METTL3-mediated RanGAP1 promotes colorectal cancer progression through the MAPK pathway by recruiting YTHDF1

Abstract

Ran GTPase activating protein 1 (RanGAP1) has been implicated in various diseases, but its role in colorectal cancer (CRC) progression remains unclear. Using tumor tissues and public databases, we found that RanGAP1 was significantly upregulated in CRC tissues and was associated with poor prognosis of patients. N6-methyladenosine (m6A) was found to play an important role in higher expression of RanGAP1. MeRIP-seq, RIP-qPCR, Luciferase reporter assays and other related experiment elucidated the molecular mechanism underlying m6A modification of RanGAP1. Besides, cell function experiments and xenograft tumor models corroborated the function of RanGAP1 in CRC progression. By RNA-seq and related analysis, RanGAP1 was verified to influent CRC progression via the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Therefore, N6-methyladenosine modification of RanGAP1 by METTL3/YTHDF1 plays a role in CRC progression through the MAPK pathway and could be a potential biomarker and therapeutic target for CRC. Schematic diagram showed that N6-methyladenosine modification of RanGAP1 promotes CRC progression via the MAPK signaling pathway.

Schematic diagram showed that N6-methyladenosine modification of RanGAP1 promotes CRC progression via the MAPK signaling pathway.

Fig. 1. RanGAP1 is highly expressed in CRC and considered as a diagnostic indicator.

A RanGAP1 mRNA expression levels were higher in multiple cancers than in normal tissues based on TCGA data. B, C The mRNA expression levels of RanGAP1 based on TCGA and GSE68468 data. D, E RanGAP1 mRNA expression levels of patients with primary and metastatic colon cancer based on GEO datasets. F–H The protein expression levels of RanGAP1 were compared between CRC tissues and matched para-cancer tissues (n = 10), and were subsequently analyzed in Tissue Microarray samples of CRC and normal tissue. I The survival analysis between RanGAP1 expression and Recurrence free survival (RFS) of CRC based on KMplot data.

Fig. 2. RanGAP1 promotes proliferation of colorectal cancer.

A The intervention effect of RanGAP1 knockdown and RanGAP1 overexpressed in CRC cells. B–E CCK8 assays and colony formation assays showed the proliferation ability of silencing and overexpressing RanGAP1 on CRC cells. F, G Flow cytometry assays showed the effects of silencing and overexpressing RanGAP1 on the cycle of CRC cells. H, I Injection of modified RanGAP1 knockdown DLD1 cells in nude mice. J Volume of tumors were recorded every four days. K Xenografts from nude mice were measured. L Weight of tumors in each group. M The proteins levels of RanGAP1 and ki67 were quantified.

Fig. 3. RanGAP1 promotes metastasis of CRC.

A–F The migration and invasion abilities were analyzed in RanGAP1 knockdown and overexpressed CRC cells. G Representative mice injected with RanGAP1 knockdown DLD1 cells and control cells were measured for CRC lung metastases. H, I Lung from nude mice were obtained and analyzed. J–L The HE-staining detected the expression of the metastatic nodules of lung in the different groups.

Fig. 4. METTL3 regulates RanGAP1 by recruiting YTHDF1 to promote the progression of CRC.

A RIP assays showed the binding between the METTL3 protein and RanGAP1 mRNA. B The protein levels of RanGAP1 in METTL3 knockdown CRC cells. C m6A motifs were identified by MeRIP-seq in METTL3 silenced HCT116 and control group. D SRAMP website showed the potential site of m6A modification of RanGAP1 mRNA. E METTL3 knockdown reduced m6A modification of RanGAP1 mRNA in HCT116 cells. F Luciferase reporter assays showed the m6A modification affect the expression of RanGAP1. G RNA stability of RanGAP1 mRNA in downregulating METTL3 CRC cells and control cells after giving actinomycin D (5 μg/mL). H RIP assays showed the binding between the YTHDF1 protein and RanGAP1 mRNA in DLD1 cells. I The protein levels of RanGAP1 in YTHDF1 silenced CRC cells. J The protein expression levels in CRC cells transfected stably with the shNC+Control, shMETTL3+Control, shMETTL3+RanGAP1, shYTHDF1+Control and shYTHDF1 + RanGAP1. K–N The downtrend of proliferation, migration and invasion by shMETTL3 or shYTHDF1 of HCT116 and DLD1 were sectionally recurred because of RanGAP1 overexpressing.

Fig. 5. RanGAP1 facilitates CRC tumorigenesis via the MAPK signaling pathway.

A The volcano plot of differential mRNA expression in RanGAP1 silenced DLD1 and control group ( | log2(fold change) |>1 and p value < 0.05). B KEGG enrichment analysis of different genes in RanGAP1 knockdown cells and control cells. C, D Expression levels of proteins associated with the MAPK signaling pathway were evaluated in CRC cells transfected stably with the shRanGAP1, shNC+Control, shMETTL3+Control, shMETTL3+RanGAP1, shYTHDF1+Control and shYTHDF1 + RanGAP1.

Fig. 6. CRABP2, acting as downstream of RanGAP1, is affected by RanGAP1 at protein levels.

A Western blotting shows change of CRABP2 protein expression levels in RanGAP1 knockdown and RanGAP1 overexpressed CRC cells. B RanGAP1 and CRABP2 proteins in xenografts from nude mice are analyzed by IHC (Fig. 2K). C–E IHC shows the relationship of RanGAP1 and CRABP2 protein expression in human colon cancer tissue microarray. F RanGAP1 and CRABP2 expression levels in CRC cells stably transfected with the shNC+Control, shRanGAP1+Control and shRanGAP1 + CRABP2 by western blotting. G–I The downtrend of proliferation, migration and invasion by shRanGAP1 of DLD1 were sectionally recurred because of CRABP2 overexpressing. J The protein expression levels associated with the MAPK signaling pathway were assessed in DLD1 cells stably transfected with shCRABP2.