m⁶A methyltransferase METTL16 mediates immune evasion of colorectal cancer cells via epigenetically regulating PD-L1 expression

Evaluation of clinical samples

Patients diagnosed with CRC (n=20) and hospitalized in Tangshan Traditional Chinese Medicine Hospital were recruited in this study. Tumor and adjacent non-tumor tissues were collected during surgical operation and made into paraffin-embedded samples or frozen into liquid nitrogen for further experiments. All patients have signed the informed consents.

Xenograft model

Male BALB/c nude mice that aged 4 to 6-week-old were bought from Vital River Laboratory (China). SW480 cells transfected with control or METTL16 overexpression vectors (5 x 10⁶ cells/site) were suspended in 50 μl saline and inoculated into the fact pat on back. Tumor size was measured and calculated every three days. After feeding for 30 days, the tumors were collected and made into paraffine-embedded samples.

IHC staining

Paraffine-embedded tissues were dewaxed and blocked with goat serum. After incubation with primary antibody against PD-L1 (1:200, Abcam, USA), METTL16 (1:200, Abcam, USA), and Ki-67 (1:200, Abcam, USA), the samples were probed with HRP-conjugated secondary antibodies and reacted with DAB for visualization. Images were taken using a light microscope (Leica, Germany).

Cell lines and transfection

CRC cell lines NCM460, RKO, HCT116, SW480, Colo320, DLD-1, HCE8693, SW620, and HT29, and the normal human colon epithelial cell line FHC were purchased from Wuhan Procell and ATCC. NCM460, DLD-1, HCE8693, and Colo320 were maintained in RPMI-1640, RKO in MEM, HCT116 and HT29 in McCoy’s 5A, SW620, SW480 in Leibovitz’s L-15 medium that supplied with 10% FBS (BI, Israel) and 1% penicillin and streptomycin (Sigma, USA) and maintained in 37° C incubator with 5% CO₂.

METTL16 proteins siRNAs and overexpression vectors were obtained from GenePharma (Shanghai, China). Cells were transfected with indicated oligonucleotides using the Lipofectamine 2000 reagent (Thermo, USA) as per manufacturer’s protocol. Transfection efficacy was examined 2 days after transfection.

Cell viability and proliferation detection

Cell viability was measured by cell counting kit-8 (CCK-8, SolarBio, China) following the manufacturer’s protocol. CRC cells were seeded into 96-well plate with 5,000 cells per well and incubated at 37° C incubator for indicated time points. Then CCK-8 reagent was added and incubated for 2 hours. The absorbance values at 450 nm were measured by a microplate detector. For colony formation assay, cells were seeded into 6-well plate and incubated for 10 days. The visible colonies were then stained with crystal violet for 20 min. Images were taken by a digital camera.

Cell apoptosis

Cell apoptosis was measured using Annexin V/PI apoptosis detection kit (Beyotime, China) in line with manufacturer’s introduction. In brief, cells were collected and suspended in binding buffer that contains Annexin V and PI reagent. After incubation for 30 minutes in dark, the samples were loaded in a flow cytometer (BD Bioscience, USA) and the portion of apoptotic cells were measured.

Transwell assay

CRC cells were suspended in serum-free medium and seeded into the upper chambers of Transwell (Costar, USA). The lower chambers were filled with complete culture medium. After incubation for 24 hours, the upper chambers were collected, and the inner side of chamber was wiped with cotton sticks. The migrated cells were then stained with crystal violet for 20 min. To measure cell invasion, the Transwell chambers were pre-coated with Matrigel that mixed with Leibovitz’s L-15 at 1:1. Images of cells were taken under a microscope (Leica, Germany) [25].

Wound healing experiment

CRC cells were seeded into 6-well plate and cultured overnight to confluence. The monolayer of cells was then scratched with a sterile 200 μl pipette and washed with PBS to remove debris. Cells were then incubated for 24 hours, and images of the wounds were taken at 0. 24, and 48 h after scratching.

Western blotting assay

Cell pellets were homogenized with RIPA lysis buffer and quantified with BCA kit. Protein samples containing 40 mg of protein were separated by SDS-PAGE gel and transferred onto PVDF membranes, followed by blocking in 5% skim milk for 2 hours. The blots were then probed with primary antibodies that target E-cadherin (1:2000, ab231303, Abcam, USA), Vimentin (1:2000, ab92547, Abcam, USA), METTL16 (1:2000, ab252420, Abcam, USA), and β-actin (1:2000, ab8226, Abcam, USA) overnight at 4° C. The membranes were then hatched with HRP-conjugated secondary antibodies and ECL reagent for visualization.

Quantitative real time PCR (qPCR)

Total RNAs were extracted using the Trizol solution (Qiagen, USA). Then a total of 1 μg RNA was subjected to reverse transcription reactions using a Reverse Transcription kit (Qiagen, USA). PCR amplification was conducted using the SYBR GREEN qPCR mix (Thermo, USA).

PD-L1 forward primer: 5’- TGGCATTTGCTGAACGCATTT-3’,

PD-L1 reverse primer: 5’-TGCAGCCAGGTCTAATTGTTTT-3’;

METTL16 forward primer: 5’-CTCTGACGTGTACTCTCCTAAGG -3’,

METTL16 reverse primer: 5’- TACCAGCCATTCAAGGTTGCT-3’.

RNA stability detection

To detect the stability of METTL16 mRNA, CRC cells were treated with α-amanitin (50 μM; Sigma, USA) for indicated hours. Then total RNA was extracted and reverse transcribed. The relative level of METTL16 RNA to 0 hour was measured by qPCR assay.

Protein degradation

CRC cells were treated with the protein synthesis inhibitor cycloheximide (CHX) for 0, 3, 6, 12 hours, then homogenized to collect total protein. The expression of PD-L1 was detected by western blot assay.

RNA immunoprecipitation (RIP)

The enrichment of METTL16 on PD-L1 RNA was measured by using EZ-Magna RIP Kit (Millipore, USA) as per manufacturer’s instruction. In brief, magnetic beads were pre-coated with anti-METTL16 antibody overnight at 4° C in rotation. Next day, the antibody-beads conjugates were hatched with the isolated total RNAs for 6 hours at 4° C. The precipitated RNAs were then eluted and purified. The level of PD-L1 was measured by qPCR.

MeRIP-qPCR assay

The RNA was collected from cells and were hatched with anti-m6A antibody (Thermo, USA) for 6 hours at 4° C. The Dynabeads (Invitrogen, USA) were blocked by 2% BSA (Thermo, USA) for 3 hours at 4° C, followed by incubation with aforementioned RNA-antibody complex at 4° C overnight. After elution with SDS buffer, the RNAs were collected and level of PD-L1 was quantified by with qPCR assay [26].

T cell activation and co-culture system

Human peripheral blood samples were obtained from donors and the peripheral blood mononuclear cells (PBMCs) were purified by Ficoll methods. For T cell activation, PBMCs were incubated with Dynabeads Human T-Activator CD3 and CD28 (Gibco, USA) in line with the manufacturer’s protocol for 3 days. For co-culture, T cells and CRC cells were placed in the upper and lower chambers of Transwell, respectively. The ratio of T cells and cancer cells was 10:1 and cultured for 3 days. To determine the activation of T cells, PMSCs were incubated with anti-CD4, anti-CD-8, and anti-PD-1 antibody and analyzed by a flow cytometer.

Statistics

All statistical analyses were performed using the SPSS and GraphPad Prism 7.0 Software. The comparison between experimental groups was conducted using two-sided Student’s t-test and one-way ANOVA followed by Dunnett’s multiple comparison. P < 0.05 was set as significant.

Expression of METTL16 and PD-L1 in clinical samples and cell lines of CRC

We first analyzed the expression and correlation of METTL16 and PD-L1 in CRC. As shown in Figure 1A, 1B, the RNA level of PDL-1 in collected CRC tumor tissues is notably elevated, whereas the level of METTL16 is significantly decreased, compared with the non-tumor tissues. The IHC analysis of protein expression also indicated elevated PD-L1 and suppressed METTL16 expression in CRC tumor tissues compared with the paired non-tumor tissues (Figure 1C). Results from qPCR indicated a negative correlation between expression of METTL16 and PD-L1 (R=0.7035, p=0.0081). Moreover, we observed significantly decreased level of METTL16 in CRC cell lines, including NCM460, RKO, HCT116, SW480, Colo320, DLD-1, HCE8693, SW620, and HT29, compared with the normal human colon epithelial cell line FHC (Figure 1E). These data indicated that METTL16 may be negatively correlated with PD-L1 expression in CRC.

Figure 1. Expression of METTL16 and PD-L1 in clinical samples and cell lines of CRC. CRC tumor and non-tumor tissues were collected from patient with CRC. The RNA levels of (A) PD-L1 and (B) METTL16 were measured by qPCR assay. (C) The protein expression of PD-L1 and METTL16 was measured by IHC experiment. (D) The Pearson correlation analysis of PD-L1 and METTL16 in tumor tissues (n=20). (E) The RNA level of METTl16 in CRC cell lines (NCM460, RKO, HCT116, SW480, Colo320, DLD-1, HCE8693, SW620, and HT29) and normal human colon epithelial cell line FHC was measured by qPCR. *p<0.05, **p<0.01, ***p<0.001.

METTL16 regulates the proliferation, migration and invasion of CRC cells

Moreover, among the included CRC cell lines, the SW480 and SW620 exhibited the lowest level of METTL16 and hence were selected for further experiments. We subjected transfection of ectopic expression vectors and siRNAs of METTL16 in these two cell lines and assess cell proliferation, migration, and invasion. The results from western blotting assay demonstrated the successful overexpression and depletion of METTL16 in SW480 and SW620 cells (Figure 2A, 2B). As shown in Figure 2C, 2D, overexpression of METTL16 led to significantly suppressed growth (Figure 2C) and colonies (Figure 2D) of SW480 and SW620 cells. Besides, results from Transwell assay indicated that overexpression of METTL16 notably suppressed the number of migrated and invaded cells (Figure 3A). The suppressed healing of wounds in Figure 3B also suggested the impeded cell migration. Results from western blotting showed that overexpression of METTL16 caused increased level of E-cadherin and decreased level of Vimentin in SW480 and SW620 cells (Figure 3C), indicating the enhanced EMT. These data indicated that METTL16 suppressed CRC cell proliferation, migration and invasion.

Figure 2. METTL16 regulates the proliferation of CRC cells. (A) SW480 and SW620 cells were transfected with METTL16 overexpression vectors, and protein level of METTL16 was measured by western blotting assay. (B) SW480 and SW620 cells were transfected with METTL16 siRNAs, then protein level of METTL16 was measured by western blotting assay. (C) Cell viability of SW480 and SW620 cells under overexpression of METTL16 was measured by CCK-8 assay. (D) Proliferation of SW480 and SW620 cells under overexpression of METTL16 was detected by using colony formation assay. **p<0.01.

Figure 3. METTL16 regulates the migration and invasion of CRC cells. SW480 and SW620 cells were transfected with METTL16 overexpression vectors. (A) Cell migration and invasion were detected by using Transwell experiment. (B) Cell migration was measured by wound healing assay. (C) Protein levels of E-cadherin and Vimentin were detected by western blotting assay.

METTL16 modulates immune invasion in a CRC cell/T cell co-culture system

To explore the effects of METTL16 on immune invasion of CRC cells, we adopted a co-culture system of CRC cells with unstimulated or activated T cells and evaluated the activation, viability and apoptosis of T cells. As shown in Figure 1A, the portions of PD-1+ cell in activated T cells were notably decreased under co-culture with METTL16-overexpressed CRC cells, compared with those co-cultured with control CRC cells. Moreover, co-culture with CRC cells notably suppressed the viability and induced apoptosis of T cells (Figure 4B, 4C), which were significantly enhanced by depletion of METTL16 in CRC cells. Besides, overexpression of METTL16 reversed the suppressive effects of CRC cells on T cell viability (Figure 4B, 4C). These data suggested that METTL16 overexpression in CRC cells could promote immune response of T cells.

Figure 4. METTL16 modulates immune invasion in a CRC cell/T cell co-culture system. CRC cells were co-cultured with unstimulated or activated T cells. (A) The portion of PD-1+/CD4+ T cells and PD-1+/CD8+ T cells was measured by flow cytometry. (B) Cell viability was assessed by CCK-8 assay. (C) Cell apoptosis was measured by flow cytometry. **p<0.01.

METTL16-mediated m⁶A modification regulates PD-L1 expression in CRC cells

METTL16 is a well-recognized regulator of RNA methylation. Hence, we wondered if METTL16 directly modulates the expression of PD-L1. We observed that overexpression of METTL16 caused significant decrease in PD-L1 mRNA level (Figure 5A), and depletion of METTL16 markedly increased PD-L1 mRNA level (Figure 5B). We validated the elevated m6A modification level in several reported target of METTL16, including the RAB11B-AS1, MAT2A, and EEF1A1 (Supplementary Figure 1). Consistent with these findings, the results from RIP experiment indicated that the PD-L1 mRNA was modified by m6A (Figure 5C). Simultaneously, the m⁶A level of PD-L1 was upregulated by METTL16 overexpression and downregulated by METTL16 knockdown (Figure 5D, 5E), suggesting that METTL16 regulated the methylation of PD-L1 in CRC cells. Moreover, m6A modification has been reported to modulate RNA stability. Here, we examined the PD-L1 RNA stability after using α-amanitin to block new RNA synthesis. The degradation of PD-L1 mRNA was notably faster in METTL16-overexpressed cells, compared with the control cells (Figure 5F), and depletion of METTL16 maintained the stability of PD-L1 mRNA (Figure 5G). Moreover, treatment with Cycloheximide (CHX), the protein synthesis inhibitor, led to degradation of PD-L1 expression, and overexpression of METTL16 enhanced the PD-L1 degradation (Figure 5H). These data indicated that METTL16 could induce the m⁶A modification of PD-L1 mRNA and decreased its stability.

Figure 5. METTL16-mediated m⁶A modification regulates PD-L1 expression in CRC cells. (A, B) SW480 and SW620 cells were transfected with METTL16 overexpression vectors or siRNAs, then RNA level of PD-L1 was measured by qPCR assay. (C) The interaction between METTL16 with PD-L1 mRNA was measured by RIP assay. (D, E) SW480 and SW620 cells were transfected with METTL16 overexpression vectors or siRNAs, and MeRIP assay was conducted to assess m⁶A enrichment. (F, G) SW480 and SW620 cells were transfected with METTL16 overexpression vectors or siRNAs and treated with RNA synthesis inhibitor α-amanitin (50 μM). The stability of PD-L1 mRNA was checked by qPCR. (H) SW480 cells were treated with CHX for indicated time points and the expression of PD-L1 was measured by western blot. **p<0.01.

METTL16 modulates CRC cell growth in vivo

We next investigated the in vivo effects of METTL16 on CRC development. A xenograft tumor model was established using SW480 cells and treated with anti-PD-1 antibody. We observed that overexpression of METTL16 suppressed the tumor size and tumor growth curve and enhanced the tumor suppressive effect of anti-PD-1 antibody (Figure 6A, 6B). Analysis on tumor tissues demonstrated that overexpression of METTL16 and blocking of PD-1 could notably suppress the expression of proliferative biomarker Ki-67 (Figure 6C). Moreover, the levels of CD4 and CD8 positive cells in tumor tissues were notably elevated by overexpression of METTL16 and anti-PD-1 treatment compared with the control group and were further increased by their combination (Figure 6D).

Figure 6. METTL16 modulates CRC cell growth in vivo. The xenograft mouse model was established using control and METTL16 overexpressed SW480 cells and treated with anti-PD-1 antibody. (A) Images of tumors were taken. (B) Tumor growth curve was recorded. (C) The expression of Ki-67 was measured by IHC assay. (D) The expression of CD8 and CD4 in tumor tissues was measured by IHC assay. **p<0.01.