IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway

Figures & data

Figure 1. METTL3 was down-regulated in CAFs. CAFs were separated from NSCLC tissues, and NFs were separated from para-carcinoma tissues. a, the morphology of CAFs and NFs was identified by IF. Scale bar = 100 μm. b, the level of METTL3, METTL16, METTL14, WTAP, FTO and ALKBH5 were analysed by TCGA database. c, m⁶A writers’ levels, including METTL14, METTL3, WTAP, METTL16, FTO and ALKBH5 were detected by qRT-PCR. d, m⁶A writers’ levels, including METTL14, METTL3, WTAP, FTO, ALKBH5 and METTL16 were detected by Western blot. e, histopathological feature of NSCLC tissues and para-carcinoma tissues was evaluated by H&E staining. Scale bar = 50 μm. f, METTL3 level in NSCLC tissues and para-carcinoma tissues was analysed by IHC. Scale bar = 50 μm. g, α-SMA and METTL3 level in CAFs and NFs. h, IL-18 expression in CAFs and tumour cells of NSCLC was detected. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Table 1. Primer sequences.

Primer name	Primer sequences
F- METTL3	5′-GAGTGCATGAAAGCCAGTGA-3′
R- METTL3	5′-CTGGAATCACCTCCGACACT-3′
F- METTL14	5′-AGGGGTTGGACCTTGGAAGA-3′
R- METTL14	5′-GAAGTCCCCGTCTGTGCTAC-3′
F- METTL16	5′-GGCAGAAGGAGGTGAATTAGAG-3′
R- METTL16	5′-TTCCCAGCATGCAGCTATAC-3′
F-WTAP	5′-TGGCAGAGGAGGTAGTGGTT-3′
R-WTAP	5′-GGGAACCCACAGTTCGATTA-3′
F-FTO	5′-ACCTCCAGCATTAGATTC-3′
R-FTO	5′-GAAACTACCGCATTTACC-3′
F-ALKBH5	5′-TGAGCACAGTCACGCTTCCC-3′
R-ALKBH5	5′-TCCGTGTCCTTCTTTAGCGACTC-3′
F-YTHDF2	5′-AGCCCCACTTCCTACCAGATG-3′
R-YTHDF2	5′-TGAGAACTGTTATTTCCCCATGC-3′
F-IL-18	5′-TGGCTGCTGAACCAGTAGAA-3′
R-IL-18	5′-ATAGAGGCCGATTTCCTTGG-3′
F-GAPDH	5′-CCAGGTGGTCTCCTCTGA-3′
R- GAPDH	5′-GCTGTAGCCAAATCGTTGT-3′

Figure 2. CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. CAFs were transfected with sh-METTL3 or oe-METTL3, CAFs that transfected with sh-NC or oe-NC served as the negative control. a-b, the levels of METTL3 and IL-18 in CAFs were detected by qRT-PCR and Western blot. c, the levels of IL-18 in CM from CAFs were detected by ELISA. d-e, PD-L1 levels in A549 and H1650 cells were assessed by Western blot and FCM. f, the levels of granzyme B and perforin in CD8⁺ T cells were detected by ELISA. g, the cytotoxicity of CD8⁺ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. IL-18 was a directly target of METTL3. a, IL-18 mRNA level was detected by qRT-PCR. b, correlation analysis of METTL3 and IL-18 in NSCLC tissues. c, RNA m⁶A dot blot assay was carried to detect the level of RNA m⁶A. d, the m⁶A motif sites of METTL3 coding region were predicted by RMBase v2.0 website. e, MeRIP-qPCR was carried to assess the level of IL-18 m⁶A. f, the direct binding relationship between METTL3 and IL-18 was confirmed by dual-luciferase reporter assay. g, IL-18 mRNA level was detected by qRT-PCR following METTL3 overexpression and actinomycin D treatment. h, the combination of YTHDF2 and IL-18 mRNA was detected by RIP assay. i, the combination of YTHDF2 and IL-18 mRNA was confirmed by RNA pulldown. CAFs were transfected with sh-YTHDF2, CAFs that transfected with sh-NC were served as the negative control. j, YTHDF2 level was detected by RT-qPCR. k, the level of YTHDF2 and IL-18 was measured by Western blot. l, the level of IL-18 was detected by ELISA. m, YTHDF2 knockdown-CAFs were treated with actinomycin D, and the degradation of IL-18 mRNA was evaluated by qRT-PCR. METTL3 overexpressed-CAFs were furtherly transfected with sh-YTHDF2. n, IL-18 was measured by Western blot. o, the level of IL-18 was detected by ELISA. All data were shown as mean ± SD. n = 3 per group. *P < 0.01, **P < 0.01, ***P < 0.001.

Figure 4. IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC. a-b, IL-18 level was detected by qRT-PCR and Western blot. c-d, PD-L1 level was assessed by Western blot and FCM. e, the levels of granzyme B and perforin were detected by ELISA. F, the cytotoxicity of CD8⁺ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. A-B, IL-18 level was detected by qRT-PCR and Western blot. C, NF-κB pathway related proteins’ expression was measured by Western blot. D-E, PD-L1 level was assessed by Western blot and FCM. F, the levels of granzyme B and perforin were detected by ELISA. G, the cytotoxicity of CD8⁺ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. The down-regulation of CAF derived METTL3 accelerated immunosuppression of NSCL. a-b, Statistical analysis of tumour volume and weight of NSCLC mice. c, the levels of METTL3 and IL-18 were detected by qRT-PCR. d, the levels of METTL3, IL-18, p65 and p-p65 were detected by Western blot. e, the distribution of CD8 was detected by IHC, Scale bar = 50 μm. f, the PD-L1 expression status in tumours by western blot. All data were shown as mean ± SD. n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Data availability statement

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.