METTL14 benefits the mesenchymal stem cells in patients with steroid-associated osteonecrosis of the femoral head by regulating the m6A level of PTPN6

Identification of BMSCs

When the cells were passaged to the second generation, they had a typical long fusiform appearance under the microscope (Figure 1A). Besides, the cells have the potential to differentiate into osteoblasts and adipocytes (Figure 1B, 1C). Furthermore, we analyzed four known surface markers associated with human bone marrow mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for CD29 and CD44, but negative for CD34 and CD45 (Figure 1D). These results indicated that the isolated cells were human BMSCs.

Figure 1. Identification of BMSCs. (A) The appearance of BMSCs cultured to the second generation. Scale bar = 100 μm. (B) The osteogenesis differentiation of BMSCs was confirmed by alizarin red staining. Scale bar = 100 μm. (C) The potential of BMSCs to differentiate into adipocytes was verified by oil red staining. Scale bar = 100 μm. (D) The surface markers of BMSCs were identified by flow cytometry.

METTL14 was responsible for the aberrant m6A modifications in BMSCs from SONFH patients

To explore the potential role of m6A modification in SONFH, we initially examined the level of m6A in the total RNA of femoral head tissues and BMSCs. As shown in Figure 2A, the m6A content in SONFH tissues and BMSCs was significantly lower than that in normal group. According to the previous studies, m6A modifications are catalyzed by m6A methyltransferase and m6A demethylase, so we next examined the mRNA levels of m6A modification associated genes, including METTL3, METTL14, WTAP, FTO and ALKBH5. Interestingly, the m6A methyltransferase METTL14 was obviously down-regulated in osteonecrosis tissues and BMSCs from SONFH patients, while the demethylase ALKBH5 was up-regulated in SONFH tissues, but not in SONFH BMSCs. The levels of METTL3, WTAP and FTO exhibited no significant differences between the two groups (Figure 2B). Thus, we hypothesized that METTL14 was a critical regulator that contributed to the abnormal m6A modifications. We further performed IHC analysis with femoral head tissues, and found that the positive rate of METTL14 in necrotic femoral head tissues was significantly lower than that in healthy femoral head tissues (Figure 2C). Moreover, western blotting indicated that protein levels of METTL14 were down-regulated in BMSCs from SONFH patients compared to those in normal BMSCs (Figure 2D). To further verify the effect of METTL14 on the m6A modification in BMSCs, we overexpressed METTL14 in SONFH BMSCs, and qRT-PCR and western blotting were used to determine the transfection effect (Figure 2E, 2F). Then, we detected the m6A content in cells. The results showed that the m6A level was increased significantly upon METTL14 overexpression (Figure 2G). Together, these data demonstrated that the m6A level was decreased in SONFH femoral head tissues and BMSCs, and that METTL14 was the regulator involving the abnormal m6A modification.

Figure 2. METTL14 was responsible for the aberrant m6A modification in BMSCs from SONFH patients. (A) The m6A content in total RNA of SONFH tissues and BMSCs (n = 20) and normal tissues and BMSCs (n = 20). (B) mRNA levels of m6A modification associated genes in SONFH tissues and BMSCs and normal tissues and BMSCs. (C) IHC assay determined the expression of METTL14 in osteonecrosis tissues and normal tissues. Scale bar = 100 μm. (D) The protein levels of METTL14 in SONFH BMSCs and normal BMSCs were measured by western blot. (E, F) The efficiency of METTL14 overexpression in SONFH BMSCs was confirmed by qRT-PCR and western blot analysis. (G) Quantitative m6A methylation assay was used to define the effect of METTL14 overexpression on the level of m6A modification in BMSCs from SONFH patients. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.

METTL14 up-regulation promoted the proliferation and osteogenesis of SONFH BMSCs

To investigate the biological functions of METTL14, we assessed the proliferation and osteogenesis abilities in SONFH BMSCs transfected with OE-METTL14. The results of CCK-8 and EdU assays confirmed that METTL14 overexpression significantly promoted the proliferation capacity of BMSCs (Figure 3A, 3B). Furthermore, METTL14 up-regulation augmented cell ALP activity (Figure 3C). Alizarin red staining and oil red staining showed that the ability to differentiate into osteoblasts was enhanced and the potential to differentiate into adipocytes was decreased when METTL14 expression was overexpressed (Figure 3D). Western blotting results demonstrated that with METTL14 overexpression, the levels of the osteogenic-related gene RUNX2 was up-regulated, while the expression of adipogenic-related genes PPARγ, C/EBPα and C/EBPβ were down-regulated in SONFH BMSCs (Figure 3E). Taken together, these results suggested that METTL14 promotes the proliferation and osteogenesis of BMSCs from SONFH patients.

Figure 3. METTL14 up-regulation promoted the proliferation and osteogenesis of SONFH BMSCs. (A) The effect of METTL14 up-regulation on the cell viability of SONFH BMSCs was analyzed by CCK-8 assay. (B) EdU assay was used to detect the influence of METTL14 overexpression on the proliferation of SONFH BMSCs. Scale bar = 100 μm. (C) The ALP activity assay provided evidence that METTL14 up-regulation enhanced the ability of osteogenic differentiation of BMSCs from SONFH patients. (D) METTL14 overexpression promoted the ability of osteogenic differentiation and suppressed the adipogenesis capacity of SONFH BMSCs were confirmed by alizarin red staining and oil red staining. Scale bar = 100 μm. (E) The expression of osteogenic-related and adipogenic-related genes in BMSCs with METTL14 overexpression were analyzed by western blot. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.

METTL14-dependent m6A modifications regulated the expression of PTPN6

M6A modification mostly exists in mRNA and assumes as a form of posttranscriptional regulation, which further affects the expression of the modified gene. To identify the downstream target of METTL14, we performed mRNA sequencing on BMSCs from SONFH patients transfected with OE-METTL14 or OE-NC. Differentially expressed genes were screened based on p-value <0.05 and false discovery rate <0.05. Compared to control cells, METTL14-overexpressing cells had 101 genes with increased expression and 142 genes were decreased, and these genes were clustered analyzed and presented as a heat map (Figure 4A). Among differentially expressed genes, the expression of PTPN6 was most significantly affected by METTL14 (fold change was the highest). The data of qRT-PCR displayed that PTPN6 mRNA levels in osteonecrosis tissues was decreased compared with control femoral head tissues (Figure 4B). IHC assay also provided consistent results, the positive staining of PTPN6 in necrotic femoral head tissues was significantly less than that in normal tissues (Figure 4C). Moreover, the qRT-PCR and western blot results indicated that the level of PTPN6 in BMSCs of SONFH was significantly lower than that in normal BMSCs (Figure 4D, 4E), and PTPN6 mRNA expression was positively correlated with METTL14 mRNA in SONFH BMSCs (Figure 4F). Besides, in SONFH BMSCs, the mRNA and protein levels of PTPN6 were increased with METTL14 up-regulation (Figure 4G, 4H). We then investigated the m6A modification status of PTPN6 by performing MeRIP-qPCR assay. The results validated that the up-regulation of METTL14 enriched the m6A modification level of PTPN6 mRNA (Figure 4I). Furthermore, we carried out the RNA stability assay to analyze the half-life of PTPN6 mRNA. As shown in Figure 4J, the half-life of PTPN6 mRNA was 5.26 hours in SONFH BMSCs, while the half-life of PTPN6 was longer than 8 hours in METTL14 overexpression cells. To further elucidate the mechanism of METTL14 regulation of PTPN6 mRNA stability, we performed the luciferase reporter assay and found that METTL14 overexpression reinforced the activity of the luciferase construct containing the wild type 3′UTR of PTPN6 mRNA. Mutation at position 859 (A to T) almost completely rendered resistance to the luciferase activity of METTL14 overexpression (Figure 4K). Collectively, METTL14-dependent m6A modification regulates the expression of PTPN6 by enhancing the stability of PTPN6 mRNA.

Figure 4. METTL14-dependent m6A modification regulated the expression of PTPN6. (A) Heat map of normalized gene expression levels of SONFH BMSCs transfected with OE-NC or OE-METTL14. Red indicates down-regulation of gene expression and Green indicates up-regulation of gene expression. (B) PTPN6 mRNA levels in normal tissues (n = 20) and osteonecrosis tissues (n = 20) were measured by qRT-PCR. (C) IHC assay detected the expression of PTPN6 in femoral head tissues of two group. Scale bar = 100 μm. (D, E) qRT-PCR and western blot quantified the abundance of PTPN6 in normal BMSCs and SONFH BMSCs. (F) The correlation between METTL14 and PTPN6 expression in SONFH BMSCs was analyzed by Pearson’s Correlation Coefficient. (G, H) qRT-PCR and western blot confirmed that PTPN6 expression increased with the up-regulation of METTL14 in SONFH BMSCs. (I) The methylated PTPN6 mRNA level in the METTL14 overexpressed BMSCs was determined by MeRIP-qPCR. (J) The mRNA half-life of PTPN6 in SONFH BMSCs transfected with OE-NC or OE-METTL14. (K) SONFH BMSCs were pre-transfected with wild-type or mutated PTPN6-3′UTR plasmids, and then treated with OE-NC or OE-METTL14. The luciferase activity was normalized to firefly luciferase activity. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.

PTPN6 knockdown abrogated the beneficial effects of METTL14 overexpression on SONFH BMSCs

To explore whether PTPN6 is critical to the effects of METTL14 on SONFH BMSCs, we silenced PTPN6 in METTL14 overexpression cells. The efficiency of PTPN6 knockdown was confirmed by qRT-PCR and western blot assays (Figure 5A, 5B). The CCK-8 assay and EdU assay indicated that silenced PTPN6 dramatically abrogated the effect of METTL14 on cell proliferation (Figure 5C, 5D). The results of alizarin red staining, oil red staining and ALP activity assays demonstrated that PTPN6 knockdown reversed the promotion of METTL14 overexpression on the osteogenic differentiation of BMSCs (Figure 5E, 5F). In addition, when we weakened PTPN6 expression in METTL14-overexpressing cells, the protein level of RUNX2 was down-regulated, and those of PPARγ, C/EBPα and C/EBPβ were up-regulated (Figure 5G). Overall, these results suggested that PTPN6 is decisive to the beneficial effect of METTL14 on BMSCs from SONFH patients.

Figure 5. PTPN6 knockdown abrogated the beneficial effects of METTL14 overexpression on SONFH BMSCs. (A, B) qRT-PCR and western blot validated the efficiency of PTPN6 knockdown in METTL14 overexpression cells. (C, D) The CCK-8 and EdU assay evaluated the reversal effect of PTPN6 knockdown on cell proliferation induced by METTL14 overexpression. Scale bar = 100 μm. (E) The ALP activity assay was used to assess the reversal effect of PTPN6 knockdown on the osteogenesis promoted by METTL14 up-regulation. (F) The effects of sh-PTPN6 on the abilities of osteogenic differentiation and adipogenic differentiation of METTL14 overexpression cells were analyzed by Alizarin red staining and oil red staining. Scale bar = 100 μm. (G) Knockdown of PTPN6 counteracted the regulatory effect of METTL14 overexpression on osteogenic and adipogenic related genes. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.

PTPN6-mediated METTL14 activated the Wnt signaling pathway

The canonical Wnt signaling pathway is the essential signal transduction involved in the osteogenic differentiation of stem cells [35–37]. To verify whether METTL14 affects the activity of the Wnt signaling pathway in SONFH BMSCs, we initially conducted TOP/FOP-Flash reporter assay. The results showed that the activity of the Wnt signaling pathway was heightened in METTL14 overexpressed cells, and PTPN6 knockdown reversed this effect (Figure 6A). The western blot indicated that METTL14 overexpression resulted in the up-regulation of total and nuclear β-catenin and the decrease of phosphorylated β-catenin, while PTPN6 knockdown diminished the abundance of β-catenin in total protein and nucleoprotein, and the phosphorylation of β-catenin protein was augmented simultaneously (Figure 6B, 6C). Moreover, the immunofluorescence staining showed lower level of β-catenin in the nuclei of the control group than METTL14 overexpression group. The up-regulation of METTL14 obviously induced the translocation of β-catenin (red) into the nucleus (blue). PTPN6 knockdown evidently attenuated the effect of METTL14, as PTPN6 knockdown almost completely blocked the nuclear translocation of β-catenin (Figure 6D). GSK-3β assumes an essential role in the degradation of β-catenin. According to co-IP experiment, we found that PTPN6 could interact with GSK-3β in BMSC (Figure 6E). In addition, in SONFH BMSCs, PTPN6 inhibition obviously reduced the phosphorylation level of GSK-3β (Figure 6F). In conclusion, PTPN6-mediated METTL14 activates the Wnt signaling pathway in BMSCs from SONFH patients.

Figure 6. PTPN6-mediated METTL14 activated the Wnt signaling pathway. (A) The activity of Wnt signaling in SONFH BMSCs was analyzed by TOP/FOP-Flash reporter assay. (B) The protein level of β-catenin in the nucleus was measured by western blot. (C) Western blot detected the total and phosphorylated β-catenin levels in SONFH BMSCs treated as indicated. (D) Immunofluorescence staining was used to analyze the distribution of β-catenin in the cytoplasm and nucleus after METTL14 overexpression and PTPN6 knockdown. Scale bar = 50 μm. (E) The interaction of PTPN6 and GSK-3β in BMSCs was verified by co-IP assay. (F) The effect of PTPN6 inhibition on the phosphorylation level of GSK-3β in SONFH BMSCs was determined by western blot. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s is no significance.

Patients

From January 2019 to December 2019, 20 patients with steroid-associated osteonecrosis of the femoral head who underwent hip arthroplasty at the Department of Orthopedics of Henan Provincial People’s Hospital were selected as the SONFH group and 20 patients with femoral neck fracture who underwent hip arthroplasty served as the control group. Bone marrow and femoral head tissues were collected during the operation. Table 1 summarizes the clinical characteristics of all patients. The present study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, and this study was approved by the Ethics Committee of Henan Provincial People’s Hospital (Approval number: 2017237). All patients signed informed consent before operation.

Bone marrow separation and cell culture

Five milliliters of bone marrow were procured from the proximal femur during hip arthroplasty. BMSCs were isolated and cultured as described in previous study [12]. Briefly, mononuclear cells in the bone marrow were centrifuged with an equal volume of 1.073 g/ml Percoll solution (Sigma, USA) at 2000 rpm for 30 minutes. Then, cells were collected, suspended in DMEM/F12 medium (HyClone, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Biosharp, China), and cultured at 37°C, 5% CO₂ and 99% relative humidity. In order to maintain the consistent biological characteristics, experiments were carried out when the cells were subcultured to the second generation and were no longer used after they were subcultured to the fifth passage.

Flow cytometry

In order to verify that cells we extracted were BMSCs, we detected the expression of cell surface markers by flow cytometry. Briefly, we collected cells at the second passage and resuspended them in PBS. Then, 2 × 10⁵ cells were incubated with PE or FITC labeled cell surface marker antibodies against CD29, CD34, CD44 and CD45 (BD Biosciences, USA), as well as isotype control antibody at 4°C for 30 minutes in the dark. After the incubation is completed, the samples were analyzed by the flow cytometer (BD Biosciences, USA).

Cell transfection

The METTL14 overexpression plasmid (termed OE-METTL14) and the negative control (termed OE-NC) were purchased from OBIO Technology (Shanghai, China). Short hairpin RNA targeting PTPN6 (termed sh-PTPN6) and the negative control (termed sh-NC) were designed by GeneChem (Shanghai, China) and cloned into GV112 lentiviral vectors (GeneChem, Shanghai). The cells were infected with the viruses in the presence of Polybrene for 48 hours. Then, positively transfected cells were selected for 7–10 days using 2 μg/ml puromycin. The overexpression and knockdown efficiencies were determined by qRT-PCR and western blot assay.

Osteogenic and adipogenic differentiation

For osteogenic differentiation, BMSCs were cultured in complete DMEM/F12 medium containing 0.25 mM ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone for 21 days.

Adipogenic differentiation of human BMSCs was performed according to an earlier study [53]. Cells were cultured in adipogenesis-inducing medium: complete DMEM/F12 medium containing 1 μM dexamethasone, 0.2 mM indomethacin, 0.5 mM isobutyl-methylxanthine and 0.01 mg/ml insulin. Three days later, the adipogenesis-inducing medium was changed to adipogenesis maintenance medium (complete DMEM/F12 medium containing 0.01 mg/ml insulin). After 24 hours, switched back to adipogenesis-inducing medium. The cells were alternately cultured in the two media for 3 cycles, after which the cells were maintained in the maintenance medium for one week.

Alizarin red S staining and oil red O staining

The cells induced by osteogenic differentiation or adipogenic differentiation were fixed with polyformaldehyde for 30 minutes and then stained with 40 nM alizarin red solution (pH 4.2) or filtered oil red O solution at room temperature for 30 minutes. The nonspecific staining was removed with PBS. Stained BMSCs were observed and photographed by an inverted microscope (Olympus, Japan).

Alkaline phosphatase (ALP) activity assay

ALP activity assay was performed with the Alkaline Phosphatase Assay Kit (Abcam, UK). Initially, 1% Triton X-100 was added to lyse cells for 20 minutes, followed by centrifugation at 10000 g for 5 minutes, and the supernatant was collected. Thereafter, the reaction mixture was added and incubated at room temperature for 60 minutes. Finally, the assay buffer was added and incubated again for 30 minutes, the absorbance at 450 nm was measured by a microplate reader (Thermo Scientific, USA).

Cell proliferation assay

Cell Counting Kit-8 (CCK-8, Beyotime, China) was used to measure the cell proliferation activity according to the instructions. In brief, the cells were cultured in 96-well plates for 0, 24, 48 and 72 hours and then exchanged for 100 μl serum-free medium, and 10 μl CCK-8 solution was added to each well. The absorbance at 450 nm was measured by a microplate reader after incubation at 37°C for 2 hours.

EdU assay

According to the precedent study described [54], the EdU assay kit (RiboBio, China) was adopted to determine the cell proliferation ability. Cells were seeded into 96-well plates at a density of 5 × 10⁵ cells per well, and cultured under standard conditions for 72 hours. Then, they were incubated with EdU buffer at 37°C for 2 hours, fixed with 4% formaldehyde for 30 minutes and permeabilized with 0.1% Triton X-100 for 20 minutes. EdU solution was added to the culture, followed by the staining of nuclei with DAPI. The results were visualized by a fluorescence microscope (Olympus, Japan).

Quantitative real-time PCR

Total RNA in femoral head tissues and BMSCs was extracted using TRIzol Reagent and used to synthesize cDNA with the One-Step RT-PCR Kit (Thermo Scientific, USA). qRT-PCR was performed using the SYBR Primer-Script RT-PCR Kit (TaKaRa, Japan) with a CFX96 Touch quantitative PCR system (Bio-Rad, USA). Primers were generated by Sangon Biotechnology (Shanghai, China), and the primer sequences were as follows: METTL14, 5′-AGAAACTTGCAGGGCTTCCT-3′ (forward) and 5′-TCTTCTTCATATGGCAAATTTTCTT-3′ (reverse); PTPN6, 5′-GCCTGGACTGTGACATTGAC-3′ (forward) and 5′-ATGTTCCCGTACTCCGACTC-3′ (reverse); METTL3, 5′-CAAGCTGCACTTCAGAC GAA-3′ (forward) and 5′-GCTTGGCGTGTGGT CTTT-3′ (reverse); WTAP, 5′-ACGCAGGGAGAAC ATTCTTG-3′ (forward) and 5′-CACACTCGGCTGC TGAACT-3′ (reverse); FTO, 5′-TTCATGCTGGAT GACCTCAATG-3′ (forward) and 5′-GCCAACTGAC AGCGTTCTAAG-3′ (reverse); ALKBH5, 5′-TCACT GCATACGGCCTCAGGACAT-3′ (forward) and 5′-T TAGAGCAGGGTCCCTGTTGT-3′ (reverse); GAPDH, 5′-TGGTATCGTGGAAGGACTCA-3′ (forward) and 5′-CCAGTAGAGGCAGGGATGAT-3′ (reverse).

Western blot

Total protein was extracted with RIPA lysate containing cocktail protease inhibitor (Beyotime, China), and nuclear protein was extracted by the CelLytic NuClear Extraction Kit (Sigma, USA). The protein concentration was detected with the BCA Protein Assay Kit (Beyotime, China). Protein lysates were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Then, PVDF membranes were blocked with 5% nonfat milk and incubated with primary antibodies. The following primary antibodies were used in this study: anti-METTL14 (ab252562, Abcam), anti-PTPN6 (ab227503, Abcam), anti-RUNX2 (ab76956, Abcam), anti-PPARγ (ab59256, Abcam), anti-C/EBPα (ab40764, Abcam), anti-C/EBPβ (ab15049, Abcam), anti-β-catenin (ab6302, Abcam), anti-p-β-catenin (ab11350, Abcam), anti-GSK-3β (ab32391, Abcam), anti-p-GSK-3β (ab68476, Abcam), anti-GAPDH (ab8245, Abcam), anti-Histone H3 (ab1791, Abcam). GAPDH and H3 were used as the internal controls for total protein and nuclear protein, respectively. Horseradish peroxidase-conjugated secondary antibody was used to develop the blots. Blots were detected with the ECL Western Blotting Substrate Kit (ab65623, Abcam) on an ECL system (Bio-Rad, USA).

m6A RNA methylation quantification

The content of m6A in total RNA was measured by the m6A RNA Methylation Assay Kit (ab185912, Abcam). Briefly, isolated total RNA and bound RNA to the assay wells, and the wells were washed and incubated with capture antibody. After that, rewashed wells and added detection antibody and enhancer solution. Finally, the color developing solution was added and measured the absorbance at 450 nm by a microplate reader.

mRNA sequencing

The high-quality total RNA was extracted using TRIzol reagents from 1 × 10⁶ METTL14-overexpressing cells and control cells per duplicate and genomic DNA removed using a DNA-free Kit (ThermoFisher, USA). Library construction was conducted using a Collibri 3′ mRNA Library Preparation Kit (ThermoFisher, USA) according to the manufacturer’s instructions. Quality control and sequencing were performed by Genenergy Bio-Technology Inc. The DEGseq software package was used to identify differentially expressed genes between the METTL14 overexpression group and the control group with parameters p-value <0.05 and false discovery rate <0.05.

MeRIP-qPCR

The m6A level of the individual gene was determined by MeRIP-qPCR assay. According to the previous study [41], purified poly (A) RNA from total RNA was obtained using the Dynabeads mRNA Purification Kit (Thermo Scientific, USA). A/G immunomagnetic beads were incubated with anti-m6A antibody (ab208577, Abcam) or anti-IgG antibody (ab172730, Abcam) in the immunoprecipitation buffer to immunoprecipitate m6A-RNA complexes. Thereafter, the complexes were digested by Proteinase K (20 mg/ml), and the methylated mRNA of PTPN6 was measured by qRT-PCR.

RNA stability

To evaluate the effect of METTL14 on the stability of PTPN6 mRNA, we detected the half-life of PTPN6 mRNA. Actinomycin D (5 μg/ml) was used to intervene in OE-NC and OE-METTL14 transfected BMSCs from SONFH patients, and total RNA was extracted at 0, 2, 4, 6 and 8 hours. The level of PTPN6 mRNA was measured by qRT-PCR, and the half-life was obtained by nonlinear regression analysis.

Luciferase reporter assay

Luciferase reporters were generated by cloning wild type 3′UTR of PTPN6 mRNA or mutant type 3′UTR of PTPN6 mRNA (mutant A to T at position 859) into psiCHECK2 (Promega, USA). Then, cells were seeded in 24-well plates and co-transfected with 150 ng of renilla reporters and OE-METTL14. 48 hours later, collected cells and luciferase activities were detected using the Dual-Luciferase Reporter Assay System (Promega, USA) according to manufacturer’s protocol.

TOP/FOP-Flash reporter assay

TOP/FOP-Flash reporter assays were used to determine the transcriptional activity of the Wnt/β-catenin pathway. BMSCs were seeded in 24-well plates and transfected with the TOP/FOP-Flash reporter (Biovector NTCC Inc., China). After 48 hours, the luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, USA). The luciferase activity of each sample was normalized to the respective Renilla luciferase activity.

Immunofluorescence staining

Cells cultured on cover slips were washed twice with cold PBS and fixed with paraformaldehyde for 15 minutes at room temperature and were permeabilized with 0.1% Triton X for 20 minutes. Cells were incubated with 1% bovine serum albumin for 40 minutes to block the nonspecific antibody binding sites and subsequently incubated with anti-β-catenin antibodies (1:200) overnight at 4°C. Afterwards, the cells were washed three times with PBS and incubated with a 1:500 dilution of an APC-conjugated secondary antibody for 1 hour. Finally, cells were stained with DAPI to visualize the nuclei. Images were captured by a fluorescence microscope.

IHC staining

The femoral head tissues were decalcified at room temperature in 15% EDTA for 5 weeks. After decalcification, the sample was embedded in paraffin. Four micrometer paraffin slides were prepared on glass slides coated with polylysine and then dried for one hour at 60°C. The slides were blocked with 5% BSA and then stained with anti-METTL14 antibody (1:100) or anti-PTPN6 antibody (1:100) at 4°C overnight. Finally, the slides were incubated with HRP-labeled secondary antibody. The slides were visualized by an inverted microscope.

Co-immunoprecipitation assay

Total protein lysates for immunoprecipitation assay were extracted with IP-lysis buffer (ThermoFisher, USA). The supernatant was collected by centrifugation at 12000 rpm for 10 minutes at 4°C. For immunoprecipitation, the supernatant was mixed with anti-PTPN6 antibody (ab227503, Abcam) or anti-IgG antibody (ab172730, Abcam) and 50 μL of protein A/G Dynabeads (ThermoFisher, USA) at 4°C overnight. After that, the beads were washed three times with pre-cooled IP lysis buffer, and immunoprecipitates were subjected to western blotting.

Statistical analysis

All experiments were independently repeated at least three times. Data in this study are expressed as the mean ± SD. GraphPad Prism 7.0 software was used for all data analysis. The difference between two groups was analyzed by t-test. One-way analysis of variance (ANOVA) was used for comparison among the multiple groups. The difference of clinical features between SONFH group and control group was analyzed via χ² analysis. The linear association between METTL14 and PTPN6 was tested by Pearson’s Correlation Coefficient. P-value <0.05 was considered statistically significant.