The human osteosarcoma cell lines MG-63 (FH0443, FuHeng Biology, Shanghai, China) and HOS (Human osteosarcoma cells) (FH0440; FuHeng Biology) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; FHD01; FuHeng Biology) containing 10% fetal bovine serum (FBS, F8318, Sigma, USA) and 1% double antibiotic solution (15140148, Gibco, USA). The cells were placed in a constant temperature incubator at 37 ℃ for culturea nd 5% CO${}_2$. When the cell confluence exceeded 80%, the cells were detached and subcultured at a 1:4 ratio. All cell lines were validated by STR profiling and tested negative for mycoplasma.

Lentiviral vectors carrying a puromycin resistance cassette and a shRNA (short hairpin RNA) sequence targeting METTL3 or CBX4 were synthesized by Shanghai GenePharma (Shanghai, China), and the shRNA sequences are shown in Table 1. We transduced MG-63 cells and HOS cells with the METTL3 and CBX4 shRNA-containing lentiviral vectors separately. After 72 hours, we measured the RNA expression levels of METTL3 and CBX4 by qPCR and measured the protein expression levels of METTL3 and CBX4 by Western blotting. The CBX4 overexpression lentivirus was produced by Shanghai GenePharma. The overexpression efficiency of CBX4 was evaluated by both qPCR and Western blotting.

Cells (HOS cells and MG-63 cells) from the experimental groups (shRNA-METTL3-1, shRNA-METTL3-2) and the control group (shRNA-NC) in logarithmic growth phase were seeded into 96-well plates at 1 $\times$ 10${}^4$ cells/well, and culture medium was added to a final volume of 100 µL in each well; the cells were then cultured in 5% CO${}_2$ at 37 °C for 24 h, 48 h, 72 h, and 96 h, and 6 replicate wells and 2 control wells were established. Cell Counting Kit-8 (CCK-8) reagent (C0048, Beyotime Biotechnology, Shanghai, China) was added, the absorbance at 450 nm was measured using a microplate reader (MULTISKAN-MK3, Thermo, MA, USA), and cell growth curves were plotted with the values in the control group set to zero, the time as the abscissa, and the absorbance as the ordinate.

Apoptosis was detected by flow cytometry using an Annexin V-FITC Apoptosis Assay Kit (AT101, MultiSciences, Hangzhou, China). After removing the intervention culture medium in each group of MG-63 cells and HOS cells (shRNA-NC, shRNA-METTL3-1, shRNA-METTL3-2), trypsin was added to detach the cells, complete DMEM culture medium was used to adjust the cell suspension concentration to 10${}^6$ cells/mL, 75% ethanol was added to a final volume of 2 mL to fix the cells, and the cells were gently mixed after standing for 5 min and were then placed in a low-temperature centrifuge for centrifugation at 12,000 rpm for 10 min. After centrifugation, the supernatant was removed, and PBS solution was added to the cell pellet to gently wash the cells three times for 5 min each. Annexin V-FITC/PI double staining was also performed according to the manufacturer’s instructions (FACSCalibur, New Jersey, BD, USA) for detection of apoptosis by flow cytometry. The apoptosis assay was repeated three times, and the numbers of cells in the early and late stages of apoptosis were quantified.

Total RNA was isolated using the MiniBEST Universal RNA Extraction Kit (9767, Takara, Beijing, China) according to the manufacturer’s protocol, and total RNA samples were reverse transcribed into cDNA using RT Master Mix (RR036A; Takara). Real-time PCR was performed in triplicate using SYBR Green PCR Master Mix (RR420A; Takara) and an ABI 7500 real-time PCR system. All procedures were performed according to the manufacturer’s instructions. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) and $\beta$-actin was used as an internal control. The primer sequences are shown in Table 2. The relative RNA expression of target genes was calculated by the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method.

Samples from each group were added to protein lysis buffer containing PMSF (protease inhibitor), placed on ice for 40 min, and centrifuged at 12,000 r/min for 40 min at 4 °C, and the supernatant was then collected. The protein concentration in the supernatant was performed by the Bradford assay using BSA (bovine serum albumin) as a standard. A 20 µg protein sample (20 µg) was separated by 10% SDS‒PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane after electrophoretic separation, and the membrane was blocked with blocking solution for 1 h, washed with PBS and incubated with primary antibodies against METTL3 (DF12020, Affinity, OH, USA), CBX4 (DF13461, Affinity), matrix metalloproteinase 2 (MMP2) (AF5330, Affinity), MMP9 (AF5228, Affinity), E-Cadherin (AF0131, Affinity), and N-Cadherin (AF5239, Affinity) overnight at 4 °C. Following three washes in TBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (H + L) (SA00001-1, Proteintech, NY, USA) as the secondary antibody for 1 h at room temperature. Bands were detected using a gel documentation system. Band intensities were quantified using ImageJ software (version1.46, U. S. National Institutes of Health, Bethesda, MD, USA), and all values were compared to that of $\beta$-actin (81115-1-RR; Proteintech) as a reference protein.

MG-63 cells and HOS cells transfected with shRNAs were plated into the upper chamber of 24-mm Transwell inserts (3412; Corning) (2 $\times$ 10${}^4$ cells per well) in 200 µL of serum-free DMEM. DMEM containing 10% FBS was added to the lower chamber. The cells were continuously cultured for 24 h and fixed in methanol for 30 min. Subsequently, 0.1% crystal violet (C0121; Beyotime Biotechnology) was added to stain the cells for 15 min, and finally, the migrated cells were imaged with an inverted light microscope (Eclipse TS100, Nikon, Japan) and counted in five randomly selected fields.

To verify the binding ability of CBX4 mRNA to METTL3, we performed RIP assays using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17-700, Millipore, MA, USA) according to a previously described method [20]. The antibodies used in the experiment were as follows: normal rabbit IgG (Cat# AC005, ABclonal, Woburn, MA, USA) and anti-METTL3 (Cat# A301-567A, Bethyl Laboratories, Montgomery, TX, USA).

The m6A immunoprecipitation (MeRIP) procedure was performed using a Magna MeRIP™ m6A kit (#17–10,499, Merck Millipore, MA, USA) according to instructions issued by the manufacturer. The steps are briefly described as follows: first, RNA was enriched and disrupted specifically; in the second step, RNA is incubated with antibodies (anti-m6A and IgG) and enriched specifically. We separated approximately 10% of the RNA in the m6A IP (immunoprecipitation) as input, and the third step was elution and reverse transcription–PCR. Next, we eluted the RNA from the anti-m6A antibody and IgG, as well as the input RNA, according to the requirements of the conventional kit, and performed reverse transcription with random primers. Through this experiment, we determined the effect of interference with METTL3 expression on the m6A level of cbx4 in HOS cells and MG-63 cells.

The experimental data are expressed as the means $\pm$ SDs, and one-way analysis of variance (ANOVA) and correlation analysis were performed using GraphPad Prism 8.0 statistical software (GraphPad Software, Inc., San Diego, CA, USA). The differences between any two groups were corrected for multiple comparisons using the Bonferroni method, with p 0.05 considered to indicate a statistically significant difference.

We found that METTL3 was highly expressed in osteosarcoma tumor samples compared with normal human osteoblasts by analyzing the expression levels of genes encoding m6A methylation-related enzymes in whole-genome sequencing data from the GSE12865 dataset (Fig. 1A), and high METTL3 expression was negatively correlated with the survival of patients with sarcoma (data from GEPIA) (Fig. 1B).

Fig. 1.

METTL3 (Methyltransferase Like Protein 3) is highly expressed in osteosarcoma and negatively correlated with the prognosis of patients with sarcoma. (A) METTL3 was highly expressed in osteosarcoma tumor samples relative to normal human osteoblasts, as determined by analyzing the expression levels of genes encoding N6-methyladenosine (m6A) methylation-related enzymes in whole-genome sequencing data from the GSE12865 dataset. **p 0.01. (B) High METTL3 expression was negatively correlated with the survival of patients with sarcoma (data from GEPIA).

We then sought to further investigate the role of METTL3 in osteosarcoma cells. We used shRNA lentiviral vectors to interfere with METTL3 expression in osteosarcoma cells. We constructed lentiviral vectors containing three shRNAs targeting METTL3 to transduce HOS cells and MG-63 cells, and 72 hours later, we used RT‒PCR to determine the silencing efficiency of the three METTL3 shRNAs on METTL3 mRNA expression (Fig. 2A). Second, Western blotting was used to verify the effect of the three shRNAs on METTL3 protein expression (Fig. 2B). HOS cells and MG-63 cells were reinfected after screening for effective silencing of METTL3 by lentiviral shRNA transduction, and we evaluated the effect of METTL3 on cell metastasis (Fig. 2C). The results showed that among the three METTL3 shRNAs, shRNA-METTL3-1 and shRNA-METTL3-2 effectively inhibited the mRNA and protein expression of METTL3 and that silencing METTL3 significantly inhibited the metastasis of osteosarcoma cells.

Fig. 2.

METTL3 deficiency inhibits the metastasis of osteosarcoma cells. (A,B) Lentiviral interference was used to silence METTL3 expression in HOS cells and MG-63 cells. We used real-time PCR and Western blotting to determine the silencing efficiency of METTL3. As shown in the figure, METTL3 RNA and protein expression was significantly inhibited. (C) The migration ability was determined using the indicated stable cells as described in the Methods section. The results showed that silencing METTL3 significantly inhibited the metastasis of osteosarcoma cells. The results are expressed as the mean $\pm$ SD of three independent experimentss. ns, no significance, *p 0.05, **p 0.01, ***p 0.001 using two-tailed Student’s t test. NC, Negative Control.

To further verify the effect of METTL3 on the biological functions of osteosarcoma cells, we examined the effect of inhibiting METTL3 expression on osteosarcoma cell proliferation by a CCK8 assay (Fig. 3A) and the effect of inhibiting METTL3 expression on osteosarcoma cell apoptosis by flow cytometry (Fig. 3B). The results showed that inhibiting METTL3 expression significantly inhibited the proliferation and promoted the apoptosis of osteosarcoma cells.

Fig. 3.

METTL3 deficiency inhibits the proliferation and promotes the apoptosis of osteosarcoma cells. (A,B) The proliferation and apoptosis abilities were evaluated using the indicated stable cells as described in the Methods section (Cell Counting Kit-8 (CCK8) assay and flow cytometric analysis of apoptosis, respectively). The results showed that silencing METTL3 significantly inhibited the proliferation and promoted the apoptosis of osteosarcoma cells (HOS cells and MG-63 cells). The results are expressed as the mean $\pm$ SD of three independent experiments. **p 0.01, ***p 0.001 using two-tailed Student’s t test.

Previous literature reported that CBX4 transcriptionally upregulates Runx2 by recruiting GCN5 to the Runx2 promoter, thereby promoting the metastasis of osteosarcoma cells [19]. Our analysis of potential target genes downstream of METTL3 (data not shown) revealed that CBX4 was a potential target gene of METTL3; we then predicted through the SRAMP prediction server that CBX4 RNA contains m6A sites [21] (Fig. 4A), and detailed information about the predicted sites is shown in Table 3. We also detected the binding of METTL3 to CBX4 mRNA using RIP-qPCR (Fig. 4B). We demonstrated that METTL3 downregulated CBX4 RNA expression after interfering with METTL3 expression in HOS cells and MG-63 cells (Fig. 4C). These results demonstrated that there is a regulatory relationship between METTL3 and CBX4. Through MeRIP-qPCR, we found that silencing METTL3 significantly reduced the methylation level of CBX4 RNA in HOS cells and MG-63 cells (Fig. 4D).

Fig. 4.

CBX4 is regulated by METTL3. (A) CBX4 messenger RNA (mRNA) contains m6A methylation sites according to the SRAMP prediction server (a representative site map). (B) The RNA Binding Protein Immunoprecipitation (RIP) assay of METTL3 showed direct binding between the METTL3 protein and the mRNA of CBX4 (three independent experiments, ***p 0.001). (C) Interfering with METTL3 expression in HOS cells and MG-63 cells inhibited the RNA expression of CBX4 (three independent experiments, **p 0.01, ***p 0.001. (D) Interfering with METTL3 expression in HOS cells and MG-63 cells reduced the methylation level of CBX4 RNA (three independent experiments, **p 0.01, ***p 0.001).

We used lentiviral shRNA transduction to interfere with CBX4 expression in osteosarcoma cells, and Fig. 5A,B show the changes in the RNA and protein expression levels after interference with CBX4 in HOS cells and MG-63 cells. The transwell assay data in Fig. 5C demonstrate that interference with CBX4 expression alone in HOS cells and MG-63 cells also inhibited osteosarcoma cell metastasis.

Fig. 5.

CBX4 deficiency inhibits the metastasis of osteosarcoma cells. (A,B) Lentiviral transduction was used to interfere with CBX4 expression in HOS cells and MG-63 cells. (C) The migration ability was determined using the indicated stable cells as described in the Methods section. The results showed that silencing CBX4 significantly inhibited the metastasis of osteosarcoma cells (HOS cells and MG-63 cells). The results are expressed as the mean $\pm$ SD of three independent experiments. *p 0.05, **p 0.01, ***p 0.001 using two-tailed Student’s t test.

METTL3 inhibits the metastasis of osteosarcoma cells through CBX4. To further demonstrate that CBX4 is involved in METTL3-mediated inhibition of osteosarcoma cell metastasis, we overexpressed CBX4 in HOS cells and MG-63 cells with METTL3 silencing. The results showed that the metastatic ability of osteosarcoma cells was attenuated after silencing METTL3 and enhanced after overexpression of CBX4, while there was no significant change in the metastatic ability of cells with overexpression of NC after silencing METTL3 (Fig. 6A,B). Negative Control (NC).

Fig. 6.

METTL3 inhibits the metastasis of osteosarcoma cells through CBX4. (A) Lentiviral transduction was used to overexpress CBX4 in HOS cells and MG-63 cells. (B) The migration ability was determined using four groups of cells (HOS cells and MG-63 cells): shRNA-NC, shRNA-METTL3-1, shRNA-METTL3-1+OE-CBX4, and shRNA-METTL3-1+OE-NC. The results showed that the migration ability of osteosarcoma cells was attenuated after silencing METTL3 and increased after overexpression of CBX4, while there was no significant change in the metastatic ability of cells with overexpression of NC after silencing METTL3. The results are expressed as the mean $\pm$ SD of three independent experiments. *p 0.05, ***p 0.001 using two-tailed Student’s t test.

The specific regulatory mechanism of METTL3 in osteosarcoma is currently unknown. In this study, we used Western blotting to measure the levels of MMP-associated proteins (MMP2 and MMP9) and epithelial-mesenchymal transition (EMT)-associated proteins (E-Cadherin and N-Cadherin). The results showed that silencing METTL3 significantly decreased the protein expression of MMP2, MMP9, and N-Cadherin and increased the protein expression of E-Cadherin in osteosarcoma cells; moreover, overexpression of CBX4 increased the protein expression of MMP2, MMP9 and N-Cadherin and decreased the protein expression of E-Cadherin in osteosarcoma cells, and silencing METTL3 did not result in significant changes in these protein levels in cells with overexpression of NC (Fig. 7A,B).

Fig. 7.

METTL3 participates in the metastasis of osteosarcoma cells by regulating matrix metalloproteinase (MMP) and epithelial-mesenchymal transition (EMT) signaling through CBX4. (A) We used Western blotting to measure the levels of MMP-associated proteins (MMP2 and MMP9) and EMT-associated proteins (E-Cadherin and N-Cadherin) in four groups of cells (HOS cells and MG-63 cells): shRNA-NC, shRNA-METTL3-1, shRNA-METTL3-1+OE-CBX4, and shRNA-METTL3-1+OE-NC. The differences in protein (MMP2, MMP9, E-cadherin, N-cadherin) expression in the four groups of cells were analyzed. The results are expressed as the mean $\pm$ SD of three independent experiments. (B) shows the calculated protein gray value. **p 0.01, ***p 0.001 using two-tailed Student’s t test.