METTL16 Promotes Lipid Metabolic Reprogramming and Colorectal Cancer Progression

Abstract

Background: Lipid reprogramming represents a pivotal stage in tumor progression. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotic cells, plays a significant role in colorectal cancer (CRC) development, though its specific involvement in lipid reprogramming remains unclear. Methods: Bioinformatics analysis of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases revealed differential expression of METTL16 (M16), which was further validated through qRT-PCR and Western blotting in CRC tissues and cell lines. The impact of M16 on CRC proliferation, metastasis, invasion, and lipid reprogramming was evaluated using both in vivo and in vitro approaches. Regulatory mechanisms underlying M16's role in CRC progression were explored using immunofluorescence (IF) staining, RNA immunoprecipitation (RIP), MERIP assay, RNA pull-down assay, total m6A measurement, RNA stability assay, protein stability analysis, and luciferase reporter assays. Results: Analysis results demonstrated a significant upregulation of the m6A methyltransferase METTL16 in CRC, closely associated with poor prognosis and abnormal lipid droplet accumulation. Functional assays revealed that M16 overexpression markedly promotes CRC cell proliferation, migration, and invasion both in vitro and in vivo, primarily by enhancing lipid reprogramming. Mechanistically, M16 induces m6A modification of TM7SF2 mRNA, stabilizing it via an IGF2BP1- and IGF2BP2-dependent pathway, thereby upregulating TM7SF2 expression and driving lipid reprogramming in CRC. Conclusion: In conclusion, these findings highlight the critical role of the M16/m6A/TM7SF2 axis in lipid metabolic reprogramming in CRC, offering potential therapeutic targets for its treatment.

Keywords: CRC; M16; m6A; metabolism.

Figure 1

Increased M16 expression in CRC is associated with abnormal lipid metabolism and poor prognosis. A. Pan-cancer analysis of M16 was performed using the TIMER 2.0 database. B-C. M16 expression levels in the TCGA-CRC and GEO-CRC (GSE9348) datasets. D-F. Correlation between M16 expression and TNM staging in patients from the TCGA-CRC dataset. G. qRT-PCR was used to determine the expression levels of M16 in CRC tumors and neighboring non-tumor tissues. H. qRT-PCR study of M16 expression in CRC tissues categorized by pathological grade (I/II/III or IV). I. Kaplan-Meier survival curves comparing overall survival (OS) in patients with CRC exhibiting high or low M16 expression. J. IHC examination of M16 expression in CRC samples and neighboring non-tumor tissues. K. Oil Red O staining to examine lipid droplet distribution in CRC tissues and surrounding non-tumor tissues. L. Western blotting study of M16 expression in CRC tumors and nearby non-tumor tissues. M-N. M16 expression assessed by qRT-PCR and Western blotting in FHC, SW480, HCT116, SW620, DLD1, and LOVO cells. O. IF staining to determine M16 subcellular localization in SW480 and HCT116 cells. Scale bar: 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

M16 promotes CRC progression in vitro and in vivo. A-D. qRT-PCR and Western blotting were used to confirm M16 knockdown and overexpression in CRC cells. E. CCK-8 assay was used to evaluate the proliferation capacity of CRC cells with M16 knockdown or overexpression. F-G. Colony formation ability of CRC cells with M16 knockdown or overexpression was assessed using plate colony formation assays. H-K. Migration and invasion capabilities of CRC cells with M16 knockdown or overexpression were analyzed by Transwell assays. L. Xenograft tumors from SW480-shNC, SW480-shM16-1, SW480-shM16-2, SW480-vector, and SW480-OE-M16. M. Growth curve of xenograft tumors was monitored and plotted over a specified period. N. Xenograft tumor weights were measured after euthanizing the mice. O. IHC analysis of M16 and Ki67 expression in xenograft tumors. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

M16 promotes lipid metabolic reprogramming in CRC both in vitro and in vivo. A-D. In comparison to control groups, the expression of neutral lipids in SW480 and HCT116 with M16 overexpression or knockdown was examined using lipid fluorescence staining. Scale bar: 25 μm. E. Cholesterol expression was measured in SW480 and HCT116 cells that had M16 knockdown or overexpression. F. Triglyceride expression was measured using M16 overexpression or knockdown in SW480 and HCT116. G. M16 knockdown or overexpression was used to measure the expression of FFAs in SW480 and HCT116. H. Cholesterol, triglyceride, and FFA levels were measured in xenograft tumors from shNC, shM16-1, shM16-2, vector, and OE-M16 groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

TM7SF2 induces lipid metabolic reprogramming in CRC cells. A. Heatmap displaying differentially expressed genes in SW480 cells following M16 knockdown. B. KEGG enrichment analysis identifying metabolic pathways associated with M16. C-D. qRT-PCR analysis identifying lipid metabolism-related genes with significant differential expression in SW480 and HCT116 cells following M16 knockdown. E. Western blotting confirming TM7SF2 protein expression in SW480 cells following M16 overexpression or knockdown. F. IHC labeling to examine the TM7SF2 protein expression in neighboring non-tumor tissues and CRC tissues. G-I. Correlation between TM7SF2 expression and TNM staging in TCGA-CRC patients. J-K. Lipid fluorescence staining assessing neutral lipid expression in CRC cells (SW480 and HCT116) with TM7SF2 knockdown compared to control groups. Scale bar: 25 μm. L-N. Assessment of cholesterol, triglycerides, and FFAs in CRC cells with TM7SF2 knockdown. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

M16 promotes TM7SF2 mRNA stability through m6A-dependent mechanisms. A. Total m6A levels were assessed to determine the impact of M16 knockdown in SW480 and HCT116 cells. B-C. RIP-qPCR experiments identified whether TM7SF2 mRNA binds with M16 in SW480 and HCT116 cells. D-E. RIP-qPCR was conducted to evaluate m6A enrichment on TM7SF2 mRNA after M16 knockdown or overexpression in SW480 cells. F-G. SRAMP website predicts possible m6A modification sites on TM7SF2 mRNA. H. Secondary structure prediction of TM7SF2 mRNA containing m6A sites. I. Primers were designed for specific TM7SF2 mRNA fragments. J-K. TM7SF2 mRNA was treated with metal ions to fragment it into 100-200 bp lengths, followed by M16-RIP-PCR to identify specific binding sites of M16 on TM7SF2 mRNA in SW480 cells. L. Creation of luciferase reporter plasmids encoding firefly and renilla luciferases. The 3' UTR of firefly luciferase was inserted with either wild-type (WT) or mutant (Mut) sequences. M. Transfection of constructed luciferase reporter plasmids into SW480 cells and assessment of firefly and renilla luciferase activities. N-O. Treatment of SW480 and HCT116 cells with actinomycin D (10 µg/ml) post M16 knockdown, and assessment of TM7SF2 mRNA stability at specific times (0h, 1h, 2h, 3h, 4h) via qRT-PCR. P. Treatment of SW480 cells post M16 knockdown with CHX (100 µg/ml), and monitoring of TM7SF2 protein stability at specific times (0h, 1h, 2h, 3h, 4h) via Western blotting. Q. Correlation between M16 mRNA and TM7SF2 mRNA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

TM7SF2 mRNA is recognized by IGF2BP1 and IGF2BP2. A. RNA pulldown with biotinylated TM7SF2 as the probe and beads alone as the control probe, followed by Western blotting, identified the readers interacting with TM7SF2 mRNA in SW480 cells. B-C. Western blotting and qRT-PCR confirmed the expression of IGF2BP1 and TM7SF2 mRNA and protein levels following IGF2BP1 knockdown. D-E. Western blotting and qRT-PCR verified the expression of IGF2BP2 and TM7SF2 mRNA and protein following IGF2BP2 knockdown. F-G. qRT-PCR and Western blotting were used to confirm the expression of IGF2BP3 and TM7SF2 mRNA and protein following IGF2BP3 knockdown. H-I. RIP-PCR showed that IGF2BP1 binds to TM7SF2 mRNA in SW480 and HCT116. J-K. RIP-PCR showed that IGF2BP2 binds to TM7SF2 mRNA in SW480 and HCT116. L. Treatment of CRC cells with actinomycin D (10 µg/ml) and measurement of TM7SF2 mRNA stability at specified times (0h, 1h, 2h, 3h, 4h) in shNC, shIGF2BP1, shIGF2BP2, and shIGF2BP1+shIGF2BP2 groups via qRT-PCR. M. Treatment of CRC cells with CHX (100 µg/ml) and measurement of TM7SF2 protein stability at specified times (0h, 1h, 2h, 3h, 4h) in shNC, shIGF2BP1, shIGF2BP2 groups via qRT-PCR. N. qRT-PCR experiments were conducted to assess the expression levels of TM7SF2 mRNA following the knockdown of M16, IGF2BP1, or IGF2BP2. O. Western blotting assays were conducted to measure TM7SF2 protein expression in the shIGF2BP1, shIGF2BP2, and shIGF2BP1+shIGF2BP2 groups. P. After knocking down IGF2BP1, Western blotting was performed to measure IGF2BP2 protein expression. Q. Following IGF2BP2 knockdown, Western blotting was utilized to assess IGF2BP1 protein expression. R. Correlation between TM7SF2 mRNA and IGF2BP1 mRNA or IGF2BP2 mRNA. S. Using IHC to analyze the correlation between TM7SF2 protein and IGF2BP1 protein or IGF2BP2 protein. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

M16/m6A/TM7SF2 axis promotes lipid metabolic reprogramming and CRC progression. A-B. Lipid fluorescence assays evaluated the effect of TM7SF2 overexpression on neutral lipid levels in SW480 cells after M16 knockdown, with quantitative analysis of the results. Scale bar: 25 µm. C-E. The impact of TM7SF2 overexpression on cholesterol, triglycerides, and FFA levels in SW480 cells following M16 knockdown was measured. F. CCK-8 assays were conducted to assess how TM7SF2 overexpression affected SW480 cell proliferation after M16 knockdown. G-H. The effect of TM7SF2 overexpression on the migratory and invasive potential of SW480 cells after M16 knockdown was evaluated using Transwell assays. I-J. Colony formation assays evaluated how TM7SF2 overexpression affected SW480 cells' colony-forming capacity after M16 knockdown. K. Xenograft tumors from SW480-shNC, SW480-shM16, SW480-shM16+vector, and SW480-shM16+OE-TM7SF2. L. Growth curves of xenograft tumors were monitored and plotted over a specific period. M. Xenograft tumor weights were measured after the mice were euthanized. N. IHC analysis of M16, TM7SF2, and Ki67 expression in xenograft tumors. O. Cholesterol, triglycerides, and FFA expression were measured in xenograft tumors from shNC, shM16, shM16+vector, and shM16+OE-TM7SF2 groups. P-Q. IHC analysis of the correlation between TM7SF2 protein and M16, IGF2BP1, or IGF2BP2 proteins. Scale bar: 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

A schematic illustration describes how M16 overexpression in CRC promotes m6A modification of TM7SF2 mRNA, leading to enhanced stability of TM7SF2 mRNA by IGF2BP1 and IGF2BP2. Increased TM7SF2 promotes lipid reprogramming and CRC progression.