N7-methyladenosine-induced SLC7A7 serves as a prognostic biomarker in pan-cancer and promotes CRC progression in colorectal cancer

Abstract

Solute transport family 7A member 7 (SLC7A7) mutations contribute to lysinuric protein intolerance (LPI), which is the mechanism of action that has been extensively studied. In colorectal cancer (CRC), SLC7A7 appears to play a role, but the features and mechanisms are not yet well understood. Survival was analyzed using the Kaplan-Meier analysis. Enrichment analysis was performed to characterize, immune infiltration, methylation, genetic instability, and crucial pathways of SLC7A7. Afterward, functional experiments were conducted in vitro to investigate how SLC7A7 affects tumor metastasis. Mechanistically, quantitative real-time PCR (qRT-PCR), western blot (WB), and methylated RNA immunoprecipitation (me-RIP) were carried out to confirm the methylation modification of SLC7A7 and related functions. High levels of expression of SLC7A7 are predictive of a worse prognosis for CRC patients. Enrichment analysis showed that SLC7A7 was significantly enriched during EMT and could be enriched in the Wnt/β-catenin signaling pathway, immune infiltration analysis of pan-cancer showed that SLC7A7 was significantly enriched in macrophages, and methylation analysis showed that SLC7A7 methylation modification affected the prognosis of specific cancers. SLC7A7 was indicated to promote the migration and invasion of CRC cells in in vitro functional experiments. Mechanistically, SLC7A7 was observed to potentially interact with the Wnt/β-catenin signaling pathway, possibly by influencing adenomatous polyposis coli (APC) expression. Furthermore, we identified that SLC7A7 undergoes N7-methylguanosine (m7G) modification, which may regulate SLC7A7 mRNA stability, with Quaking (QKI) potentially playing a role in this process by recognizing the m7G modification. Our results indicate that SLC7A7 may promote CRC metastasis through the SLC7A7/APC/Wnt/β-catenin signaling pathway. Moreover, m7G modification might be involved in regulating SLC7A7 mRNA stability, highlighting a novel layer of regulation.

Keywords: Colorectal cancer; Invasion; Migration; SLC7A7; m7g modification.

Fig. 1

Diagnostic and prognostic values of SLC7A7 expression in pan-cancer. (A) The mRNA expression levels of SLC7A7 among 31 different human cancer tissues. (B) Kaplan–Meier survival analysis indicated that the OS and DFS of patients with high expression of SLC7A7 were all shorter than those of patients with low expression. (C) SLC7A7 expression in colon cancer tissues was analyzed by GEO and TCGA databases (GSE21510, GSE71187, GSE87211, and TCGA). (D) SLC7A7 expression was analyzed by GSE106584 concerning OS and RFS. (E) Survival maps show the prognosis of different tumors. (F). Enrichment of GSEA results based on SLC7A7 expression of the KEGG and Hallmark pathway.

Fig. 2

Enrichment and immune infiltration analysis of SLC7A7 expression in CRC. (A) Distribution is summarized into three broad categories: biological processes (BP), molecular functions (MF), and cellular components (CC). (B) Several important GSEA results are presented of SLC7A7 expression in GSEA pathways. (C) Summary of nTPM for all single-cell types, SLC7A7mrna Expression in Cells. (D) Analysis of SLC7A7 immune infiltration in colon cancer. (E) Listing of cells in the TCGA dataset positively and negatively correlated with SLC7A7mrna expression.

Fig. 3

SLC7A7expression is associated with methylation in pan-cancer. (A) Enrichment analysis of SLC7A7 among eight immune cells in different tissues. (B) Single-cell sequencing results from colon cancer tissues were analyzed, and the vast majority of SLC7A7 was expressed in macrophages. (C) The expression of SLC7A7 methylation in different human cancer types. (D) The expression of SLC7A7 methylation in colorectal cancer. (E) Association of SLC7A7 methylation with prognosis in various cancers. (F) Methylation was negatively correlated with SLC7A7 expression in several tumors, including colon cancer.

Fig. 4

SLC7A7 genomic alterations and genomic instability. (A) SLC7A7 SNV were analyzed for pan-cancer. (B) Evaluated the COSMIC database for mutation types in SLC7A7. (C) In colon cancer, the SLC7A7 Somatic Mutation Rate: is 1.23%. (D) Correlations of CNV with mRNA expression. (E) CNV percentage in each cancer and Survival difference between CNV groups. (F) Variation in gene mutation, Gain, and Loss of high and low SLC7A7 expression in colon cancer.

Fig. 5

(A)SLC7A7 promotes CRC invasion and migration in vitro and in vivo. Transwell shows that after the knockdown of SLC7A7, both invasion and migration are reduced compared to NC. (B) The efficiency of SLC7A7 knockdown by qRT-PCR and WB in HCT116 and SW480 cell lines. (C) The statistic graph of invasion and migration in HCT116, SW480 cell lines transwell statistic graph. (D) Wound healing assay of HCT116 and SW480 cell lines at 0 h, 24 h, and 48 h, compared to the NC group, siSLC7A7 resulted in diminished cell migration. (E) The migration statistic graph of wound healing assay in HCT116 and SW480 cell lines. (F) Bioluminescent images of lungs for each experimental group. HE staining of lung sections displayed metastatic nodules of the lungs (magnification, × 100, scale bar,100 μm).

Fig. 6

SLC7A7 activation of Wnt/β-catenin signaling pathway promotes colon cancer progression through the EMT process and m7g modification promotes SLC7A7 mRNA stabilization. (A) Enrichment analysis results showed that SLC7A7 significantly enriched in EMT Apoptosis Hormone ER et al. (B) Activity of EMT pathway between high(n = 243) and low(n = 243) SLC7A7 expression groups in COAD. (C) The APC expression of si-SLC7A7 was analyzed by qRT-PCR and WB in HCT116 and SW480 cells. (D) The expression of E-cadherin, ZO-1 β-catenin in HCT116 and SW480 cells transfected with SLC7A7 siRNA was detected by Western blot. (E) The m7g-hub website predicts the SLC7A7 mRNA m7g modification site. (F) The m7g antibody specifically enriches SLC7A7 mRNA. (G) The efficiency of QKI siRNA was detected using qRT-PCR and WB in SW480 cells. (H) Analysis of SLC7A7 mRNA levels in down-regulated QKI SW480 cells incubated with actinomycin D for 0 h, 4 h, 8 h, 12 h, and 24 h by qRT-PCR. (I)The efficiency of OV METTL1 was detected using qRT-PCR, analysis of SLC7A7 mRNA levels in overexpression METTL1 SW480 cells incubated with actinomycin D for 0 h, 4 h, 12 h, and 20 h by qRT-PCR.analysis of SLC7A7 mRNA levels in OV-METTL1-mutant SW480 cells incubated with actinomycin D for 0 h, 4 h, 12 h, and 20 h by qRT-PCR.