Background
Osteosarcoma (OS) is a frequent aggressive malignant bone tumor [
1], and it is the second leading cause of cancer-related death among children and young adolescents [
2]. The standard treatment for OS is surgery in combination with chemotherapy; however, OS has high recurrence and drug resistance and the 5-year survival rate is still less than 70% [
3,
4]. Some recent studies revealed that the molecular pathogenesis of OS was highly associated with OS stem cells (OSCs) [
5,
6]; however, the effective therapeutic targets and diagnostic markers for OS have not been identified yet [
7]. Therefore, it is highly critical to better understand the OS pathology and the mechanism underlying the initiation and recurrence of the disease in order to develop strategies and new therapeutic methods in improving the prognosis of patients with osteosarcoma.
MicroRNAs (miRNAs) are a species of the non-protein coding RNA family, represented by short, single-stranded RNA approximately 18–22 nucleotides in length, and are the key regulators of post-transcriptional gene expression [
8,
9]. Importantly, studies suggested a high association between abnormal miRNA expressions and the development and progression of numerous cancer types [
9‐
12]. MiR-23b-3p, one of the many in miRNA family, has been considered as a biomarker for assisting the diagnosis and prognosis of different types of cancers such as ovarian cancer [
13], prostate cancer [
14], and hepatocellular carcinoma (HCC) [
15]. In 2018, Xian et al. found that upregulating miR-23b-3p level could effectively inhibit gastric carcinoma cell proliferation, migration, and invasion through targeting cannabinoid receptor 1 (CB1R) [
16]. Furthermore, miR-23b-3p could also inhibit cell proliferation in lung carcinoma by regulating the expression of cyclin G1 (CCNG1), which has been reported as an oncogene in OS development [
17,
18]. These studies suggest that miR-23b-3p may also have a suppressive effect on OS cell proliferation by regulating CCNG1 expression.
Farnesoid X receptor (FXR, NR1H4), which serves as a ligand-activated transcription factor with multiple functions, is mainly expressed in the liver, intestine, kidneys, and adrenal glands [
19,
20]. In the recent years, growing studies showed that the downstream targets of FXR such as miRNAs have a potent effect on multiple cancer progression, for example, Qiao et al. demonstrated that the upregulation of FXR by GW4064, an agonist of FXR, could significantly inhibit human colorectal cancer cell proliferation via miR-135A1/CCNG2 pathway [
21]. In this study, we found a positive correlation between FXR expression and miR-23b-3p level. We also investigated whether FXR expression participated in OS development via miR-23b-3p/CCNG1 pathway.
Methods
Cell culture
Normal human osteoblasts (hFOB1.19) and osteosarcoma cell lines (MG-63, HOS, U2OS, SAOS2, and SJSA1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco) and maintained in a 5% CO2 humidified incubator. The culture environment of normal osteoblasts was set at 34 °C, whereas osteosarcoma cells were cultured at 37 °C.
Quantification of RNA
Total RNA including miRNAs was isolated using TRIzol (Invitrogen, CA, USA). The synthesis of cDNA was carried out using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) in a 10-μl reaction system. The reaction parameters were set at 65 °C for 5 min and then at 42 °C for 60 min. The relative mRNA levels were determined using the SYBR-Green PCR Master Mix kit (Takara, Dalian, China) on ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The thermocycling parameters were set at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s, and the primers for qPCR are listed in Table
1. The relative expression levels of miR-23b-3p and cellular genes were normalized to U6 and GAPDH, respectively. The result was calculated by 2
−ΔΔCt method [
22].
Table 1
Primers for RT-qPCR
FXR | Forward: 5′-GATTGCTTTGCTGAAAGGGTC-3′ |
Reverse: 5′- CAGAATGCCCAGACGGAAG-3′ |
miR-23b-3p | Forward: 5′-GAGCATCACATTGCCAGGG-3′ |
Reverse: 5′-GTGCAGGGTCCGAGGT-3′ |
CCNG1 | Forward: 5′- TTACCGCTGAGGAGCTGCAGTC-3′ |
Reverse: 5′- CAGCCATCCTGGATGGATTCAG-3′ |
GAPDH | Forward: 5′- CACCGTCAAGGCTGAGAAC-3′ |
Reverse: 5′- GGTGAAGACGCCAGTGGA-3′ |
U6 | Forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ |
Reverse: 5′- CGCTTCACGAATTTGCGTGTCAT-3′ |
Cell treatment
GW4064, a FXR agonist, was purchased from Sigma-Aldrich (#5172, Merck KGaA, Darmstadt, Germany). In order to study the correlation between FXR and miR-23b-3p, MG-63 cells were incubated with GW4064 at different concentrations. In the blank group, MG-63 cells were cultured in dimethyl sulfoxide vehicle, while in the GW4064 group, MG-63 cells were respectively treated by 0.5 and 5 μM GW4064. After 48 h of incubation, the cells were harvested for expression analysis.
Luciferase reporter assay
TargetScan predicted that the sequence of CCNG1-3′-untranslated regions (UTR) contains a binding site of miR-23b-3p. PGL3-CCNG1 luciferase vector was constructed by cloning the fragment of CCNG1-3′-UTR, which contained putative binding sites for miR-23b-3p in the pGL3-Basic (Promega, Madison, WI, USA). HEK293T cells (ATCC) were plated onto 12-well plates (5 × 105 cells/well) before the cell transfection. Subsequently, pGL-3 firefly luciferase reporters (1 μg per well) were respectively co-transfected with mimics control, miR-23b-3p mimics, and mutant (50 nM) using Lipofectamine 2000 reagent (Invitrogen). After 48 h of transfection, the luciferase activities were determined by dual-luciferase reporter assay system (Promega).
Cell transfection
SiRNA for FXR (siFXR: 5′-CCUCAGGAAAUAACAAAUATT-3′), and non-specific scrambled siRNA (siNC: 5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). MG-63 cells at logarithmic phase were divided into four groups, and the cells were transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, USA). In the siNC group, cells were transfected with siNC (50 nM). In siNC + GW4064 group, after transfection for 24 h, the cells were treated with GW4064 (5 μM) for 48 h. In the siFXR + GW4064 group, the cells with FXR knockdown were treated with GW4064 (5 μM) and incubated for 48 h. After the treatment, the cells were harvested for detecting transfection efficiencies by quantitative PCR (qPCR) and Western blot.
MiR-23b-3p inhibitor (anti-miR-23b-3p, 5′-UCUCAGUGCUUCUGGCUGC-3′) and inhibitor NC (anti-miR-NC, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′) were synthesized by GenePharma. The miR-23b-3p inhibitor and inhibitor control were transfected into MG-63 cells following the protocols of Lipofectamine 2000 (Invitrogen). The cells were assigned to four groups, namely, (A) IC group, in which MG-63 cells were transfected with inhibitor control; (B) IC + GW4064 group, in which cells were cultured with GW4064 (5 μM) for 48 h based on the IC group; (C) I group, in which MG-63 cells were transfected with miR-23b-3p inhibitor; and (D) I + GW4064 group, in which the transfected cells were treated with GW4064 (5 μM) for 48 h based on the I group. We further determine the levels of CCNG1 mRNA and protein to determine the transfection efficiencies of inhibitor control and miR-23b-3p inhibitor.
Cell viability assay
Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was used for cell viability detection according to the manufacturer’s protocol. Cells from different treatment groups were seeded in a 96-well plate (5 × 103 cells/well). CCK-8 reagent was added to each well to react and then incubated for 2 h. Absorbance value was determined using an enzyme microplate reader at 450 nm (Bio-Tek, Winooski, VT, USA).
The ability of cell proliferation was assessed by soft agar clone formation assay as described previously [
23]. Briefly, the cells (1 × 10
3) were suspended in 1 ml DMEM containing 10% FBS and 0.3% low-melting agar (GE Healthcare, USA) on 6-well plates, which had been pre-coated by a solidified bottom layer made of 0.6% agarose in DMEM medium. After incubation for 2 weeks, cell culture was terminated when macroscopic apophyses were observed. The cells were fixed with 20% methanol for 15 min. Then, Giemsa solution was added to stain the cells for 40 min. After washing and air-drying, the clones were counted using a cloning counter.
Annexin V/PI staining assay for apoptosis
Fluorescein isothiocyanate (FITC)-labeled annexin V (Annexin-V-FITC) apoptosis detection kit (Sigma-Aldrich) was performed to determine cell apoptosis on a flow cytometry (Gallios, Beckman Coulter, Inc., Brea, CA, USA). In brief, the transfected cells from each group were trypsinized and centrifuged at 1000×g for 5 min. Cells were washed twice by phosphate-buffered saline (PBS) and then incubated with 100 μL Annexin V-FITC and 5 μL propidium iodide (PI) in the dark for 15 min. The apoptosis rates were determined by flow cytometry.
Cell cycle analysis
After 24 h of transfection, the transfected cells were cultured in 5 μM GW4064 and incubated for 48 h. Then, the cells were trypsinized and collected by centrifuging at 1000×g for 5 min. The cells were washed by PBS and fixed in 70% ethanol at 4 °C overnight and subsequently suspended in 400 μL buffer containing PI and RNase (BD Pharmingen, San Diego, CA, USA) for 30 min. The rates of cell cycle distribution were determined by flow cytometry (Gallios).
Western blot analysis
Total proteins were extracted from the cells by RIPA protein extraction reagent (Thermo Fisher Scientific, Inc.). Then, cell lysate was subjected to 10% sodium lauryl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (PVDF, Bio-Rad, Hercules, CA). After blocking the membranes by 5% non-fat milk for 1 h at room temperature, the membranes were cultured with primary antibodies. GAPDH (Mouse, #ab8245, 1:20000, 36 KD, Abcam, Cambridge, MA) served as an internal control. After incubation with secondary antibodies (goat anti-rabbit (1:20,000, 42 KD, #ab205718, Abcam), goat anti-mouse IgG (1:20,000, 52 KD, #ab205719, Abcam)) for 1 h at 4 °C, the specific signals were visualized by a LI-COR Odyssey Infrared Imaging System. Primary antibodies against CCNG1 (Mouse, 1:1000, #8016, 34 KD, Santa Cruz Biotechnology, Inc., Dallas, USA), FXR (Mouse, 1:1000, #25309, 70 KD, Santa Cruz Biotechnology, Inc.), B-cell lymphoma-2 (Bcl-2, Rabbit, #ab59348, 1:1000, 26 KD Abcam), Bax (Rabbit, #ab32503, 1:2000, 21 KD, Abcam), and Cleaved Caspase-3 (Rabbit, #ab2302, 1:1000, 17 KD, Abcam) were used.
Statistical analyses
All data were expressed as the mean ± SEM. Pearson’s correlation coefficient (PCC) was determined in Microsoft Excel according to the expressions of FXR and miR-23b-3p. Student’s t test was used for evaluating the difference between two group values, and ANOVA analysis followed by Dunnett’s t test was used for determining statistical significance of multiple groups. P < 0.05 was considered as statistically significant.
Discussion
We observed that both FXR and miR-23b-3p expressions decreased observably in OS cell lines, compared with normal osteoblasts (hFOB1.19) cells. Then, by treating the cells with FXR agonist GW4064, we confirmed a positive correlation between the expressions of FXR and miR-23b-3p in human OS cells. CCNG1 was confirmed as a target for miR-23b-3p, and FXR participated in the regulation of CCNG1 via miR-23b-3p. In addition, we also observed that the upregulated FXR by GW4064 could reduce cell proliferation, enhance apoptosis, and induce cell cycle arrest in OS cells. Therefore, based on the positive correlation between FXR and miR-23b-3p, the anti-cancer effects of FXR on OS cell development were associated with the regulation of miR-23b-3p/CCNG1 pathway.
Growing studies have reported that miR-23b-3p functions as a predictor and prognostic molecular marker for different cancer types such as esophageal squamous cell carcinomas [
17], pancreatic ductal adenocarcinoma [
24], and HCC [
15]. In our study, we found a positive correlation between miR-23b-3p and FXR and that GW4064 treatment could induce the upregulation of FXR and miR-23b-3p. Therefore, we reasonably speculated that miR-23b-3p was involved in the upregulation of FXR by GW4064 in suppressing cell proliferation and promoting apoptosis and cell cycle arrest in OS. These data are consistent with previous researches, in which enhancing miR-23b-3p expression mediated several targets to effectively affect tumor cell development, proliferation, and metastasis, and ultimately inhibited cancer development and progression [
13,
25,
26].
The regulatory effects of miRNAs on the progression of cancer largely rely on directly regulating the expressions of their target genes. Previous study has revealed that miR-23b-3p specifically targets CCNG1 to regulate HCC cell proliferation and invasion [
17]. We also confirmed the close relationship between CCNG1 and miR-23b-3p. CCNG1 is a pivotal component of commanding cyclin G1/murine double minute gene 2/tumor protein p53 (CCNG1/Mdm2/p53) axis [
27]. P53 functions as a key regulator of cell cycle progression [
28]. CCNG1 could stimulate and propel PP2A catalytic activity toward E3 ubiquitin-protein ligase (Mdm2), which has increased or abnormal expression in numerous cancer types including OS [
29,
30]. Then, activation of Mdm2 antagonizes the p53 tumor suppressor function by forming a physical complex with p53 [
27]. Previous studies have demonstrated the promoting effects of CCNG1 on cell growth and that the inhibition of CCNG1 could effectively suppress the growth of human tumor xenografts in a nude mouse model [
18,
31]. Currently, the potential of companion diagnostics of CCNG1 pathway in the staging, prognosis, and treatment of cancers is widely recognized. In the current study, our data showed that FXR could negatively regulate the expression of CCNG through miR-23b-3p, indicating that the inhibitory effects of FXR on OS cell proliferation and growth were highly associated with the downregulated CCNG1 level.
In recent years, FXR has attracted increasing attention as a therapeutic target in the treatment of cancers [
32]. Data from previous reports indicated that FXR deficiency could induce the formation of spontaneous liver tumors and small intestine adenocarcinoma in APCmin mice and promote colon cell proliferation [
33,
34]. These studies showed that FXR plays a tumor-suppressive role in certain cancers [
35,
36]. In our study, the expressions of FXR in the OS cell lines were also generally lower than that of normal osteoblasts, while the upregulation of FXR by GW4064 showed a potent effect on OS cell proliferation, apoptosis, and cell cycle distribution. The downstream factors of FXR, including miRNAs, have been proved to participate in multiple carcinogenesis [
37]. In 2013, Song et al. demonstrated a novel role of peroxisome proliferator-activated receptor gamma (PPARγ), that is, it could epigenetically induce the upregulation of miRNA transcription [
38]. FXR shares a number of characteristics with PPARγ and it could bind to retinoid X receptor α (RXRα) as a monomer, and subsequently recruits different cofactors to induce the expressions of target genes in HCC cells [
32,
39]. However, whether FXR can epigenetically induce miR-23b-3p expression as PPARγ is still unclear; however, this may be partially explained by the positive correlation between miR-23b-3p and FXR; however, the molecular mechanism requires further study. It should be noted that in vivo model was not constructed in this study, which was a limitation in this study.
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