circUSP34 accelerates osteosarcoma malignant progression by sponging miR‐16‐5p

Abstract Osteosarcoma (OS) is a primary and highly malignant mesenchymal tissue tumor. The specific pathological mechanism underlying disease initiation or progression remains unclear. Circular RNAs (circRNAs) are a type of covalently circular RNA with a head‐to‐tail junction site. In this study, we aimed to investigate the sponging mechanism between circRNAs and microRNAs (miRNAs) in OS. Based on the inhibited effect of miR‐16‐5p reported on OS, circUSP34 was analyzed as a sponge of miR‐16‐5p via Starbase. We found that circUSP34 promoted the proliferation, migration, and invasion of OS in vitro and in vivo. circUSP34 increased but miR‐16‐5p decreased in OS by qRT‐PCR. Function assays showed that the malignancy of OS cells, including proliferation, migration, and invasion, was inhibited after knocking out circUSP34. Western blotting results showed that the expression level of vimentin and Ki‐67 decreased. Similarly, miR‐16‐5p mimic compromised the proliferation, migration, and invasion of OS cells. FISH assay results indicated that circUSP34 and miR‐16‐5p were colocalized in the cytoplasm. The sponging mechanism of circUSP34 and miR‐16‐5p was verified by dual‐luciferase reporter assay, RNA immunoprecipitation (RIP), and RNA pull down assays. Interestingly, the miR‐16‐5p inhibitor partly reversed the inhibitory effect of sh‐circUSP34 on the malignancy of OS cells. Further, mice tumors for IHC indicated that vimentin, N‐cadherin, and Ki‐67 protein expression decreased, but E‐cadherin protein expression increased. Collectively, circUSP34 promoted OS malignancy, including proliferation, migration, and invasion, by sponging miR‐16‐5p. It can serve as a potential therapeutic target and biomarker.


| INTRODUC TI ON
OS is a primary mesenchymal malignant tumor that commonly occurs in adolescents and young adults. [1][2][3][4] Under estimation, the OS incidence in teenagers is approximately 5.6/1 000 000. 5 Long tubular bones, such as tibia, fibula, and femur, suffer from OS lesions most commonly. 6 Studies have shown that the combination of surgery and chemotherapy significantly improves patients' prognosis. 7 However, due to its high malignancy and frequent distant metastases, most patients with advanced-stage disease have a terrible prognosis. 8 And more notably, it is still a tough problem to tackle lung metastasis, which remarkably declines patients' survival. Obviously, the positive treatment of metastasis can improve the OS prognosis significantly. 5 The initial cause of OS remains unclear, hindering its diagnosis and early treatment. Therefore, investigating its pathophysiologic mechanisms and exploring novel therapeutic targets are urgently needed.
CircRNA is a kind of head-to-tail closed circular RNA without 5′-and 3′-terminals. It is formed by back-splicing pre-mRNA and is usually over 200 nucleotides long. 9,10 CircRNA can regulate a variety of physiological and pathophysiological activities by interacting with transcript factors, miRNA, and RNA-binding protein (RBP). As the covalent combination, circRNA can be detected in body fluid.
Therefore, it can be used as a diagnostic and prognostic biomarker. 11 Increasingly, studies have reported that abnormally expressed cir-cRNAs are associated with tumorigenesis, proliferation, metastasis, and invasion. 12,13 They are often aberrantly expressed in a variety of tumors, such as non-small cell lung cancer, colorectal cancer, and hepatocellular cancer. [14][15][16] Some circRNAs have oncogenic function, whereas others have anti-oncogenic function. 17 It is well known that circRNA sponging miRNA is a classic interaction in tumors. For example, circ_0000285 promotes the proliferation, migration, and invasion but inhibits the apoptosis of osteosarcoma by sponging miR-409-3p. 18 Upregulated circ_0028171 in cells competes endogenously with miR-218-5p to promote osteosarcoma progression. 19 Based on above literatures, we speculate that circUSP34 plays an oncogenic role in OS by sponging miR-16-5p. It was reported that miR-16-5p inhibited OS malignancy. Herein, circUSP34 was predicted as the sponge of miR-16-5p by Starbase. Previously, circUSP34 was not involved in any tumors. The regulatory mechanisms of circUSP34 in OS by sponging miR-16-5p need to be further explored.

| RNase R treatment
Total RNA extracted from 143B cells was treated with RNase R (Epicenter) under 3 U/μg. Then, the mix was incubated at 37℃ for 20 minutes. The product was examined by quantitative real time PCR (qRT-PCR) and nucleic acid electrophoresis.

| EdU assay
The proliferation ability of cells was evaluated by EdU assay

| Wound-healing assay
KHOS and 143B cells transfected with sh-circUSP34 and sh-NC were seeded in six-well plates at 8 × 10 5 cells per well. When the cell confluency reached ~80%, a wound line was scratched using a P-200 pipette tip. Next, images were taken using a microscope (Leica) at 0 hour and after 24 hours. The distance between the injury lines was measured and analyzed using Image J.

| Transwell assay
Briefly, 200 μL serum-free medium containing 5 × 10 5 cells was added to the upper chamber (Corning) in triplicate. The bottom chamber was filled with 600 μL of medium having 20% FBS. All chambers were incubated at 37°C for 24 hours. The next day, the chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells in the upper membrane of the chamber were slightly wiped using a swab. Finally, the migrating cells in five different areas were counted under a microscope.

| RNA pull down
Biotinylated miR-16-5p was synthesized by GenePharma. Briefly, KHOS cells were collected and then lysed. M280 streptavidin dynabeads (Invitrogen) were incubated with the probe. Then the probedynabeads complex was incubated with cell lysate overnight at 4℃.
RNA conjugated with dynabeads was eluted and purified using TRIzol (Invitrogen) for further qRT-PCR analysis.

| Fluorescence in situ hybridization (FISH) assay
FAM-labeled circUSP34 probes and Cy3-labeled miR-16-5p probes were designed and synthesized by GenePharma. Hybridization was performed overnight at 4°C with circUSP34 and miR-16-5p probes according to the manufacturer's instructions. Images were acquired on a Nikon Eclipse Ti scanning confocal microscope.

| Immunohistochemistry
Tissue samples were fixed in 4% paraformaldehyde, embedded in The results were photographed under a microscope.

| Statistical analyses
SPSS software (version 22.0) and GraphPad Prism 7 were used for statistical analyses. The data were analyzed via Student's t-test or one-way ANOVA, and the results are presented as the mean ± SD.

| The characteristics and expression level of circUSP34 in OS cells and tissues
To better understand the characteristics of circUSP34, its location on the chromosome was identified. Data from circBank (http:// www.circb ank.cn/index.html) and UCSC (http://genome.ucsc. edu/) showed that circUSP34, which is present on chr.2p14, exhibited a circular structure via post-transcriptional head-to-tail splicing ( Figure 1A). Reportedly, circRNAs, including circUSP34 which is relatively stable, are resistant to RNase R digestion. 20 Subsequently, divergent and convergent primers were designed to verify the stability of circUSP34 with and without RNase R treatment using qRT-PCR. The result showed that circUSP34 was not digested, but USP34 mRNA was digested ( Figure 1B). Agarose gel electrophoresis was performed to examine the product of qRT-PCR, and we confirmed that circUSP34 was resistant to RNase R digestion ( Figure 1C), as opposed to USP34 mRNA. qRT-PCR results revealed that circUSP34 had upregulated expression in both 143B and KHOS cells and tissues ( Figure 1D,E). Therefore, we verified the circular structure and stability of circUSP34 and found that it was upregulated in OS.

| CircUSP34 promotes OS cells' malignancy, especially proliferation and migration
143B and KHOS cells were transfected with sh-circUSP34 #1 and #2 to knock down circUSP34. qRT-PCR results showed that the expression level of circUSP34 significantly decreased in both cell lines ( Figure 2A and Figure S1A). To determine the role of circUSP34 in proliferation viability in vitro, CCK8 and EdU assays were performed. All results revealed that interfering circUSP34 compromised the proliferation ability of 143B and KHOS cells ( Figure 2B,C and Figure S1B,C). Moreover, the colony formation assay also indicated that interfering circUSP34 suppressed the proliferation of OS cells, which was consistent with the above results ( Figure 2D and Figure S1D). Overall, these results suggested that circUSP34 can promote the proliferation of OS cells.
Transwell migration as well as invasion and wound-healing assays were performed to further explore the effect of circUSP34 on the migration and invasion of OS cells. A wound-healing assay showed that migration capacity was inhibited in 143B and KHOS cells transfected with sh-circUSP34 compared with that in the NC group ( Figure 2E and Figure S1E). Transwell migration and invasion assays demonstrated that both were inhibited ( Figure 2F). Therefore, these results demonstrated that circUSP34 can advance both migration and invasion ability.
Furthermore, western blot results indicated that vimentin expression decreased after knocking out circUSP34 in KHOS cell.
Additionally, Ki-67 expression level was also downregulated in KHOS cell( Figure 2G and Figure S1F). Taken together, our data indicated that circUSP34 can promote the progression of OS in vitro.

F I G U R E 3
CircUSP34 acts as a sponge to regulate miR-16-5p . A, The potential target site between circUSP34 and miR-16-5p was predicted by Starbase. B, The cellular location of circUSP34 (green) and miR-16-5p (red) in cells was observed by performing FISH (magnification: 400×, scale bar: 100 μm). C, miR-16-5p expression level was confirmed by qRT-PCR after transfecting sh-circUSP34. D, The relative luciferase activity was detected in the KHOS cells after cotransfecting with circUSP34-WT or circUSP34-MUT and mimics or NC, respectively. E, RNA immunoprecipitation (RIP) assay was performed in KHOS cells after transfecting with miR-16-5p mimic or NC respectively, with qRT-PCR and nucleic acid electrophoresis. F, The enrichment of circTLK1 was measured by qRT-PCR in RNA pull down assay. Data are shown as mean ± SD. ns, no significance;*P < 0.05, **P < 0.01

F I G U R E 4 miR-16-5p inhibits malignancy of OS in vitro.
A, The expression level of miR-16-5p was confirmed by qRT-PCR in OS cells and tissues. B and C, CCK8 and EdU assays showed that proliferation viability was inhibited by mimic in both OS cells. D and E, Woundhealing and transwell assays indicated that migration and invasion abilities were suppressed in vitro. F, Colony formation results showed that proliferation ability was suppressed. Data are shown as mean ± SD. *P < 0.05, **P < 0.01

| miR-16-5p inhibits the malignancy of OS in vitro
We next explored miR-16-5p expression in OS cells as well as tissues. qRT-PCR data showed that miR-16-5p expression was significantly downregulated in OS cells compared with HUVEC and in OS tissues compared with non-cancerous tissues( Figure 4A). To confirm the potential anti-tumorous effect of miR-16-5p, gain-offunction assays were performed. CCK-8 assay results showed that miR-16-5p mimic suppressed the proliferation of KHOS and 143B cells ( Figure 4B). For additional verification, the EdU assay was used, and the result showed increased miR-16-5p inhibited OS cells in vitro ( Figure 4C). Similarly, the colony formation assay showed that the proliferation ability was inhibited in vitro ( Figure 4F). miR-16-5p mimic comprised the migration ability of KHOS and 143B cells, as confirmed by the wound-healing assay ( Figure 4D).
Transwell assay results revealed that migration and invasion abilities were inhibited by miR-16-5p mimic ( Figure 4E). Our results showed that miR-16-5p plays a suppressive role in malignant progression in vitro.

| miR-16-5p inhibitor partly reversed the antitumor function of sh-circUSP34
To clarify the specific relationship between miR-16-5p and circUSP34, OS cells treated with sh-circUSP34 were transfected with miR-16-5p inhibitor. The efficacy of transfection was quantified by qRT-PCR. The result showed that the expression level of miR-16-5p decreased but that of circUSP34 increased in OS cells ( Figure 5A). EdU and CCK8 results indicated that miR-16-5p inhibitor rescued the effect ofcircUSP34 on OS cell viability ( Figure 5B,C). Herein, miR-16-5p inhibitor promoted the oncogenic effect of circUSP34 on proliferation. Wound-healing assay results indicated that the function of circUSP34 after knocking out was rescued by the miR-16-5p inhibitor ( Figure 5D). Collectively, these results showed that circUSP34 served as a sponge of miR-16-5p.
F I G U R E 5 miR-16-5p inhibitor reversed the antitumor function of sh-circUSP34. A, The expression level of miR-16-5p and circUSP34 with inhibitor and sh-circUSP34 was confirmed by qRT-PCR, respectively. B and C, CCK8 and EdU assays showed that the oncogenic function of circUSP34 was rescued by the miR-16-5p inhibitor . D, The effect of the miR-16-5p inhibitor on migration in OS cells transfected by sh-circUSP34 was verified by wound-healing assay. Data are shown as mean ± SD. *P < 0.05, **P < 0.01

| CircUSP34 promotes proliferation and metastasis of OS cells in vivo
To confirm the oncogenic function of circUSP34 in vivo, we constructed five BALB/c nude mice models of every group. Every week, the tumor volume of every mouse was measured, and results showed that tumor weight and volume in the sh-circUSP34 Ki-67 protein expression decreased, but E-cadherin protein expression increased ( Figure 6F). Simultaneously, Western blot results also indicated that OS malignancy was markedly compromised. Vimentin, N-cadherin, and Ki-67 expression levels decreased significantly; however, E-cadherin expression level increased ( Figure 6G). These results suggest that circUSP34 acts as an oncogene to promote OS progression.

| D ISCUSS I ON
OS is a primary malignant tumor derived from mesenchymal tissue, but its high-grade malignancy, especially distant metastasis, cannot be neglected. The combination of surgery and chemotherapy has markedly improved patients' prognosis. 21 However, local invasion, metastasis, and drug resistance phenotypes still need to be resolved. 22 Exploring an effective therapeutic target is an urgent and critical issue. Increasingly, studies have shown that circRNAs are aberrantly expressed in various tumors including OS. 23,24 For instance, high expression of circRNA can promote the invasion ability of gastrointestinal cancer, lung cancer, and liver cancer. [25][26][27] The above prompted us to further investigate the potential role of circUSP34 in OS. Thus, we identified circUSP34 using Starbase as an upstream "sponge" of miR-16-5p, which was reported to inhibit OS. 28 In this present study, the expression level of circUSP34 was confirmed by qRT-PCR, which was elevated in OS cells compared with HUVEC. to promote uveal melanoma development. 33 It is worth noting that not every circRNA can sponge miRNAs to modulate their biological functions, as their activity has several requirements, such as a consistent subcellular location. Although miR-16-5p has been previously reported to impede tumor growth, 28 in our study we found that it has an inhibitory function. The low expression level was quantified by performing qRT-PCR, but it significantly increased after circUSP34 knockdown. This result further supported the hypothesis advocating an interaction between circUSP34 and miR-16-5p. Similarly, gain-of-function assay results showed that overexpression of miR-16-5p impeded OS development. Therefore, it is probable that cir-cUSP34 can sponge miR-16-5p. To better understand this activity, a rescue experiment was performed. Importantly, Zhang et al. 34 reported that Smad3 acts as a target of miR-16-5p in chordoma cells.
Therefore, we hypothesize that miR-16-5p inhibits OS malignancy by targeting Smad3. Further studies are required to explore the underlying mechanisms. Our results showed that circUSP34 promotes OS proliferation, migration, and invasion by sponging miR-16-5p.
However, the expression of circUSP34 was negatively correlated with tumor size in vivo.
In summary, our findings revealed that the highly regulated cir-cUSP34 plays a role in oncogenes and underlying biomarkers in OS.
Furthermore, circUSP34 may be a potential therapeutic target for OS.
Ours is the first study to demonstrate that circUSP34 promotes OS proliferation, migration, and invasion by sponging miR-16-5p. However, our study requires further exploration of the mechanism of circUSP34 and its clinicopathological correlation with patients with OS.

ACK N OWLED G EM ENTS
Not applicable.

D I SCLOS U R E
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request to the authors.