suggested that circATP2A2 silencing decreased OS growth in vivo. Therefore, we inferred that circATP2A2 acted as a promoter in OS and might be a biomarker and target for OS diagnosis and treatment. A characteristic of cancer cells is that their glucose metabolism changes from mainly oxidative respiration to aerobic glycolysis [29]. The elevation of glycolysis accelerates cancer cell angiogenesis and metastasis [30]. HK2 and PKM2, the rate-limiting enzymes in glycolysis, are involved in the conversion of glucose to pyruvate [24]. Herein, circATP2A2 inhibition reduced HK2 and PKM2 protein levels in OS cells in vitro and xenograft tumors in vivo. These data manifested that circATP2A2 promoted OS cell glycolysis.
Based on the ceRNA hypothesis that circRNAs can adsorb miRs by MREs, thus regulating the targets of miRs [31], we uncovered circATP2A2 as a miR-335-5p decoy. Wang et al. reported that DANCR adsorbed miR-1972 and miR-335-5p to elevate ROCK1 expression, resulting in facilitating OS cell growth and metastasis [32]. Also, TUG1 elevated ROCK1 expression by serving as a miR-335-5p decoy, thereby accelerating OS cell invasion and migration [33]. These results illustrated that miR-335-5p played an antitumor tumor activity in OS. Our data also presented that miR-335-5p silencing reversed the suppressive impacts of circATP2A2 silencing on OS cell malignancy and glycolysis. Thus, we concluded that circATP2A2 acted as a miR-335-5p decoy and promoted OS cell malignancy and glycolysis by sequestering miR-335-5p.
In addition, MYH9 was validated as a miR-335-5p target. Wang et al. discovered that MYH9 could activate the MAPK/AKT pathway, resulting in colorectal cancer cell growth and metastasis [34]. Also, the negative feedback loop of miR-6089/MYH9/β-catenin/c-Jun constrained the growth of ovarian cancer [35]. Zhou et al. showed that MYH9 expression was strikingly increased in OS, and high MYH9 expression was associated with OS cell invasion and metastasis [36]. Also, MRPL23-AS1 accelerated carcinogenesis and tumor growth via increasing MYH9 by sponging miR-30b in OS [37]. In our study, MYH9 elevation reversed the inhibitory impacts of the miR-335-5p mimic on OS cell malignancy and glycolysis. Additionally, MYH9 was regulated by the circATP2A2/miR-335-5p axis. Therefore, we concluded that circATP2A2 promoted OS cell malignancy and glycolysis through upregulating MYH9 expression by sponging miR-335-5p (Graphical abstract image).

Conclusion
circATP2A2 played a carcinogenic role in OS. Furthermore, circATP2A2 promoted malignant behaviors and glycolysis by serving as a miR-335-5p decoy and elevating MYH9 expression. This research provided a new mechanism of circATP2A2 responsible for tumor growth and glycolysis in OS. Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.