ALKBH5 involves in osteosarcoma tumor progression by mediating Notch signaling

ALKBH5 is the major demethylase of ribonuclease m6A and exerts these multiple biological functions in cancer. In this study, we mainly explore the use of ALKBH5 in the development and progression of osteosarcoma and elucidate the potential molecular mechanisms by which it functions. Relative to human osteoblast cell lines (NHOst), we detected ALKBH5 expression in osteosarcoma cell lines (HOS, U2OS, and MG‐63) by RT‐PCR and western blot. By overexpression and knockout of ALKBH5 gene, to observe the effect of ALKBH5 on apoptosis, invasion and epithelial‐mesenchymal transition (EMT) of osteosarcoma cells, as well as the effect of Notch signaling pathway. We found that ALKBH5 was significantly higher in osteosarcoma cell lines than in osteoblastic cell lines. In addition, ALKBH5 overexpression promoted the proliferation, migration, and invasion of osteosarcoma cells, and simultaneously activated Notch signaling pathway and up‐regulated E‐cadherin protein. Knockout of ALKBH5 inhibited these effects in osteosarcoma cells. It was also found that up‐regulation of ALKBH5 could promote the proliferation of osteosarcoma cells in vitro and in vivo. ALKBH5 can be involved in osteosarcoma development through Notch signaling pathway regulation. ALKBH5 is therefore expected to be a new target in the treatment of osteosarcoma.

composed of methyltransferase-like 3, 14, and 16 that functions on mammalian nuclei, and WTAP interacts with it and affects methylation. 8,9 AlkB homolog 5(ALKBH5) is a demethylase that plays an important role in m6A modification. ALKBH5 has been found to be highly expressed in a variety of cancer studies, such as breast, glioblastoma, ovarian, and gastric cancers. [10][11][12] In terms of osteosarcoma, it has been shown that ALKBH5 expression is positively correlated with lncRNA PVT1 expression. 13 ALKBH5 was also found to be down regulated in osteosarcoma tissues and inhibited osteosarcoma by targeting YAP. 14 Therefore, the exact role of ALKBH5 in osteosarcoma development warrants further investigation. Here, we show that down-regulation of ALKBH5-induced M6a demethylation suppresses growth, migration, and invasion of human osteosarcoma cells by activating Notch signaling.

| Cell culture and treatment
Normal osteoblast cell lines (NHOST) and human osteosarcoma cell lines (U2OS, HOS, and MG63) were cultured in complete medium containing 10% FBS and 1% double antibody (DMEM for U2OS and MG63, EMEM for HOS, and DMEM/F-12 for NHOST) and then placed in a 37 C constant temperature incubator containing 5% CO 2 .
All cell lines were tested for mycoplasma contamination. The ALKBH5 siRNA sequence (5 0 -CGGACGTTGCAGTGATA-3 0 ) was designed and synthesized by Shanghai Sangon Biotech; all cell culture reagents were from Thermo Fisher Scientific, USA.
1.2 | m6A enzyme-linked immunosorbent assay (ELISA) Cell samples were processed according to the m6A ELISA kit instructions (ab185912, Abcam, UK) and absorbance at 450 nm was measured using a microplate reader to draw a standard curve and calculate concentration.

| Quantitative real-time PCR
Total RNA was extracted using TRIzol according to the manufacturer's protocol, and recovered RNA was purified in strict accordance with the RNA recovery kit's instructions. cDNA from a reverse transcription reaction was used, and we performed real-time PCR in triplicate using Power SYBR green polymerase chain reaction (PCR) master mix and an ABI 7500 real-time PCR system. All procedures were performed according to the manufacturer's recommendations. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the target gene primer sequences were as follows: ALKBH5-F:CGGCGAAGGCTACACTTACG, ALKBH5-R:CCACCAGCTTTTGGATCACCA, GAPDH-F:GAGGCTGAA CCTTAAGGT, GAPDH-R:AGGGCCGCTGGTCAGAAGTT with GAPDH used as an internal control. The relative expression of target genes was calculated based on the 2-CT value.

| Western blot
The osteosarcoma cells with different treatment for two days were

| Colony-formation assay
Cells infected with plasmids or siRNAs were seeded at 1000 cells/well in 6-well plates and cultured normally. Air colonies were performed and counted after 2 weeks fixation with formaldehyde, crystal violet staining, and repeated three times.    Figure 2B).

| Effect of ALKBH5 on apoptosis, proliferation and growth of osteosarcoma cells in vitro and in vivo
Flow cytometry showed that knockdown of ALKBH5 significantly increased apoptosis, but had little effect on overexpression ( Figure 3A); in addition, elevated ALKBH5 could reduce or silence increased colony formation ability in MG-63 cells ( Figure 3B). In vitro, ALKBH5 silencing decreased the oncogenic behavior of MG-63 cells ( Figure 4A), and xenograft growth and liver metastasis of MG-63 cells injected with silenced ALKBH5 vector were significantly reduced ( Figure 4B). Thus, ALKBH5 impairs not only proliferation but also colonization of osteosarcoma cells, thereby inhibiting tumor growth and metastasis in vivo.

| ALKBH5 inhibits osteosarcoma progression by mediating EMT through Notch signaling
We found that expression of Notch1Notch2 and Notch2 was significantly reduced in the ALKBH5 knockdown group and significantly increased in the ALKBH5 overexpression group. It has been shown that Notch signaling can promote osteosarcoma development by enhancing EMT. 15 We investigated the expression of EMT-related proteins (E-cadherin and N-cadherin) in ALKBH5-overexpressing or silenced cells. The results showed that ALKBH5 knockdown showed decreased expression of E-cadherin and increased expression of N-cadherin ( Figure 5).  second, silencing ALKBH5 can inhibit osteosarcoma cell growth and invasion, and overexpression has an annoying effect, thus exerting anti-tumor effects. In addition, overexpression of ALKBH5 can activate Notch signaling pathway and promote proliferation and metastasis of osteosarcoma, while down-regulation of ALKBH5 inhibits these effects. Therefore, we suggest that ALKBH5 is a tumor-inducing gene and that ALKBH5 knockdown may be a novel alternative therapy for osteosarcoma.
Panneerdoss S et al. reported that silencing at ALKBH5 resulted in decreased cell proliferation, migration, and invasion in breast, cervical, liver, and prostate cancers. 16 We found that ALKBH5 was significantly highly expressed in osteosarcoma cell lines. Silencing of AlKBH5 significantly inhibited the growth and invasion of osteosarcoma cells, and overexpression had the opposite effect. Similarly, animal experiments showed that tumors were smaller after ALKBH5 silencing than in controls. These results illustrate that ALKBH5 is an oncogene in osteosarcoma. Zhang C et al. showed that ALKBH5 impairs tumor formation and reduces BCSC in breast cancer, 17 while Fukumoto T found that ALKBH5 silencing promotes PARPI resistance by increasing Wnt signaling above m6A modification in FZD10 mRNA. 18 These results suggest that ALKBH5 is involved in tumor development and has an oncogenic effect in osteosarcoma. Therefore, it is significant to explore ALKBH5 downstream targets in osteosarcoma studies. Notch receptor is a transmembrane protein composed of a variety of protein modules, which regulates the body's response to environmental signals. 19 It plays an important role in the development of various tumors such as lung, colon, liver, and gastrointestinal tract. [20][21][22] Notch1/2 has been found to activate EMT to promote invasion and metastasis in osteosarcoma studies and is associated with poor patient prognosis. 15 Engin F found that Notch1/2 was highly expressed in osteosarcoma and promoted osteosarcoma cell proliferation. 23 However, the relationship between ALKBH5 and Notch in osteosarcoma has not been investigated. To clarify the association among them, we found that Notch signaling pathway was significantly enriched in the ALKBH5 high expression group.
Through in-depth study, it was found that silencing ALKBH5 inhib-

ACKNOWLEDGMENTS
The authors thank all students and technicians in the laboratory for their cooperation.

FUNDING INFORMATION
This study was financially sponsored by the Shanghai Pudong New Area Science and Technology Development Fund (PKJ2017-Y44).

CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.

ETHICS STATEMENT
All animals were kept in a pathogen-free environment and fed ad lib. The procedures for care and use of animals were approved by the Ethics Committee of the Animal Protection and Utilization Committee of Fudan University and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.
F I G U R E 5 ALKBH5 is involved in osteosarcoma progression through EMT and Notch signaling pathways. (A) Effect of ALKBH5 on Notch1, Notch2, Hes-1, E-cadherin, and N-cadherin proteins in MG-63 cells. (B) Western blot statistical analysis. *p < .01 and**p < .01, n = 3 relative to control and NC.