FTO is increased expression in OS
To evaluate the expression of FTO, we investigate the expression pattern of FTO in OS cells and the normal osteoblast cells (Nhost) by qPCR. And results showed that the mRNA levels of FTO in OS cells were elevated (Fig. 1a). We also detected the mRNA levels of FTO in OS and adjacent tissues. Similarly, mRNA levels of FTO were frequently increased in cancer tissues compared with matched normal tissues (Fig. 1b). IHC staining was also used to detect the FTO expression in OS and matched normal tissues (Fig. 1c). According to the quantitative analysis of IHC staining of tissue samples, we divided the FTO expression into high expression and low expression. 64.3% of OS tissues showed high expression, whereas the low expression rate of FTO in normal tissues was only 26.2% (Fig. 1d).
We further analyzed the relationship between the FTO expression and clinicopathological parameters and postoperative survival of OS. Results from the IHC assay showed that there was a significant correlation between the high expression of FTO and several clinicopathological features (Table 1), such as tumor size, metastasis, and TNM stage, but not with the gender and age. In order to further understand the relationship between FTO and survival of OS, according to the FTO expression, we performed Kaplan-Meier survival curve analysis. The FTO expression was negatively correlated with the 5-year overall survival of patients with OS (Fig. 1e).
FTO promotes OS cell proliferation, migration and invasion
In order to further study the effect of FTO expression on the proliferation of OS cells, FTO knockdown or overexpression was used to detect the proliferation ability of OS cells at 24h, 48h, 72h, respectively. Transfection efficiencies of FTO knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 2a). Results showed that overexpression of FTO in OS cells promoted cell proliferation (Fig. 2b), while knockdown of FTO inhibited cell proliferation (Fig. 2b). As shown in (Fig. 2c), Clone formation also supported the role of FTO in contributing to proliferation of OS cells, and FTO downregulation weakened the cell proliferation. In addition, enforced expression of FTO inhibited OS cell apoptosis, however, blockade of FTO expedited apoptosis (Fig. 2d).
Scratch healing assay was used to verify the migration ability of FTO to OS cells. Amplification of FTO accelerated scratch healing of OS cells (Fig. 2e). Transwell chamber assay showed that FTO attenuates the invasion of OS cells; inversely, silencing FTO attenuated the invasive ability (Fig. 2f). These findings indicated FTO promoted proliferation, migration and invasion of OS cells.
FTO inhibits KLF3 expression in OS
As the first discovered demethylase FTO, its discovery reveals the dynamic and reversible process of m6A modification, and it can reverse the m6A modification on RNAs [22]. Previous results indicated that KLF3 is decreased expression in sarcomas [19]. It has been reported that the tumor suppressor KLF4, another member of the KLF family, could be regulated in an m6A dependent manner bladder cancer [23]. METTL3-mediated m6A methylation directly promotes the mRNA decay of KLF4 through m6A binding protein YTHDF2 [23]. Similarly, we identify whether the role of KLF3 in OS is regulated by m6A eraser FTO. Therefore, we subsequently analyzed the profile of m6A content in OS tissues. Results from m6A methylation quantification showed that m6A modification is significantly decreased in the total RNAs of OS tissues compared to adjacent normal tissues (Fig. 3a). m6A methylation quantification and dot blot assays were employed to detect the effect of FTO on m6A methylation in OS cells. As expected, m6A level was down-regulated in OS cells expressing FTO (Fig. 3b-c). Western blotting confirmed that FTO contributed to decrease KLF3 expression in OS cells, and silencing FTO conferred by siRNA inversely increased KLF3 (Fig. 3d), which were consistent with results from qRT-PCR (Fig. 3e). IHC staining also supported the decreased expression of KLF3 in OS tissues compared with normal tissues (Fig. 3f). Correlation analysis revealed that there was a significant negative correlation between FTO and KLF3 mRNA levels in OS tissues (Fig. 3g). Thus, we speculated that FTO can negatively regulate the expression of KLF3.
FTO regulates KLF3 expression in an m6A-dependent manner
The functional interaction between m6A methylase and demethylase determines the dynamic and reversible regulation of m6A modification. And m6A binding proteins can bind to mRNA containing m6A, thus affecting the fate of target mRNAs [24]. Me-RIP qPCR suggested that knockdown of FTO augmented the m6A enrichment on KLF3 mRNAs in OS cells (Fig. 3h). FTO can negatively regulate the content of KLF3 mRNAs (Fig. 3e). We carried out the stability assay of KLF3 mRNAs. The results suggested that, after treatment with Actinomycin D (Transcription inhibitor), the residual percentage of KLF3 mRNA in FTO group was significantly lower than that in control group (Fig. 3i). Previous studies have confirmed that m6A binding protein YTHDF2 affects the stability of m6A modified RNA by locating them at mRNA decay sites [24]. Transfection efficiencies of YTHDF2 knockdown or overexpression were analyzed by qPCR in OS cells (Fig. 3j). YTHDF2 attenuated the mRNA and protein levels of KLF3 (Fig. 3k-l). And YTHDF2 reduced the half-life of KLF3 mRNA (Fig. 3m). RIP-qPCR assay proved the interaction between YTHDF2 protein and KLF3 mRNA in OS cells, which was enhanced in OS cells expressing FTO (Fig. 3n). Silencing YTHDF2 abrogated FTO-mediated the decrease of KLF3 mRNA and protein levels in OS cells (Fig. 3o-p). qRT-PCR results also verified that YTHDF2 knockdown reversed FTO-induced mRNA degrading of KLF3 (Fig. 3q). Overall, Silencing FTO accelerated the YTHDF2-involved decay of KLF3 mRNA in OS cells.
KLF3 impairs the FTO-induced proliferation and invasion of OS cells
To ascertain whether KLF3 is involved in FTO function, OS cells transfected with FTO plasmids showed lower expression of KLF3, which could be reversed by overexpressing KLF3 (Fig. 4a). Moreover, FTO dramatically accelerated the proliferation of OS cells, whereas enforced expression of KLF3 reversed the FTO-induced promotion effects (Fig. 4b). Cloning formation efficiencies were significantly lower in OS cells expressing FTO and KLF3 than that of OS cells expressing FTO (Fig. 4c). FTO inhibited apoptosis, which was severely impeded in OS cells with ectopic expression of KLF3 (Fig. 4d). Analogously, KLF3 dramatically suppressed FTO-induced cell invasion (Fig. 4e). Cyclin D1 and p21 is the key protein of cell cycle regulation. FTO increased Cyclin D1 expression and decreased p21 expression in OS cells (Fig. 4f), and KLF3 could reverse the alternation. EMT also plays an important role in the process of tumor metastasis. FTO could enhance N-cadherin and Vimentin expression and suppress E-cadherin expression (Fig. 4f), which were compromised by KLF3 amplification. Thus, our findings indicated that FTO promoted cell proliferation and invasion by abating KLF3 in OS cells.