METTL1-WDR4 promotes the migration and proliferation of gastric cancer through N7-methylguanosine

Abstract

Background: Gastric cancer (GC) is one of the most common malignant tumor worldwide. Metastasis is leading cases of cancer-related death of GC. It has been found that N⁷-methylguanosine (m7G) modifications play an important role in cancer. However, the role of m7G modifications within mRNA and its "writer" METTL1 and WDR4 in tumors, particularly GC, has not been revealed.

Methods: RT-qPCR, WB and IHC were used to detect the expression of METTL1 and WDR4 in GC cells and tissues. Function-based experiments were performed using METTL1-WDR4 knockdown and overexpression cell lines in vitro and in vivo, including CCK8, colony formation, transwell and nude mice models. Mechanistically, RNA-seq, MeRIP-seq, MeRIP-qPCR, western blot, dot blot, co-IP, ChIP and IHC stainings were performed.

Results: METTL1 and WDR4 were upregulated in GC patients. High expression of METTL1 and WDR4 were associated with poor prognosis. Silencing METTL1-WDR4 inhibited GC cell migration and proliferation in vitro and vivo. Mechanistically, METTL1-WDR4 can enhance the mRNA stability of PIK3C2B and AKT by promoting their m7G levels, which leading the overexpression of p-AKT. Interestingly, we also found that on the one hand, the transcription factor YY1 can promote the mRNA transcription expression of METTL1 and WDR4 at the same time, and on the other hand, METTL1-WDR4 can promote YY1 expression by increasing the level of m7G. This regulation presents positive feedback. Above all, METTL1 and WDR4 ultimately up-regulate the level of m7G and promote the malignant progression of GC.

Conclusions: These findings suggest that METTL1-WDR4 might serve as a potential diagnostic and prognostic biomarker and a therapeutic target for GC treatment by regulating m7G level.

Keywords: Gastric cancer; METTL1; N7-methylguanosine; WDR4.

Fig. 1

METTL1 and WDR4 were upregulated in GC. A. Western Blot was utilized to analyze the METTL1 and WDR4 expression in 40 pairs of GC tissues and corresponding adjacent non-tumoral tissues. B. Quantitative analysis of 40 pairs of METTL1 and WDR4 protein in gastric cancer and paracanceroma. C. RT-qPCR was utilized to analyze the METTL1 and WDR4 expression in 40 pairs of GC tissues and corresponding adjacent non-tumoral tissues. D. The expression of METTL1 and WDR4 were applied by IHC staining assay in GC and corresponding adjacent non-tumoral tissues. The results showed that METTL1 and WDR4 expressed higher in tumor tissues. E. RT-qPCR was applied to analyze the METTL1 and WDR4 expression in 40 pairs of GC patients and general people plasm. F. The online database Starbase (http://starbase.sysu.edu.cn/) is used to predict METTL1 and WDR4 expression in GC and normal samples. G. RT-qPCR was applied to analyze the METTL1 and WDR4 expression in 40 pairs of GC pre-surgery and post-surgery patients plasm. H. ROC analysis of the prognostic sensitivity and specificity for GC patients and general people detected by the expression of METTL1, WDR4 and METTL1-WDR4 in plasm. I. Kaplan-Meier overall survival curves according to METTL1 and WDR4 expression in our cohort. *P < 0.05, **P < 0.01 and ***P < 0.001. N: Normal, T: Tumour

Fig. 2

METTL1 and WDR4 regulated in GC cells and enhanced the proliferation and invasion of GC cells. A and B. RT-qPCR and Western Blot indicated the METTL1 and WDR4 expressed higher in GC cell lines (AGS, BGC-823, SGC-7901 and HGC-27) than in the normal gastric epithelium cell line (GES-1). C and D. The transwell assay was performed for invasion and migration of GC cells. E. The CCK-8 assay was performed for proliferation of GC cells. F. The colony formation assay was performed for proliferation of CRC cells. *P < 0.05, **P < 0.01 and ***P < 0.001. M1: METTL1, W4: WDR4

Fig. 3

METTL1-WDR4 accelerates GC progression through regulating PIK3C2B and AKT m7G level. A and B. Dot Blot assay showed that METTL1 and WDR4 can upregulate m7G level of GC cells. C. MeRIP-seq results showed genes with differential m7G levels after downregulation of METTL1 and WDR4. D. MeRIP-seq results showed that METTL1 and WDR4 had connection with focal adhesion. E. RNA-seq results showed that after knocking down METTL1 and WDR4, the expression levels of key adhesion pathway proteins PIK3C2B and AKT were significantly down-regulated. F. co–IP showed that METTL1 and WDR4 are able to bind to each other. G. RT-qPCR results showed that METTL1 and WDR4 had positive regulation on PIK3C2B and AKT. H. Western Blot results showed that METTL1 and WDR4 had positive regulation on PIK3C2B, AKT and p-AKT. I. Western Blot results showed that PIK3C2B had positive regulation on p-AKT but not AKT. J. Western blot analysis showed that down-regulated p-AKT expression due to METTL1-WDR4 knockdown can be rescued by PIK3C2B and AKT. K. MeRIP-qPCR assay showed that the m7G level of PIK3C2B and AKT can be positively regulated by METTL1-WDR4. L. Transcript half-life of PIK3C2B and AKT in GC cells treated with the transcription inhibitor actinomycin D. *P < 0.05, **P < 0.01 and ***P < 0.001. M1: METTL1, W4: WDR4, M1W4: METTL1-WDR4

Fig. 4

METTL1-WDR4 accelerates GC cells migration, invasion and proliferation through regulating PIK3C2B and AKT. A and B. The transwell assay demonstrated that cell migration and invasion abilities of GC cells transfected with sh-METTL1-WDR4 were counteracted when co-transfected with PIK3C2B or AKT. C. The colony formation assay demonstrated that cell migration and invasion abilities of GC cells transfected with sh-METTL1-WDR4 were counteracted when co-transfected with PIK3C2B or AKT. D. The CCK-8 assay demonstrated that cell migration and invasion abilities of GC cells transfected with sh-METTL1-WDR4 were counteracted when co-transfected with PIK3C2B or AKT. *P < 0.05, **P < 0.01 and ***P < 0.001. M1W4: METTL1-WDR4

Fig. 5

YY1 may function as the upstream of METTL1 and WDR4. A. Venny showed that YY1 may regulate METTL1 and WDR4 transcription. B. GEPIA database exhibited that YY1 is upregulated in GC. C. GEPIA database showed that YY1 has a positive correlation with METTL1 and WDR4 in GC. D. RT-qPCR results showed that the level of METTL1 and WDR4 mRNA were decreased when YY1 was knockdown in GC cells. E. P1-P10 showed the regions of METTL1 and WDR4 promoter detected by the paired primers. ChIP assay revealed that YY1 can partly bind to METTL1 and WDR4 promoter. F. Diagram of predicted transcription factor YY1 binding sites to the METTL1 promoter region. The activities of serially truncated METTL1 promoter reporter vectors in the 293 T cells cotransfected with pGMLV-CMV-YY1. G. Diagram of predicted transcription factor YY1 binding sites to the WDR4 promoter region. The activities of serially truncated WDR4 promoter reporter vectors in the 293 T cells cotransfected with pGMLV-CMV-YY1. H. MeRIP-seq results showed that the m7G level of YY1 decreased after knocking down METTL1-WDR4. I. RNA-seq results showed that after knocking down METTL1-WDR4, the expression levels of YY1 were significantly down-regulated. J. MeRIP-qPCR assay showed that the m7G level of YY1 can be positively regulated by METTL1-WDR4. K. RT-qPCR results showed that METTL1-WDR4 had positive regulation on YY1. L. Western Blot results showed that METTL1-WDR4 had positive regulation on YY1. M. Transcript half-life of YY1 in GC cells treated with the transcription inhibitor actinomycin D. *P < 0.05, **P < 0.01 and ***P < 0.001. TFBS: transcription factor binding sites

Fig. 6

METTL1-WDR4 enhances tumour growth and metastasis in vivo. A–C. AGS cells stably transfected with sh-METTL1-WDR4 or vector were injected subcutaneously into the BALB/c nude mice. Tumour volume and weight were dramatically decreased in sh-METTL1-WDR4 group. D. Western Blot showed that p-AKT and YY1 expressed lower in experimental group. E. IHC assay showed that p-AKT, YY1 and Ki67 expressed lower in experimental group. F. After AGS cells with knockdown METTL1-WDR4 injected into the tail vein of nude mice, in vivo fluorescence imaging, the gross lesion in lung tissues, H. E staining metastatic nodules in the lungs were observed. G. The hypothetical model depicts the roles of METTL1-WDR4 in the promotion of GC. *P < 0.05, **P < 0.01 and ***P < 0.001. N: NC group, S: sh group, M1W4: METTL1-WDR4