METTL16 promotes cell proliferation by up‐regulating cyclin D1 expression in gastric cancer

Abstract N6‐methyladenosine (m6A) is a well‐known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT‐qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down‐regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock‐down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16‐mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.


| BACKG ROU N D
Gastric cancer (GC) is a global health issue, with more than 1 million new cases worldwide every year. Although the incidence and mortality of GC have declined globally in the last 50 years, it currently remains as the third leading cause of cancer-related deaths with the fifth highest incidence in all of cancer. 1 Currently, surgery is the only possible radical cure. However, most patients lost the opportunity for surgical treatment due to the fact that they were diagnosed in the mid to advanced stages. Although some progress has been made in the pathogenesis and treatment targets of GC, it is not enough to meet the clinical demands for improving the diagnosis and treatment of GC. 2 Therefore, exploring the pathogenesis of GC, finding better treatment targets and optimizing treatment strategies are issues that we urgently need to resolve.
As the most abundant internal modification in eukaryotic mRNA, N6-methyladenosine (m6A) modification affects the splicing, transcription, translation, localization, metabolism and stability of RNA. 3 m6A modification plays key roles in a multitude of biological processes, such as the development of nervous system, circadian rhythm, DNA damage response, heat shock response, cell signal transduction and tumorigenesis. 4 There is growing evidence that RNA modification pathways function in the regulation of human cancers and they may be ideal targets for cancer treatment. 5 Changes of m6A levels in malignant tumours may play a role in promoting or suppressing cancer development through affecting related tumour markers, for example maintaining proliferation signals, evading growth inhibitors, resisting apoptosis, making replicates immortal, inducing angiogenesis, activating invasion and metastasis, reprogramming energy metabolism, promoting genome instability and mutation, inducing evasion of immune surveillance and cancerpromoting inflammation. [6][7][8] METTL16 is the second RNA m6A methyltransferase discovered so far, which can modify certain coding and non-coding RNAs with m6A. 9 It can regulate MAT2A mRNA level in cell to maintain the steady state of S-adenosylmethionine (SAM), 10,11 and it is also indispensable for mouse development. 12 However, the specific role of METTL16 in the development of cancer, especially GC, and related regulatory mechanism are still unclear.
In our study, we investigated the expression of METTL16 in GC and found that the proliferation of GC cells was significantly inhibited after knocking down METTL16, and the cell cycle was blocked in G1/S phase. By search target of this effect, we found cycle D1 as a key downstream target for this RNA modification. Our results suggested that METTL16 might be a potential therapeutic target for the treatment of human GC.

| Clinical specimens and ethical approval
We had collected 231 paraffin-processed GC samples with its paired normal adjacent tissues (NATs) from patients who underwent radical GC surgery from January 2008 to December 2013 in the First Affiliated Hospital of Sun Yat-sen University. The 5 µm paraffin sections were completed in the pathology department, along with complete follow-up data provided by the GI surgical department of the hospital. Follow-up period was once every 3 months in the first 2 years, and once every 6 months from the 3rd to 5th years, with a mean follow-up period of 49.1 months. Total survival time was defined as from the day of surgery to the time of death or date of last follow-up. Clinicopathological characteristics of the patients can be found in Table 1. We have also collected 16 fresh GC samples with its paired NATs from patients who underwent radical GC surgery from June 2019 to September 2019 in the Seventh Affiliated Hospital of Sun Yat-sen University, including 10 male cases and 6 female cases, with a median age of 59 years (47-72 years). These fresh samples underwent rapid freezing with liquid nitrogen upon excision and then stored in −80°C refrigerator until use. Research protocol was approved by both Ethics Committee of the First and Seventh Affiliated Hospital of Sun Yat-sen University.

| Immunohistochemistry (IHC)
We employed immunohistochemistry to detect expression levels of METTL16 in GC tissues and its paired NATs. Firstly, the sections were deparaffinized and rehydrated. 1X Tris-EDTA buffer was used for antigen retrieval at 100°C for 8 minutes, and then, the sections were treated with 3% hydrogen peroxide for 20 minutes, followed by 5% goat serum for 30 minutes. Rabbit anti-METTL16 polyclonal antibody (1:200; cat. no. A118157; SIGMA) was used overnight at 4°C. Subsequently, after washing with PBS, the section was incubated for 1h at room temperature with horseradish peroxidase (HRP) (goat anti-rabbit, cat. no. A0208; Beyotime Institute of Biotechnology). After DAB staining and haematoxylin staining, METTL16 expressions in the tissues were observed under a light microscope. An improved H scoring system was used to semi-quantitate the expression of METTL16. Formula: maximum staining intensity (0, negative -; 1, weak positive +; 2, moderately positive++; 3, strong positive +++) multiplied by the percentage of positive tumour cells (0%-100%) equal corrected H score (range 0-300). 13 The immunohistochemical score was scored independently by two pathologists following the scoring method above. They were blinded to the clinical information of the patient, and the final H score was averaged. METTL16 staining was classified as high or low expression according to the median H score.

| METTL16-targeting short hairpin RNA (shRNA) and lentivirus packaging
Three targeted shRNA and 1 non-targeted scrambled RNA sequence were sub-cloned into GV493 lentiviral vector by Shanghai GeneChem Co., Ltd. The target sequence of shMETTL16 is

| Cell cycle analysis
The treated cells were collected and fixed with 75% pre-chilled ethanol at 4°C overnight. After removing the ethanol, phosphate buffer was used to wash the cells twice, and then, the cells are incubated with propidium iodide staining solution in the cell cycle analysis kit (Beyotime, CAT#C1052) for 30min at room temperature. Cell cycle distribution was analysed through flow cytometry (Beckman CytoFlex).

| CCK8 cell proliferation assay and colony formation assay
In the cell proliferation assay, 2 × 10 3 cells/well were seeded into 96-well plates. After the cells adhered, 10 ul of CCK8 reagent (Fude Biological, CAT#FD3788) was added to each well on day 1, 2, 3, 4 and 5, and the absorbance was measured by spectrophotometry at 450 nm wavelength after 2 hours.
In the colony formation assay, 500 cells/well were seeded in a 6-well culture dish. After 2 weeks, the cells were fixed in 4% paraformaldehyde, stained with crystal violet (Beyotime Biotechnology, CAT#C0121) and counted microscopically.

| Western blotting and antibodies
Cells were collected and placed into a protein lysis buffer on ice for 30 minutes. BCA protein assay kit (KeyGEN BioTECH, CAT#KGP903) was used to quantify protein concentration. The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Next, the sample was transferred to a polyvinylidene difluoride

| Statistical analysis
All results were expressed as mean±standard deviation. The differences between categorical variables were tested via χ2 test, and the differences between two groups were tested via Student's t test.
Kaplan-Meier curve and log-rank test were used for statistical analysis of OS other survival-related data of GC patients. SPSS 17.0 software was used for statistical analysis. P value < .05 was considered statistically significant.

| METTL16 is up-regulated in GC tissues and GC cell lines
In order to investigate whether METTL16 is abnormally expressed in GC, we first compared the expression profiles between GC tissues and the normal adjacent tissues using mRNA expression data set from The Cancer Genome Atlas (TCGA) database. The results showed that the level of METTL16 in GC tissues was significantly higher than that in paired NATs ( Figure 1A), and the mRNA level of METTL16 in different stages (N0-N4) of GC patients was significantly higher than that in normal tissues ( Figure 1B). Western blotting and qPCR analysis were then used to detect the expression of METTL16 in 16 cases of GC and its paired NATs. We found that the protein ( Figure 1C) and mRNA levels ( Figure 1D,E) of METTL16 were higher in most GC tissues compared with the paired NATs. In addition, we detected METTL16 levels in several GC cell lines and found that both mRNA level ( Figure 1F) and protein level ( Figure 1G) of METTL16 were elevated in most GC cell lines when compared with the normal gastric mucosal epithelial cells GES-1. The above data indicated that METTL16 level was increased significantly in GC tissues and GC cell lines.

| High expression of METTL16 in GC suggests a poor prognosis
In order to explore the impact of the high expression of METTL16 on the prognosis of GC patients, immunohistochemistry was used to detect expression level of METTL16 in GC tissues from 231 GC patients with complete follow-up data. METTL16 was expressed in both cytoplasm and nucleus of cancer cells, and the expression level of METTL16 in gastric cancer cells was much higher than that in surrounding non-cancer cells (Figure 2A,B). We then used the H score to quantify the staining intensity of METTL16 in these tissues, and the results indicated that the expression of METTL16 in GC tissues was significantly increased compared with the NATs (Figure 2B,C).
We also studied the relationship between METTL16 expression and clinicopathological variables in Table 1, and it is worth noting that patients with larger tumour size (≥5 cm) and regional lymph node metastasis tended to show higher level of METTL16 in tumour (Table 1). log-rank test, P = .001; Figure 2D]. We further analysed METTL16 transcription data set from Kaplan-Meier plotter database to verify the relationship between METTL16 level and prognostic effect of GC patients. The results indicated that high expression of METTL16 was associated with poor OS ( Figure 2E).

| Down regulation of METTL16 inhibits GC cell proliferation through restraining G1/S phase
In order to study the role of METTL16 in GC development, we first verified the METTL16 level in GC cell lines and found that

| METTL16 promotes tumour growth in mice
To confirm the function of METTL16 in vivo, METTL16 knocked down MGC803 cells (shMETTL16-2) or normal control MGC803 cells (shNC) were injected subcutaneously into nude mice to establish tumour xenograft models. When compared with control, we found that METTL16 depletion inhibited tumour growth ( Figure 5A,B), along with reduced tumour volume and mass ( Figure 5C,D). HE staining showed that there were obvious tumour cells in both groups ( Figure 5E). The expression of Ki67, a marker of proliferation, also decreased after METTL16 knockdown ( Figure 5F). These results showed that tumour growth is promoted by METTL16 in vivo.

| METTL16 regulates the GC cell cycle through mediating the expression of cyclin D1
In order to clarify the mechanism of the down-regulation of METTL16 leading to G1-S phase arrest, we detected the expression of cyclin D1, cyclin E1, p21, p27, CDK2 and CDK6, which are involved in transition of G1/S phase. We found that in METTL16 knocked down AGS, MGC803 and SNU719 cells, the expression of cyclin D1 was significantly reduced ( Figure 6A). Similarly, the qPCR results showed that the mRNA level of cyclin D1 was significantly     Figure 7A). Subsequently, we conducted RNA degradation assays and found that the degradation rate of cyclin D1 mRNA in METTL16 knocked down GC cells was significantly faster than that in normal control group after treated with actinomycin D, meaning that the half-life of cyclin D1 mRNA was significantly shortened ( Figure 7B). Western blotting results also showed that the cyclin D1 expression was decreased in METTL16 knocked down GC cells with treatment of actinomycin D for 8 hours ( Figure 7C).
These results showed that METTL16 can promote cyclin D1 mRNA stability in GC cells, thereby promoting the expression of cyclin D1.
Besides, we used methyltransferase inhibitors to treat GC cells (AGS, MGC803 and SNU719) and found that the m6A levels were reduced ( Figure 7D), indicating that METTL16 functions as a RNA methyltransferase to regulate the cyclin D1 expression in GC cells.  Figures S3 and S4).
In addition, methyltransferase inhibitor was used to study whether cyclin D1 expression is regulated by methyltransferase. The results showed that both mRNA and cyclin D1 protein levels were inhibited in GC cells after treated with DAA which is a methyltransferase inhibitor ( Figure 7E,F), suggesting that methyltransferase plays a significant role in regulating the expression of cyclin D1 in GC cells.
The above results suggested that METTL16 could enhance the stability of cyclin D1 mRNA in GC cells through m6A modification. was negatively associated with OS in GC patients, similar with the results of METTL3 in GC. 19 In contrast, elevated expression of METTL16 predicts higher OS in liver cancer patients. 18 We believe that the difference of prognostic significance of METTL16 between GC and liver cancer is due to the fact that the same gene encoding the methyltransferase plays different roles in different cancers. 20 For example, in the study of glioblastoma stem cells, researchers found that knocking out METTL3 could greatly promote the growth, self-renewal and tumorigenesis of human glioblastoma stem cells. 21 However, in a study of acute myeloid leukaemia, METTL3 was found to be necessary for the growth of acute myeloid leukaemia cells and down-regulation of METTL3 could lead to cell cycle arrest, leukaemia cell differentiation, and even unable to induce leukaemia in immunodeficient mice. 22,23 This is why the research on m6A modification is unique, and the study we conducted is meaningful.

| D ISCUSS I ON
In this study, we found that METTL16 knock-down could inhibit GC cell proliferation and tumour growth in mice, and the total m6A level of RNA was reduced in METTL16 knocked down or methyltransferase inhibitor-treated GC cells, including AGS, MGC803 and SNU719. In addition, it has been proved that knocking down METTL16 could lead to a reduction in the overall level of cellular RNA methylation. 24 One previous study also showed a significant rise in the m6A methylation of total RNA in GC cells and tissues. 19 These results indicated that METTL16 played a significant role in promoting cell proliferation by increasing the enzymatic activity of m6A in GC cells. has an additional role in the pre-mRNA splicing process, enabling METTL16 to be both the 'writer' of m6A and the 'reader' of m6A. 34 We have confirmed that the half-life of cyclin D1 mRNA was shortened and its stability was also significantly reduced in METTL16 knocked down GC cells compared with the control group. We also found that the mRNA and protein levels of cyclin D1 were inhibited after treatment with methyltransferase inhibitors. These results suggested that METTL16 enhanced the stability of cyclin D1 mRNA through its methyltransferase activity, thereby increasing cyclin D1 expression to promote the proliferation of GC cells.
However, we are not yet certain that cyclin D1 mRNA is the direct substrate of METTL16, nor is it clear whether METTL16 can splice the pre-mRNA of cyclin D1 and promote translation of cyclin D1. If METTL16 acts as a 'writer' to modify cyclin D1 mRNA with m6A, whether there is a recognition protein or 'reader' and a demethylase or 'eraser' for subsequent processing, these questions should be answered with further research.

| CON CLUS ION
In summary, the present study reveals that METTL16 has a cancerpromoting effect in GC and high expression of METTL16 indicates poor prognosis of GC. METTL16 functions as an m6A methyltransferase to promote GC cell proliferation through enhancing the stability of cyclin D1 mRNA ( Figure S5). Our findings enrich the functions of m6A methylation in tumour markers and shed light to a potential way to explore effective strategies for the treatment of GC.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest.

E TH I C S S TATEM ENT
Ethical committees of the First Affiliated Hospital of Sun Yatsen University and the Seventh Affiliated Hospital of Sun Yat-sen University approved this study. Written consent was obtained from all individual participants included in the study.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.