hsa-miR-199b-3p suppresses osteosarcoma progression by targeting CCDC88A, inhibiting epithelial-to-mesenchymal transition, and Wnt/beta-catenin signaling pathway

The present study investigated microRNA (miR)-199b-3p expression in osteosarcoma (OS) and aimed to identify its potential mechanism of action contributing to the development of this disease. Firstly, miR-199b-3p and coiled-coil domain containing 88A (CCDC88A) expression data were evaluated from Gene Expression Profiling Interactive Analysis and Kaplan Meier plotter was used to assess the survival data. By analyzing the GSE65071 dataset from gene expression omnibus, it was found that miR-199b-3p was expressed at a low level. By using reverse transcription-quantitative PCR analysis in OS cells and tissues, CCDC88A was found to be expressed at a high level. Moreover, TargetScan predicted CCDC88A to be a downstream target of miR-199b-3p. Luciferase reporter assays were used to verify this prediction. In vitro overexpression of miR-199b-3p decreased the invasive and proliferative activity of OS cells. Mechanistic studies indicated that decreased miR-199b-3p resulted in increased expression of CCDC88A. Concomitantly, it impeded the Wnt/beta-catenin pathway and the epithelial-to-mesenchymal transition process. Overall, the results of the present study emphasized the pivotal role of the miR-199b-3p in the formation and progression of OS, suggesting that it could be used as a potential tumor biomarker.

Inclusion and exclusion criteria. Inclusion criteria were as follows: osteosarcoma patients with an expected survival over 3 months without radiotherapy or chemotherapy before surgery. Exclusion criteria were as follows: (i) osteosarcoma patients who received radiotherapy or chemotherapy before surgery; and (ii) patients who failed to cooperate sign the informed consent form.
Ethical statement. The proposal was reviewed and approved by the Ethics Committee of our hospital prior to implementation (October 2020; approval no. 2020119). Written informed consent was obtained from the guardians of the patients included in the present study.
Reverse transcription-quantitative PCR (RT-qPCR) assays. TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract the total RNA from cells and tissues in accordance with the instructions provided by the kits. β-actin and U6 were used as the internal reference controls of CCDC88A and miR-199b-3p, respectively. The primers used are listed in Table 1. All experiments were performed in triplicate. Table 1. The primer sequences used for the RT-qPCR analysis. RT-qPCR reverse transcription-quantitative PCR, miR microRNA, CCDC88A coiled-coil domain containing 88A. www.nature.com/scientificreports/ Small interfering RNA (siRNA) transfection. miR-199b-3p mimics, miR-199b-3p inhibitor, siRNA targeting CCDC88A, and their corresponding control groups were purchased from Shanghai Genepharma Inc. The method of siRNA transfection was performed as described previously 21 . The sequences used are listed in Table 2. Each experiment was conducted in triplicate.
Cell proliferation assay. Post-transfection, U2OS, and MG63 cells were seeded at a density of 1000 cells per well in 96-well plates. A total of 5 wells were used as replicates. A total of 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml) was incubated with the cells (0, 24, 48, and 72 h groups) at 37 °C for 2 h. Subsequently, 150 μl dimethyl sulfoxide was added to the wells. A microplate reader was used to measure the absorbance at a wavelength of 490 nm. All experiments were performed in triplicate.
Transwell assay. The Transwell chamber was used to investigate OS cell migratory ability. A total of 200 μl cell suspension with only DMEM was plated into the upper chamber and 500 μl DMEM containing 20% FBS was added to the lower chamber. Subsequently, the cells were incubated for 48 h and the cells that did not penetrate through the membrane surface were removed with a cotton swab. The remaining cells were rinsed with PBS and paraformaldehyde was used to fix the cells for 10 min, followed by staining with 0.5% crystal violet. The number of cells that penetrated through the membrane was counted by an inverted microscope. Each experiment was conducted in triplicate.
Luciferase reporter assay. MG63 and U2OS cells were seeded into 24-well plates. Subsequently, wildtype (wt) or mutant 3′-untranslated region (UTR) of CCDC88A, miR-199b-3p mimic/inhibitor, and negative control (NC) were transfected to the aforementioned cells using a Lipofectamine™ 2000 kit (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection, the cells were cultured for 48 h and the cell lysate was collected to measure luciferase activity using a dual-luciferase reporter gene assay system. The mimic/inhibitor and NC sequences used are listed in Table 2. Each experiment was conducted in triplicate (Supplementary Information).
Western blot analysis. RIPA buffer (Biosharp Life Sciences) was used to extract the total proteins from MG63 and U2OS cells and the Bradford protein assay (Bio-Rad Laboratories, Inc.) was used to conduct protein quantitation. Electrophoresis was performed using 12% SDS-PAGE gels and the proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, they were blocked with 5% fat-free milk at room temperature for 2 h. The membranes were cropped and incubated with primary antibodies at 4 °C overnight and with a secondary antibody for 1 h at room temperature. The bound antibodies were developed using enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientifc, Inc.). ImageJ software (version 1.42; National Institutes of Health) was used to measure the gray values. The antibodies used in the present study are shown in Table 3. Each experiment was conducted in triplicate.  www.nature.com/scientificreports/ Statistical analysis. The data were analyzed by GraphPad Prism 8.0 (GraphPad Software, Inc.). The unpaired or paired Student t-tests were used for the determination of the differences in the quantitative data. The categorical variables were assessed by the χ 2 or Fisher's exact tests. The prognostic significance was determined by Kaplan-Meier and log-rank analyses. Receiver operating curve (ROC) and the area under the curve (AUC) were used for biomarker prediction. Pearson's correlation was used to assess the strength of the linear correlation between two continuous variables. P < 0.05 was considered to indicate a significant difference.
Ethics approval and consent to participate. The protocol was approved by the Ethics Committee of the Second People's Hospital of Lianyungang (Lianyungang, China) in accordance with the guidelines of the ethics committee. And all experiments were performed in accordance with above relevant guidelines and regulations.
Patient consent for publication. All patients and/or their legal guardians signed an informed consent form.

Results
The expression of CCDC88A is upregulated and miR-199b-3p expression is decreased in OS. Elevated expression of miR-199b-3p was associated with the overall survival of patients with OS as determined by the Kaplan-Meier plotter (Fig. 1A). The relative expression of CCDC88A was found to be significantly higher in OS tissues following analysis of the GEPIA public database (Fig. 1B); it was also shown to be associated with poor overall survival (Fig. 1C). Based on GSE65071, the miR-199b-3p expression levels were dramatically decreased in OS samples (Fig. 1D). Differentially expressed miRNAs were analyzed using a volcano plot between OS and normal samples (Fig. 1E). The heat map depicts the partial differential miRNA expression in OS (Fig. 1F).

Correlation of miR-199b-3p and CCDC88A expression levels with the clinical characteristics of patients with OS.
The expression levels of CCDC88A were higher in tumor tissues from clinical samples ( Fig. 2A), whereas those of miR-199b-3p were lower (Fig. 2B). CCDC88A levels were increased in tumor cells compared with those of the normal osteoblast cell line hFOB1.19 (Fig. 2C). However, the expression levels of miR-199b-3p were decreased in OS cell lines (Fig. 2D). A negative correlation between CCDC88A and miR-199b-3p expressions was noted in patients with OS (P = 0.005, r = − 0.39; Fig. 2E). The best cut-off value was obtained by ROC analysis and two groups were selected, based on the expression levels of miR-199b-3p (P = 0.004, AUC = 0.67; Fig. 2F). Subsequently, the high expression group (n = 15) and low expression group (n = 34) were obtained (Table 4). Tumor size, distant metastasis, and clinical stage were found to be significantly associated with low expression levels of miR-199b-3p (P = 0.015, P = 0.047, and P = 0.029, respectively). Low miR-199b-3p expression levels were associated with a poor prognosis in patients with OS (P = 0.024) (Fig. 2G).

miR-199b-3p inhibits OS cell proliferation and migration.
A miR-199b-3p mimic or inhibitor was transfected to MG63 and U2OS cells in order to induce upregulation or downregulation of miR-199b-3p expression. RT-qPCR was used to detect the efficiency of the transfection process (Fig. 3A,B). The results of the MTT assays indicated that the proliferation of the OS cell lines was significantly decreased following transfection of the cells with miR-199b-3p mimics, while that of the OS cell lines was promoted following transfection of the cells with miR-199b-3p inhibitors (Fig. 3C,D). Transwell assays indicated that the migratory activity levels of the OS cell lines were significantly decreased following transfection of the cells with miR-199b-3p mimics, while transfection of the cells with the miR-199b-3p inhibitors promoted the migratory activity of OS cells (Fig. 3E).

miR-199b-3p targets CCDC88A and decreases CCDC88A expression in OS cell lines. Previous
experiments revealed that the CCDC88A 3′UTR and miR-199b-3p had a putative binding site as determined by the TargetScan database (Fig. 4A). Luciferase reporter gene assay demonstrated in the CCDC88A-wt group that the luciferase activity was significantly inhibited by miR-199b-3p mimics (Fig. 4B,C), while in the CCDC88A-wt group, the luciferase activity was promoted by miR-199b-3p inhibition (Fig. 4D,E). Subsequently, CCDC88A levels were measured by RT-qPCR to determine if miR-199b-3p could alter CCDC88A expression. In addition, the expression levels of CCDC88A were decreased in the miR-199b-3p mimics group and increased in the miR-199b-3p inhibitor group compared with those of the NC group (Fig. 4F,G). Therefore, it was concluded that miR-199b-3p had a significant influence on OS by targeting CCDC88A.
CCDC88A knockdown reverses the miR-199b-3p-mediated capacity of proliferation and invasion. The knockdown efficiency of CCDC88A was explored by RT-qPCR analysis (Fig. 4H,I). It was found that CCDC88A expression was increased following transfection with miR-199b-3p inhibitors in MG63 and U2OS cells compared with those of the control group; however, it was inversely increased when cotransfected with miR-199b-3p inhibitors (Fig. 4J,K). The results of the MTT assays revealed that the proliferation of OS cells could be reversed by knockdown of CCDC88A expression (Fig. 4L,M). The results of the Transwell assay indicated that the invasion of OS cells could be reversed by knockdown of CCDC88A expression (Fig. 4N).   www.nature.com/scientificreports/ The miR-199b-3p-CCDC88A axis regulates the malignant behavior of OS cells via the EMT and Wnt/beta-catenin signaling pathways. The protein levels of β-catenin, E-cadherin, c-myc, cyclin D1, vimentin, and N-cadherin were detected in OS cells overexpressing miR-199b-3p. It was shown that when miR-199b-3p expression was suppressed in OS cells, the expression levels of all of the aforementioned key proteins were increased except for E-cadherin (Fig. 5A). Taken together, the results indicated that CCDC88A regulated by miR-199b-3p induced tumor proliferation and invasion in OS via the EMT and Wnt/beta-catenin signaling pathways (Fig. 5B).

Discussion
As a novel regulator, miR-199b-3p, has particular relevance to tumorigenesis in a wide range of tumors 14,15 . In addition, overexpression of miR-199b-3p hindered the proliferation of colorectal cancer cells and induced their apoptosis by decreasing cysteine-rich motor neuron 1 (CRIM1) via the Wnt/beta-catenin pathway 15 . In prostate cancer, it has been reported that miR-199b-3p can target phospholipase c epsilon and consequently suppress malignant proliferation 8 . It was also shown that lower miR-199b-3p expression correlated with poor prognosis of patients with prostate cancer 14 .
In the present study, GSE65071 was analyzed from the GEO database and the data demonstrated that the expression levels of miR-199b-3p were downregulated in OS. This finding was subsequently verified in OS cell lines. In the 49 cases with OS, the expression levels of miR-199b-3p were also significantly lower in cancer tissues compared with those noted in paracancerous tissues. Based on the patient tissue data, ROC curve analysis was used to obtain the value of AUC, which was estimated to be 0.67, indicating that miR-199b-3p was a sensitive diagnostic predictor of OS. In order to investigate the biological functions of miR-199b-3p, MTT and Transwell assays were implemented. It was revealed that the proliferation and invasion were inhibited following the upregulation of miR-199b-3p in MG63 and U2OS cell lines. According to the Kaplan-Meier analysis, lower miR-199b-3p expression was associated with a shorter overall survival time. Previous studies have demonstrated that miR-199b-3p exerts its function as a tumor suppressor in prostate cancer, colorectal carcinoma, and bladder cancer whereas it acts as an oncogene in pancreatic cancer 14,15,22 . Based on this evidence, the present study investigated the potential target of miR-199b-3p in OS.
In the present study, miR-199b-3p was found to possess a potential binding site with CCDC88A by TargetScan. CCDC88A has been reported to play an important role in tumor progression and it has also been confirmed to function as an oncogene in tumors 23 . Based on these studies, it is deduced that CCDC88A is involved in tumor progression of several cancer types, such as breast, colon, and cervical cancer [24][25][26] . It also has been reported that CCDC88A is involved in actin cytoskeleton formation and enhances Akt phosphorylation, and it also acts downstream of the PI3K/Akt signalling pathway and is directly activated by Akt 27 . CCDC88A also be reported can bind to and activate Gαi3, which further activates the PI3K/Akt signalling pathway 28 . A researcher about glioblastoma have also confirmed that CCDC88A expression is closely related to tumour malignancy, including the histological grade and metastasis, as well as progression-free survival and overall survival 29 . In human colorectal cancer, CCDC88A was associated with TNM tumor stage and the rates of liver metastasis and other distant metastases 30 . However, the effect of CCDC88A on OS progression and its regulatory mechanism remain unclear. To date, CCDC88A has not been studied in OS. In our study the relative expression of CCDC88A was found to be significantly higher in OS tissues and it was also shown to be associated with poor overall survival. www.nature.com/scientificreports/ The link between CCDC88A and miR-199b-3p was attested by luciferase reporter assays in the present study. More importantly, it was observed the CCDC88A expression levels were decreased in OS cell lines with upregulated miR-199b-3p levels. This demonstrated that miR-199b-3p and CCDC88A had a negative association, which was consistent with the findings in the clinical specimens. Strikingly, it was found that CCDC88A could partially reverse the inhibitory effect caused by the induction of miR-199b-3p. The present study demonstrated that miR-199b-3p could bind to CCDC88A and reverse its inhibitory effect on the proliferation and invasion of the OS cell lines. Subsequently, the molecular mechanism of the regulation of OS aggressiveness by the miR-199b-3p/ CCDC88A axis was also investigated. It has been reported that miR-199b-3p may modulate proliferation and invasion in colorectal cancer via the Wnt/beta-catenin signaling pathway by targetting CRIM1 15 . It has also been shown that the Wnt/beta-catenin pathway can facilitate cancer cell proliferation and differentiation, which play a fundamental role in tumorigenesis 31 . In the present study, the data indicated that miR-199b-3p may inhibit OS cell proliferation via the Wnt/β-catenin pathway. These findings are consistent with the results reported in a previous study. EMT is considered to be an embryonic process due to decreased cell-cell adherence complexes. It endows cells with enhanced migratory and invasive properties and promotes tumor metastasis 32 . Cancer cells may exhibit EMT alterations due to low expression levels of E-cadherin and high expression levels of N-cadherin and vimentin 33 . In 2021, it was reported that miR-199b-3p could inhibit EMT and cause dysfunction of the renal tubules in diabetic nephropathy 34 . In the present study, it was found that miR-199b-3p may affect cell invasion via EMT in OS.
Overall, the present study demonstrated for the first time that the miR-199b-3p/CCDC88A axis regulated OS development via the EMT process and the Wnt/beta-catenin pathway. However, the present study contains certain limitations. It was a retrospective study, which was conducted in vitro, may not reflect the behavior of OS in vivo; and only 49 cases were included, the sample size was small and generalizability may be limited. Therefore, future studies should address these limitations.
In conclusion, the results of the present study indicated that the miR-199b-3p/CCDC88A axis could play an important role in OS progression by affecting the EMT and the Wnt/beta-catenin signaling pathways. The mRNA levels of CCDC88A in MG63 cells co-transfected with CCDC88A siRNA and the miR-199b-3p inhibitor were detected by RT-qPCR. (K) The mRNA levels of CCDC88A in U2OS cells co-transfected with CCDC88A siRNA and the miR-199b-3p inhibitor were detected by RT-qPCR. (L) The proliferative activity of MG63 cells co-transfected with CCDC88A siRNA and the miR-199b-3p inhibitor was detected by the MTT assay. (M) The proliferative activity of U2OS cells co-transfected with CCDC88A siRNA and the miR-199b-3p inhibitor was detected by the MTT assay. (N) The invasive activity of MG63 and U2OS cells co-transfected with CCDC88A siRNA and the miR-199b-3p inhibitor was determined by the Transwell assay. *P < 0.05, **P < 0.01, and ***P < 0.001. miR microRNA, CCDC88A coiled-coil domain containing 88A, UTR untranslated region, siRNA small interfering RNA, RT-qPCR reverse transcription-quantitative polymerase chain reaction, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, KD knockdown, NC negative control, OD optical density, wt wild-type, mut mutant, ns not significant, CDS coding sequence. Each experiment was conducted in triplicate. The miR-199b-3p/CCDC88A axis regulates OS cell malignant behavior via the Wnt/β-catenin pathway and the EMT process. *P < 0.05, **P < 0.01, and ***P < 0.001. miR microRNA, CCDC88A coiled-coil domain containing 88A, OS osteosarcoma, EMT epithelial-to-mesenchymal transition, UTR untranslated region, EMT epithelial-mesenchymal transition. Each experiment was conducted in triplicate. Note: during development the western blotting members, due to exposure and contrast adjustment, the edges of some members were not clearly displayed in pictures.