Abstract
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.
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Introduction
Osteosarcoma (OS) is a disease that affects most frequently children and adolescents. It is considered to be one of the most universal primary malicious bone sarcomas with a poor prognosis1. Surgical resection of all tumor sites, radiation therapy, and chemotherapy are the three main therapies used for the treatment of patients with OS2. Despite significant efforts made in OS treatment, the 5-year survival rate remains between 50 and 80% and the survival rates remain unchanged since 19803,4. Therefore, in order to increase patient survival rate and inhibit OS progression, the identification of novel therapeutic targets and their molecular mechanisms is urgently required.
MicroRNAs (miRs) are one type of non-coding small RNAs with an approximate length of 21–23 nucleotides. Certain miRs have been reported to contribute to OS progression and may be used in the diagnosis, prognosis, and treatment of this disease5,6,7. Some studies have also found that miR affects the metastasis and invasion of osteosarcoma8. For example, down-regulation of miR-34a can activate epithelial–mesenchymal transition (EMT) and promote the metastasis of osteosarcoma9. LINC00210 modulated the radiosensitivity of osteosarcoma cells via the miR-342-3p/GFRA1 axis, making LINC00210 a novel target for improving radiotherapy efficiency in osteosarcoma10. Upregulated expression of miR-664a could have an inhibitory effect on MEG3 gene expression and migration of osteosarcoma cells11. MiR-659-3p inhibited osteosarcoma tumor progression and lung metastasis by inhibiting SRPK1 expression and potentially downstream cell proliferation, and epithelial-to-mesenchymal transition genes12. The high expression of miR-183 can inhibit the Wnt/beta-catenin pathway and inhibit the metastasis of osteosarcoma13. Previous research studies have found that miR-199b-3p can affect proliferation, differentiation, and apoptosis in a variety of tumors14,15. However, the role of miR-199b-3p in OS has not been previously reported.
Coiled-coil domain containing 88A (CCDC88A) can bind to the actin cytoskeleton. Akt is a serine/threonine kinase, which is required for directional cell migration, ultimately leading to invasion and metastasis in cancer cells16. Several reports have suggested that CCDC88A is involved in tumor progression. Knockdown of CCDC88A expression suppresses human pancreatic, skin, and breast cancer cell migration and invasion, and significantly inhibits primary tumorigenesis in vivo17,18,19, suggesting that CCDC88A has a key role in cancer development.
The present study we first showed that miR-199b-3p had a tumor-suppressive function in OS by suppressing OS cell growth, migration, and invasion. Then, we characterized CCDC88A as a direct target of miR-199b-3p. MiR-199b-3p interacted with the 3′UTR of CCDC88A to inhibit the EMT and Wnt/beta-catenin signaling pathway to mediate its tumor-suppressive effect on the proliferation and invasion of OS cells. These findings may be useful for the development of possible clinical approaches for OS treatment.
Materials and methods
Bioinformatic analysis
The overall survival of the patients with OS and different levels of miR-199b-3p expression was analyzed by Kaplan Meier plotter (http://kmplot.com/analysis/). The CCDC88A expression data and prognostic information were obtained by the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn). GSE65071 was used to acquire the miR-199b-3p expression data from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The potential association between miR-199b-3p and CCDC88A was obtained from TargetScan (http://www.targetscan.org/vert 80/).
Patients and specimens
A total of 49 patients were included, who underwent surgical treatment from 2010 to 2020 in our hospital. A total of 49 pairs of primary OS and adjacent non-cancerous bone tissues were obtained. All the tissues were collected and immediately cryopreserved at − 80 °C during the operation. The World Health Organization histological criteria and the American Joint Committee on Cancer (AJCC) staging system were used to confirm the diagnosis of OS. The AJCC staging system was recommended for the evaluation of the prognosis and the survival of patients with OS20.
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.
Cell culture
All cell lines, including hFOB, MG63 (RRID: CVCL_0426), and U2OS were purchased from Suzhou Culture Collection. All the cell lines were authenticated by STR analysis. DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), streptomycin (100 μg/ml), and penicillin (100 U/ml) were used for cell culture. The cells were incubated in a humidified incubator containing 5% CO2 at 37 °C.
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.
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 previously21. 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, wild-type (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.
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 χ2 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).
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 tumors14,15. 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 pathway15. In prostate cancer, it has been reported that miR-199b-3p can target phospholipase c epsilon and consequently suppress malignant proliferation8. It was also shown that lower miR-199b-3p expression correlated with poor prognosis of patients with prostate cancer14.
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 cancer14,15,22. 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 tumors23. Based on these studies, it is deduced that CCDC88A is involved in tumor progression of several cancer types, such as breast, colon, and cervical cancer24,25,26. 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 Akt27. CCDC88A also be reported can bind to and activate Gαi3, which further activates the PI3K/Akt signalling pathway28. 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 survival29. In human colorectal cancer, CCDC88A was associated with TNM tumor stage and the rates of liver metastasis and other distant metastases30. 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. 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 CRIM115. It has also been shown that the Wnt/beta-catenin pathway can facilitate cancer cell proliferation and differentiation, which play a fundamental role in tumorigenesis31. 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 metastasis32. Cancer cells may exhibit EMT alterations due to low expression levels of E-cadherin and high expression levels of N-cadherin and vimentin33. In 2021, it was reported that miR-199b-3p could inhibit EMT and cause dysfunction of the renal tubules in diabetic nephropathy34. 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.
Data availability
The datasets generated in the present study may be requested from the corresponding author.
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H.Q., D.Z., and H.Z. designed the experiments. D.Z. performed the experiments. H.Q. collected the patients’ samples. D.Z. prepared the manuscript. D.Z., and H.Z. revised the manuscript critically for important intellectual content. D.Z. aided with the statistical analysis. D.Z. and H.Z. confirm the authenticity of all the raw data. All authors confirmed the authenticity of all the raw data, and approved the final version of the manuscript.
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Zhu, D., Qi, H. & Zhu, H. hsa-miR-199b-3p suppresses osteosarcoma progression by targeting CCDC88A, inhibiting epithelial-to-mesenchymal transition, and Wnt/beta-catenin signaling pathway. Sci Rep 13, 12544 (2023). https://doi.org/10.1038/s41598-023-39537-0
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DOI: https://doi.org/10.1038/s41598-023-39537-0
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