Circular RNA ROCK1, a novel circRNA, suppresses osteosarcoma proliferation and migration via altering the miR-532-5p/PTEN axis

As the most prevalent bone tumor in children and adolescents, the pathogenesis and metastasis of osteosarcoma (OS) remain largely unclear. Here, we investigated the expression and function of a novel circular RNA (circRNA), circROCK1-E3/E4, which is back-spliced from exons 3 and 4 of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) in OS. We found that circROCK1-E3/E4, regulated by the well-known RNA-binding protein quaking (QKI), was downregulated in OS and correlated with unfavorable clinical features of patients with OS. Functional proliferation and cell motility assays indicated that circROCK1-E3/E4 serves as a tumor suppressor in OS cells. Mechanistically, circROCK1-E3/E4 suppressed proliferation and migration by upregulating phosphatase and tensin homolog (PTEN) through microRNA-532-5p (miR-532-5p) sponging. In the constructed nude mouse model, circROCK1-E3/E4 inhibited tumor growth and lung metastasis in vivo. This study demonstrates the functions and molecular mechanisms of circROCK1-E3/E4 in the progression of OS. These findings may identify novel targets for the molecular therapy of OS.


INTRODUCTION
Osteosarcoma (OS) is the most frequent malignant bone neoplasm, particularly among children and adolescents. The prediction site of osteosarcoma is most often in the metaphysis of long bones 1 . Owing to its aggressive characteristics, the prognosis of osteosarcoma is unfavorable. According to a previous study, the mortality rate of osteosarcoma was approximately 1.3% per year over the years 1990-2004 in the USA 2 . It was reported that approximately 20% of patients presented with pulmonary metastasis at their first visit due to the vicious biological behavior 2 . Consequently, it is compelling to seek out novel metastasis-related molecules and identify their underlying mechanism in osteosarcoma.
Circular RNAs (circRNAs), produced by precursor mRNA backsplicing of exons, are a distinguished kind of closed-ring structured transcripts without 5ʹ-end caps and 3ʹ-end poly A tails 3 . CircRNAs exhibit multiple functions, such as acting as miRNA sponges, interacting with proteins, being translated into proteins and regulating transcription 3 . Circular RNAs, serving as pseudogenes, regulate parental gene expression via a competitive endogenous RNA (ceRNA) mechanism [4][5][6] . CircRNAs are extensively implicated in various diseases, including osteosarcoma 7,8 . It has been reported that circular RNA TADA2A (circTADA2A), derived from exons 5 and 6 of TADA2A mRNA, increases CREB3-mediated malignant tumor behavior by readily sponging miR-203a-3p in osteosarcoma 9 .
Rho-associated coiled-coil containing kinase 1 (ROCK1) is a member of the AGC family of serine/threonine kinases, and ROCK1 was identified as an effector of RhoA 10,11 . ROCK1 has manifold functions in cancer-related pathogenesis, including cell contraction, migration, apoptosis, survival, and proliferation 12 . Previous studies from our group have also uncovered the oncogenic roles of ROCK1 in osteosarcoma [13][14][15][16] . According to Rybak-Wolf A and Salzman J's study, a total of 53 circRNAs are derived from the human ROCK1 gene [17][18][19] . However, the expression and function of any ROCK1-back-spliced circRNAs in osteosarcoma remain largely unknown. In the present study, we concentrated on investigating the expression and function of hsa_circ_0108024, a circular RNA derived from exons 3 and 4 of the ROCK1 gene, in osteosarcoma. We found that hsa_circ_0108024, which we named circROCK1-E3/ E4, serving as a tumor suppressor, inhibited proliferation and migration in osteosarcoma.

MATERIALS AND METHODS Patients and tissue samples
A total of 50 osteosarcoma tissue specimens and paired paratumor tissue specimens were collected in Central Hospital Affiliated to Shenyang Medical College (Shenyang, China) and in Cancer Hospital of China Medical University/Liaoning Cancer Hospital & Institute (Shenyang, China) during surgical resections. The patients were clinically diagnosed with osteosarcoma according to a definite pathological diagnosis, and written informed consent was collected from these patients. Permission for this study was granted by the Institute Research Medical Ethics Committee of Central Hospital Affiliated to Shenyang Medical College.

Actinomycin D assay and RNase R treatment
To determine the stability of circROCK1-E3/E4, an actinomycin D assay and an RNase R assay were performed. The actinomycin D assay was carried out as previously described 20 . In brief, 2 μg/ml actinomycin D (Merck KGaA) was added. Total RNA from different time points (0, 4, 8, 12, and 24 h) was extracted, and the expression of circROCK1-E3/E4 and linear ROCK1 mRNA was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR).
RNase R treatment was carried out as previously described 21 . Total RNA (2 mg) was incubated for 30 min at 37°C with or without 5 U/μg RNase R (Epicenter Technologies Pvt. Ltd) and subsequently purified using an RNeasy MinElute Cleaning Kit (Qiagen, Inc.). Analysis was then performed by RT-qPCR.

Nucleic acid electrophoresis
The cDNA and gDNA PCR products were investigated using 2% agarose gel electrophoresis with TAE running buffer. DNA was separated by electrophoresis at 120 V for 30 min. The DNA marker used was Super

RNA-fluorescence in situ hybridization (FISH)
A FISH assay was employed to determine the subcellular localization of circROCK1-E3E4 in OS tissues and cell lines as previously described 22 .
Paraffin-embedded OS tissues were sliced, dewaxed and gradient dehydration was performed. OS cell coverslips were rinsed and fixed with 4% paraformaldehyde at room temperature for 30 min. The tissue slices or cell coverslips were digested with 20 μg/ml proteinase K (Solarbio, Beijing, China) and washed three times with PBS for 5 min each. Hybridization buffer was added to tissue slices or cell coverslips and incubated at 37°C for 1 h. One hour later, the prehybridization solution was removed, and the labeled fluorescent probes were added to the tissue slices or cell coverslips and incubated at 37°C overnight. The next day, the tissue slices or cell coverslips were washed in a gradient with 2 × SSC (supplemented with 50% formamide, 5 min), 1 × SSC (5 min) and 0.5 × SSC (10 min) at 37°C. The tissue slices or cell coverslips were incubated with 4',6-diamidino-2phenylindole (DAPI) for 8 min in the dark to counterstain nuclei, washed with PBS, air-dried, mounted, and photographed using a Leica SP5 confocal microscope (Leica Micosystems, Mannheim, Germany).

RNA extraction, reverse transcription, and qPCR
The procedures were performed as previously described 23 . Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For qualification of circRNAs, 3U RNase R enzyme per 1000 ng RNA was used to digest linear RNAs at 37°C for 10 min. Isolated RNAs (circRNA, mRNA, and pre-mRNA) were reverse transcribed into cDNAs using a Goscript TM Reverse Transcription System (Promega, Madison, WI, USA), and qPCR was carried out using GoTaq® qPCR Master Mix (Promega) according to the manufacturers' instructions. GAPDH was used as an internal control for circRNAs and mRNAs, and U6 was used as an endogenous control for miRNAs. The relative expression levels of the genes were determined using the 2 -ΔΔCt method. The primer sequences are presented in Table 1.

Western blot analysis
The procedure was carried out as previously described 21 . In brief, proteins from cells or tissues were extracted by a total protein extraction kit (Abcam) and qualified by a BCA Protein Assay Kit (Abcam). Twenty micrograms of protein samples were subjected to electrophoresis, transferred onto a polyvinylidene difluoride membrane (EMD Millipore) and blocked with 5% skimmed milk for 60 min at room temperature. The membrane was incubated with primary antibody (Abcam) solution at 4°C overnight, followed by 15 min of TBST washing three times. The membrane was incubated with secondary antibody at room temperature for 60 min. Signals of targeted proteins were detected using an enhanced chemiluminescence detection system.

IHC and in situ hybridization (ISH)
Osteosarcoma tissues were fixed with 10% formalin, dehydrated via gradient, paraffin-embedded, sliced, dewaxed and rehydrated. Osteosarcoma Section (4-μm thick) were retrieved by antigen retrieval buffer, blocked with 3% hydrogen peroxide and sealed with 3% BSA. For IHC detection, the sections were incubated with primary antibodies (anti-QKI, cat. no. ab126742; anti-PTEN, cat. ab267787; Abcam, Cambridge, MA, UK) at 4°C overnight. The next day, the sections were incubated with secondary antibodies (cat. no. ab6112, Abcam) for 30 min at room temperature, stained with 0.05% 3,3-diaminobenzidine for 60 s, counterstained with 10% hematoxylin for 3 min, dehydrated with graded ethanol, and observed under a light microscope. For ISH detection, the sections were incubated with prehybridization solution for 1 h at 37°C and then incubated with hybridization solution containing the miR-532 probe at 4°C overnight. The next day, the sections were steeply washed with 2 × SSC (5 min, 37°C), 1×SSC (5 min, 37°C) and 0.5 × SSC (10 min, room temperature) and blocked with 5% BSA (30 min, room temperature). The sections were then incubated with mouse anti-digoxigenin-labeled peroxidase (anti-DIG-HRP) at 37°C for 40 min. Then, the sections were stained with 0.05% 3,3-diaminobenzidine for 60 s, counterstained with 10% hematoxylin for 3 min, dehydrated with graded ethanol, and observed under a light microscope.

Cell counting kit-8 (CCK8) assay
The procedure was performed as previously described 16 . Briefly, HOS and U2OS cells with diverse interventions were incubated in 96-well plates (2 × 10 3 ) supplemented with 200 µl culture medium and conditioned at 37°C with 5% CO 2 . On Days 1, 2, 3, 4, and 5, 20 μl CCK-8 solution was added to each well and then incubated for 2 h. Absorbance was measured at an optical density of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). Experiments were repeated in triplicate.

5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
The EdU incorporation assay was performed according to the protocol of a Click-iT® EdU Kit (Cat. no: C10338; Invitrogen, USA). HOS and U2OS cells (5 × 10 5 ) with diverse interventions were seeded onto glass coverslips (18 × 18 mm) and cultured overnight. The next day, 1 ml of the medium was removed, an equal volume of EdU labeling solution (concentration of 20 µM) was added to each coverslip, and the coverslips were incubated under appropriate growth conditions. Two hours later, the culture medium was discarded, and the coverslips were fixed with 3.7% formaldehyde for 15 min at room temperature. The coverslips were washed twice with 1 ml of 3% BSA in PBS and then permeabilized with 0.5% Triton® X-100 for 20 min at room temperature. The permeabilization buffer was discarded, and the coverslips were washed twice with 3% BSA in PBS. The coverslips were incubated with 0.5 ml of Click-iT® reaction cocktail and rocked in the dark for 30 min at room temperature. The coverslips were washed with 3% BSA in PBS and labeled with 1× Hoechst® 3334 in the dark for 30 min at room temperature. The coverslips were observed under a fluorescence microscope (Leica, Wetzlar, Germany). The nucleus is blue after labeling with DAPI. Positive cells are presented in green. Images were analyzed using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The quantitative data are expressed as the percentage of EdU-positive nuclei relative to the total number of nuclei counted.

Transwell assay
The procedure was performed as previously reported 15 . HOS and U2OS cells with diverse interventions (with an incubation density of 5 × 10 4 ) were incubated in the upper chambers (Corning). Culture medium without and with 10% FBS was added to the upper and lower chambers, respectively. After 12 h, nonmigrated cells were wiped out, while migrated HOS and U2OS cells were fixed, stained and counted using an inverted microscope (Olympus Corporation).

RNA immunoprecipitation (RIP) assay
The RIP assay was performed strictly according to the instructions of a Magna RIP RNA-Binding Protein Immunoprecipitation kit (EMD Millipore, USA). In brief, HOS and U2OS (2 × 10 7 ) cells were harvested, and the cell pellet was lysed with RIP lysis buffer combined with a protease inhibitor cocktail and RNase inhibitor. Ten microliters of the supernatant of the cell lysate was used as the input. Magnetic beads were resuspended and vortexed twice with RIP wash buffer. The magnetic beads were incubated with anti-AGO2 antibody (cat. no. ab186733, Abcam) or anti-IgG antibody (cat. no. ab172730, Abcam) under rotation for 30 min at room temperature. The labeled magnetic beads were harvested, and 900 μl of RIP immunoprecipitation buffer and 100 μl of cell lysate were added to the labeled magnetic beads and incubated Fig. 1 CircROCK1-E3/E4 is downregulated and associated with poor prognosis in patients with osteosarcoma. a The expression of certain ROCK1-derived circRNAs in osteosarcoma tissue specimens and paired paratumor tissue specimens was determined by an RT-qPCR assay. b Representative images of circROCK1-E3/E4 expression in osteosarcoma and paratumor tissues as displayed by a FISH assay. c The expression of circROCK1 in 50 osteosarcoma and paired paratumor tissue specimens was detected by RT-qPCR assay. **** p < 0.0001. d CircROCK1-E3/E4 was downregulated in osteosarcoma cell lines, as confirmed by an RT-qPCR assay. **** p < 0.0001. e CircROCK1-E3/E4 is circularized from exons 3 and 4 in ROCK1, as demonstrated by a schematic illustration. f CircROCK1-E3/E4 was amplified by divergent primers in cDNA but not gDNA by using GAPDH as a negative control, as validated by an RT-qPCR assay. g Compared with oligo (dT) 18 primers, the expression of circROCK1-E3/E4 was significantly decreased when random hexamer primers were applied. n.s p > 0.05 and ** p < 0.01. h Compared with linear ROCK1 mRNA, the expression changes of circROCK1-E3/E4 were unremarkable in the presence of RNase R. n.s p > 0.05 and ** p < 0.01. i The RNA levels of circROCK1-E3/E4 and ROCK1 mRNA after 2 μg/ml actinomycin D treatment were quantified at different time points by qRT-PCR assays. ** p < 0.01. j CircROCK1-E3/E4 was mainly localized in the cytoplasm of U2OS and HOS cells. k CircROCK1-E3/E4 is frequently downregulated in patients with lymph node metastasis (N1 and N2). * p < 0.05, **** p < 0.0001. l CircROCK1-E3/E4 is downregulated in patients with distant metastasis (M1 stage). **** p < 0.0001. m ROC curve analysis of the area under the curve (AUC) and 95% confidence interval (CI) of circROCK1-E3/E4 in patients with osteosarcoma. n A low expression level of circROCK1-E3/E4 was correlated with shorter survival rates in patients with osteosarcoma as determined by Kaplan-Meier analysis. All data are presented as the mean ± SD from three independent experiments. with rotation for 3 h overnight at 4°C. RNeasy MinElute Cleanup kit (Qiagen, Inc.) was used to extract the immunoprecipitated RNA, and extracted RNAs were reverse transcribed and subjected to qPCR to detect the abundance of targeted circRNAs.

GEO database reanalysis and prediction of miRNAs
The differentially expressed miRNA data in osteosarcoma were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE65071). The circRNA-miRNA interaction networks were predicted using circBank 24 , and the mRNA-miRNA interaction networks were predicted using TargetScan (http://www.targetscan.org/vert_71/) and miRDB 25 according to the instructions of the websites. Overlapping miRNAs in all the mentioned databases or websites were selected for further research.

Dual-luciferase reporter assay
The procedure was performed as previously described 26  In vivo nude mouse model The procedure was performed as previously reported 16

Statistical analysis
Statistical analysis was performed by GraphPad Prism 7.0. All numerical data are presented as the mean ± SD for multiple samples with at least three replicates. Differences between groups were evaluated by Student's t test or one-way analysis of variance, as appropriate. Overall survival differences between patients with high or low levels of circROCK1-E3/E4 expression were analyzed using the Kaplan-Meier method and log-rank test. * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered to indicate statistically significant differences.

CircROCK1-E3/E4 is downregulated and associated with poor prognosis of patients with osteosarcoma
It is widely accepted that most circRNAs are back-spliced from exons of their parental genes. In our previous research, we demonstrated that ROCK1 plays key roles in osteosarcoma progression [13][14][15][16]27 . In the current research, we focused on identifying potential circRNAs that might be derived from the ROCK1 gene. According to the online circRNA database circBase 18 , a total of 53 circRNAs are derived from the human ROCK1 gene (Supplementary Table 1 and Supplementary Fig. 1a). Among them, we chose 20 exonic circRNAs with lengths shorter than 500 bp for further detection in 5 osteosarcoma tissues and paired paratumor tissues. Based on the results of RT-qPCR displayed in Fig. 1a, hsa_circ_0108024, which we named circROCK1-E3/E4 because it was derived from exons 3 and 4 of the ROCK1 gene, was stably downregulated in the collected 5 osteosarcoma tissues. Through an RNA-FISH assay, we demonstrated that circROCK1-E3/E4 was downregulated in osteosarcoma tissues (Fig. 1b). Further RT-qPCR detection also indicated that circROCK1-E3/E4 was downregulated in osteosarcoma tissues (Fig. 1c). Additionally, we found that circROCK1-E3/E4 was downregulated in four osteosarcoma cell lines, MG63, U2OS, HOS and 143B, compared with that in the normal osteoblast cell line hFOB1.19 (Fig. 1d). By comparing the RNA sequence of its parental gene ROCK1 through analysis of circBase and the online circRNA annotation software circPrimer 28 , circROCK1-E3/E4 was back-spliced from exon 3 and exon 4 of the ROCK1 gene ( Fig. 1e and Supplementary Fig. 1b). Furthermore, convergent and divergent primers were designed to verify the head-to-tail splicing model of circROCK1-E3/E4. As the results of nucleic acid electrophoresis displayed in Fig. 1f, circROCK1-E3/E4 could be detected in the cDNA of U2OS and HOS cells but not in gDNA. To further test the circular structure of circROCK1-E3/E4, a random hexamer primer and an oligo (dT) 18 primer were reverse transcribed with RNAs extracted from U2OS and HOS cells. The expression of circROCK1-E3/E4 and ROCK1 mRNA was detected. As the results displayed in Fig. 1g show, compared with random hexamer primers, the expression of circROCK1-E3/E4 was aberrantly decreased in the oligo (dT)18 primer group. Meanwhile, the expression of ROCK1 mRNA changed insignificantly. This phenomenon suggested that circROCK1-E3/E4 had no poly-A tail. Next, we performed an RNase R assay and an actinomycin D assay to verify the stability of circROCK1-E3/E4, and the results are shown in Fig.  1h, i. circROCK1-E3/E4 was more stable than ROCK1 mRNA after RNase R and actinomycin D digestion. Furthermore, via an RNA-FISH assay, we confirmed that ROCK1-E3/E4 was mainly located in the cytoplasm of U2OS and HOS cells (Fig. 1j). Clinically, we demonstrated that circROCK1-E3/E4 was more frequently downregulated in osteosarcoma patients with lymph node metastasis and distant metastasis (Fig. 1k, l). Additionally, we applied ROC curve analysis to evaluate the clinical value of circROCK1-E3/E4. As presented in Fig. 1m, the area under the curve (AUC) was 0.9048 (with a 95% confidence interval of 0.8503-0.9593), indicating that circROCK1-E3/E4 might be a valuable prognostic biomarker for the diagnosis of osteosarcoma. According to the median value of circROCK1-E3/E4, the 50 collected cases were divided into a circROCK1-E3/E4 high group (n = 25) and a low group (n = 25). According to a Pearson chi-square test and Fisher's exact test, we found that a low expression level of circROCK1-E3/E4 was closely correlated with advanced staging (IIB/III, p = 0.007) and distant metastasis (p = 0.023) and tumor size (p = 0.038) in patients with osteosarcoma (Table 2). Last, we demonstrated that a lower expression level of circROCK1-E3/E4 was intimately correlated with shorter overall survival rates in patients with osteosarcoma (p = 0.083) (Fig. 1n).

CircROCK1-E3/E4 expression is partially regulated by QKI
It is widely accepted that circRNAs are usually generated from long flanking introns with inverted complementary sequences 29 . Therefore, we attempted to explore whether circROCK1-E3/E4 was formed via a similar mechanism. First, by blasting the complementary sequences of intron 2 and reverse sequences of intron 4 of ROCK1, we found very highly similar sequences (83% identity over 294 nt, Fig. 2a). The highly similar sequences in intron 2 of ROCK1 (position of Chr18: 18649177-18649468, hg19, which we named flanking intron segments 2, FIS2) and the sequence spanning from exon 3 of ROCK1 (position of Chr18: 18666972-18667072) to the highly similar sequences in intron 4 of ROCK1 (position of Chr18:18670654-18670944, hg19, which we named flanking intron segments 4, FIS4; in total of 3972 bp) were cloned into pZW1-snoVector 30 (vector 1#). Moreover, different deletion constructs were cloned (vector 2# -4#) separately (Fig. 2b, upper panel). All the constructed vectors were transfected into U2OS and HOS cells. Then, the expression levels of circROCK1-E3/E4 were quantified by qRT-PCR. As shown in Fig. 2b (lower panel), only vector #1 promoted circROCK1-E3/E4 expression in U2OS and HOS cells. It has been reported that the biogenesis of circRNAs is regulated by RNA-binding proteins (RBPs). Quaking (QKI), a wellknown RBP, promotes circRNA formation by binding to its consensus target single-stranded RNA (ssRNA) motif in introns flanking circRNA-forming consensus target exons 31 . We attempted to explore whether QKI was also implicated in the biogenesis of circROCK1-E3/E4. We knocked down QKI via an RNAi technique (Fig. 2c) and assessed the expression of circROCK1-E3/E4 and ROCK1 mRNA by an RT-qPCR assay. As shown in Fig. 2d, knockdown of QKI suppressed the expression of circROCK1-E3/ E4 but not ROCK1 mRNA. To confirm the targeted binding effect between QKI and sequences of intron 2 and intron 4, an RNA immunoprecipitation (RIP) assay was performed. As illustrated in Fig. 2e, compared with β-actin, partial sequences of intron 2 and intron 4 were abundantly enriched in QKI proteins rather than IgG.
CircROCK1-E3/E4 suppresses proliferation and migration in vitro. In this section, we attempted to assess the functional role of circROCK1-E3/E4 in osteosarcoma cells. We designed specific Very highly similar sequences (83% identity over 294 nt) were found, as illustrated by using BLAST (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). b Four vectors (vector #1 containing FIS2 and FIS4; vector #2 with FIS2 deletion; vector #3 with FIS4 deletion; vector #4 with FIS2 and FIS4 deletion) were transfected into U2OS and HOS cells, and the expression level of circROCK1-E3/E4 was checked by an RT-qPCR assay. **** p < 0.0001. c QKI was knocked down as determined by a western blot assay. d Expression of QKI mRNA, circROCK1-E3/E4 and ROCK1 mRNA after transfection of QKI siRNAs was analyzed by an RT-qPCR assay. n.s p > 0.05 and ** p < 0.01. e By using IgG as a control, partial segments of intron 2 and intron 4 of ROCK1 rather than β-actin were abundantly enriched in QKI, as measured by an RIP assay. n.s p > 0.05 and **** p < 0.0001. All data are presented as the mean ± SD from three independent experiments.
siRNAs targeting the back-splice sequence of circROCK1-E3/E4. As shown in Fig. 3a, b, circROCK1-E3/E4 was successfully knocked down, while a change in ROCK1 mRNA was not detected in U2OS and HOS cells. Additionally, the circROCK1-E3/E4 overexpression vector was utilized to upregulate the expression of circROCK1-E3/ E4 in U2OS and HOS cells. As shown in Fig. 3c, d, circROCK1-E3/E4 was successfully upregulated in U2OS (clones 5 and 9) and HOS (clones 3 and 6) cells, while ROCK1 mRNA changed insignificantly. Subsequently, functional cell proliferation and motility assays were performed in the aforementioned cells. The CCK8 assay results shown in Fig. 3e, f revealed that downregulation of circROCK1-E3/ E4 promoted proliferation in U2OS and HOS cells. In contrast, overexpression of circROCK1-E3/E4 suppressed the proliferation ability of U2OS and HOS cells (Fig. 3g, h). Additionally, the outcomes of the EdU assay indicated a similar tendency (Fig. 3i). Finally, a functional transwell chamber migration assay was performed to detect the migration ability changes in U2OS and HOS cells. As the representative photos displayed in Fig. 3j, k Fig. 3 CircROCK1-E3/E4 suppresses osteosarcoma cell proliferation and migration in vitro. a, b CircROCK1-E3/E4 expression levels after transfection of specific siRNAs were measured by an RT-qPCR assay. n.s p > 0.05 and ** p < 0.01. c, d Expression levels of circROCK1-E3/E4 and ROCK1 mRNA were analyzed by an RT-qPCR assay. n.s p > 0.05 and ** p < 0.01. e-h Changes in cell proliferation ability after various circROCK1-E3/E4 interventions were determined by a CCK8 assay. **** p < 0.0001. i An EdU assay was performed to evaluate changes in cell proliferation ability after up-and downregulation of circROCK1-E3/E4. **** p < 0.0001. j, k Cell motility ability changes after different circROCK1-E3/E4 interventions were determined by a transwell assay. **** p < 0.0001. All data are presented as the mean ± SD from three independent experiments.
MiR-532-5p promotes proliferation and migration by targeting PTEN in U2OS and HOS cells. As a well-known tumor suppressor, PTEN is extensively implicated in ncRNA-mediated cancer progression in multiple malignancies [35][36][37][38] . We first indicated that up-and downregulation of miR-532-5p inversely regulated PTEN expression (Fig. 5a, b). Functionally, as the data presented by the CCK8 assay and EdU assay displayed in Fig. 5c-f and Supplementary Fig.  3a-d, upregulation of miR-532-5p (transfection of miR-532-5p mimics) facilitated the proliferation of HOS and U2OS cells. This facilitative effect was significantly reversed by further upregulation of PTEN (cotransfection of miR-532-5p mimics and PTEN overexpression plasmids). In contrast, downregulation of miR-532-5p inhibited cell proliferation, and the suppressive effect was partially abolished by PTEN siRNA (cotransfection of miR-532-5p and siPTEN). Additionally, regarding the cell motility changes, the transwell assay indicated a similar tendency (Fig. 5g, h). Finally, through a luciferase assay, we confirmed that miR-532-5p targets PTEN by directly binding to the PTEN 3'UTR (Fig. 5i).
CircROCK1-E3/E4 suppresses proliferation and lung metastasis in vivo. To finally confirm the role of circROCK1-E3/E4 in tumor growth and lung metastasis, orthotopic xenograft mouse models were introduced. As the representative photographs displayed in Fig. 7a, b show, compared with the vector group, overexpression of circROCK1-E3/E4 significantly suppressed osteosarcoma tumor growth. Additionally, 18 F-FDG PET/CT scans were used to assess the tumor growth of osteosarcoma in vivo. As the representative images show in Fig. 7c, the standard uptake values (SUVs) of the circROCK1-E3/E4 group were remarkably lower than those of the vector group (p = 0.0016). Furthermore, the expression of circROCK1-E3/E4, miR-532-5p and PTEN mRNA in the formatted subcutaneous nodes was determined by qRT-PCR assays. As shown in Fig. 7d-f, upregulated circROCK1-E3/E4 and PTEN mRNA were found in the circROCK1-E3/E4 group compared with the vector group. However, the expression changes of miR-532-5p were unremarkable. A further Western blot assay and IHC assay suggested that overexpression of circROCK1-E3/E4 promoted PTEN protein expression in the formatted subcutaneous nodes (Fig. 7g, h). Moreover, a pulmonary metastasis model of osteosarcoma in mice was constructed. As the representative photos demonstrate in Fig. 7i, overexpression of circROCK1-E3/ E4 suppressed metastatic node formation in the lungs. Additionally, 18 F-FDG PET/CT scans suggested that the SUVs of the circROCK1-E3/E4 group were significantly lower than those of the vector group (p = 0.0004) (Fig. 7j). Last, as illustrated in Fig. 7k-m, overexpression of circROCK1-E3/E4 promoted PTEN but had no effect on miR-532-5p expression in metastatic nodes in the lung. Together, these results confirmed that suppression of circROCK1-E3/E4 inhibited the lung metastasis of osteosarcoma in vivo.
Emerging studies have indicated that circRNAs are extensively implicated in various pathological processes, including neuronal function, innate immune responses, cell proliferation, and pluripotency 3 . Circular RNAs regulate their parental gene function by absorbing common miRNAs to increase miRNA availability or reduce free miRNA levels 6 . For example, circular RNA forkhead box O 3 (circ-Foxo3) and Foxo3 pseudogene (Foxo3P) sponge several miRNAs and increase Foxo3 translation 39 . In the current study, we focused on circular RNAs that might be back-spliced from the ROCK1 gene. We provided evidence that circROCK1-E3/E4, which is produced from exons 3 and 4 of ROCK1 precursor mRNA, was downregulated in osteosarcoma. Additionally, we found that downregulated circROCK1-E3/E4 was closely correlated with poor prognosis in patients with osteosarcoma. Mechanistically, we demonstrated that the introduction of circROCK1 was partially regulated by QKI, a well-known RBP belonging to the STAR family of KH domain-containing RBPs 40 . The biogenesis of circRNAs can be regulated at different levels, including precursor RNA transcription, post-or cotranscriptional Fig. 4 CircROCK1-E3/E4 sponges miR-532-5p in U2OS and HOS cells. a, b Compared with circANRIL, circROCK1 was abundantly enriched in AGO2 by using IgG as an internal control. n.s p > 0.05 and *** p < 0.001 c A Venn diagram exhibiting overlapping miRNAs that might interact with circROCK1-E3/E4 and presented in GSE65071. d Expression of miR-532-5p in osteosarcoma tissues was illustrated by an in situ hybridization assay. e Expression of miR-532-5p in osteosarcoma tissues and paired healthy tissues in the osteosarcoma-related genome-wide study GSE65071. Probe ID 49 represents miR-532-5p. f Spearman correlation analysis showed that circROCK1-E3/E4 was inversely correlated with miR-532-5p. r = -0.5406 and p < 0.0001. g Expression levels of miR-532-5p in osteosarcoma cell lines were confirmed by an RT-qPCR assay. h The subcellular localization of circROCK1-E3/E4 and miR-532-5p was confirmed by a FISH assay. i The targeted binding effect and binding sites between circROCK1-E3/E4 and miR-532-5p were tested by a luciferase assay. n.s p > 0.05 and * p < 0.05. j, k Expression of miR-532-5p after up-and downregulation of circROCK1-E3/E4 was analyzed by RT-qPCR. n.s p > 0.05 and ** p < 0.01. l, m Relative expression of circROCK1-E3/E4 after different miR-532-5p interventions was measured by RT-qPCR. n.s p > 0.05. All data are presented as the mean ± SD from three independent experiments.  5 MiR-532-5p promotes proliferation and migration by targeting PTEN in U2OS and HOS cells. a, b Expression of PTEN after treatment with different miR-532-5p concentrations was determined by a western blot assay and an RT-qPCR assay. **** p < 0.0001. c-f Cell proliferation ability changes in HOS and U2OS cells were determined by CCK8 and EdU assays, respectively. **** p < 0.0001. g, h Cell migration and invasion ability changes in HOS and U2OS cells were measured by a transwell assay. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. i The targeted binding effect between miR-532-5p and PTEN was confirmed by a luciferase assay. The left panel shows the online prediction result by using TargetScan (http://www.targetscan.org/vert_72/), and the middle and right panels display the results of relative luminance changes. n.s p > 0.05 and * p < 0.05. All data are presented as the mean ± SD from three independent experiments. processing and turnover 3 . The QKI protein contains multiple proline-rich putative Src-homology 3 (SH3)-binding motifs, and QKI is implicated in RNA metabolism by binding to signaling proteins that contain SH3 domains 41 . As a classic nuclear RNAbinding protein, QKI regulates the formation of circRNAs by binding sites in introns flanking circRNA-forming exons 31 . In hepatocellular carcinoma (HCC), knockdown of QKI significantly reduced circular RNA zinc-finger protein with KRAB and SCAN domains 1 (circZKSCAN1) expression in HCC cell lines SMMC7721 and HCC-LM3 42 . In this study, we provided proof that QKI Fig. 6 CircROCK1-E3/E4 suppresses proliferation and migration via regulation of the miR-532-5p/PTEN axis in U2OS and HOS cells. a, b Expression of PTEN protein was analyzed by western blot assay. **** p < 0.0001. c, d PTEN protein expression after different circROCK1-E3/E4 and miR-532-5p interventions was measured by western blot. **** p < 0.0001. e, f Cell proliferation ability changes were checked by a CCK8 assay and an EdU assay. **** p < 0.0001. g, h Cell motility changes were evaluated by a transwell assay. **** p < 0.0001. All data are presented as the mean ± SD from three independent experiments. promoted circROCK1-E3/E4 formation posttranscriptionally by binding to flanking sequences (intron 2 and intron 4) of ROCK1 pre-mRNA.
CircRNAs function via multiple mechanisms, such as serving as miRNA sponges, interacting with proteins, being translated into proteins and regulating transcription 3 . In the present research, by functional in vivo and in vitro assays, we found that circROCK1-E3/E4 promoted proliferation and migration by sequestering miR-532-5p, thereby promoting PTEN-mediated proliferation and migration in osteosarcoma. MiRNAs are extensively implicated in multiple aspects of cancer progression, such as proliferation, apoptosis, angiogenesis, migration, invasion, drug resistance and EMT [43][44][45][46][47] . The functional role of miR-532-5p in cancer remains controversial. MiR-532-5p serves as an oncogene in various malignancies, such as colorectal cancer, lung cancer, ovarian cancer and renal cancer [48][49][50][51] . Furthermore, it functions as an oncogenic microRNA and promotes gastric cancer cell proliferation and migration 52 . The current research first illustrates the role of miR-532-5p in osteosarcoma. Although the mechanism of ceRNAs in the cancer field is no longer a trending topic, our present study still provides novel evidence of miR-532-5p in osteosarcoma. We found that miR-532-5p can act as a bridge between circROCK1/ E3E4 and PTEN. Additionally, our results indicated that both circROCK1/E3E4 and PTEN were targets of miR-532-5p.
PTEN, a well-known tumor suppressor, is widely involved in the progression of various cancers, including cell metabolism, cell motility, cell polarity and the tumor microenvironment 53 . It negatively regulates the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in osteosarcoma. Our present work showed that PTEN serves as a downstream target of miR-532-5p and is closely implicated in cell proliferation and migration in osteosarcoma.
In conclusion, as shown in the diagram displayed in Fig. 7n, our findings in the present research are the first to reveal that circROCK1/E3E4, regulated by QKI, suppressed cell proliferation and migration by altering the miR-532-5p/PTEN axis in osteosarcoma. Our findings might lay the foundation for further functional, diagnostic and therapeutic research of circRNAs in osteosarcoma. Fig. 7 CircROCK1-E3/E4 suppresses proliferation and lung metastasis in vivo. a Macroscopic appearance of nude mice injected with osteosarcoma cells with stable overexpression of circROCK1-E3/E4 or with control cells (vector). b Overexpression of circROCK1-E3/E4 obviously restricted subcutaneous nodule formation. ** p < 0.01. c Representative 18 F-FDG micro positron emission tomography/computed tomography (PET/CT) images of formatted subcutaneous nodes in mice with diverse circROCK1-E3/E4 expression. p = 0.0016. d-f Expression of circROCK1-E3/E4, miR-532-5p and PTEN mRNA in the formatted subcutaneous nodes was analyzed by RT-qPCR. n.s p > 0.05, and p-values are presented in the figures accordingly. g, h PTEN protein expression in formatted subcutaneous nodes was determined by Western blot and IHC. * p < 0.05 and ** p < 0 . 01. i Macroscopic appearance of formatted lung metastatic nodes in mice. **** p < 0.0001. j Representative 18 F-FDG micro positron emission tomography/computed tomography (PET/CT) images of formatted lung metastatic nodes in mice with diverse circROCK1-E3/E4 expression. p = 0.0004. k H&E staining of formatted lung metastatic nodes; scale bars, 200 and 50 µm for magnifications of ×100 and ×400, respectively. l Expression levels of circROCK1-E3/E4, miR-532-5p, and PTEN mRNA in the formatted lung metastatic nodes were analyzed by RT-qPCR. n.s p > 0.05, ** p < 0.01, and **** p < 0.0001. m PTEN protein expression in formatted lung metastatic nodes was determined by western blot. **** p < 0.0001. n Diagram indicating that circROCK1-E3/E4, back-spliced from exons 3 and exon 4 of the ROCK1 gene, which is regulated by QKI, suppressed cell proliferation and migration by altering the miR-532-5p/PTEN axis in osteosarcoma. All data are presented as the mean ± SD from three independent experiments.