NOP2-Mediated m5C Methylation Modification of LMNB2 mRNA Facilitates Colorectal Cancer Progression

Abstract

Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality globally, yet current therapies exhibit suboptimal efficacy with limited prognostic improvement. RNA 5-methylcytosine (m5C), a posttranscriptional modification, has been implicated in tumorigenesis and progression across malignancies. In our previous study, the m5C methyltransferase NOP2 has been shown to promote proliferation, migration, and invasion of CRC cells, however, the underlying mechanism is still elusive.

Methods: An integrated multi-omics strategy was employed, combining transcriptomic sequencing, RNA immunoprecipitation sequencing (RIP-seq), and methylated RNA immunoprecipitation sequencing (MeRIP-seq) to explore NOP2-regulated downstream genes mediating CRC progression via m5C methylation. Functional validation included in vitro and in vivo assays to assess tumor growth and metastasis. Rescue experiments were performed by overexpressing LMNB2 in NOP2-silenced CRC cells.

Results: NOP2-dependent m5C modification of LMNB2 mRNA enhanced its stability, leading to elevated LMNB2 protein levels. This mechanism drove CRC tumor growth and metastasis both in vitro and in vivo. Overexpression of LMNB2 effectively rescued the suppressed malignant phenotypes induced by NOP2 knockdown, confirming LMNB2 as a critical downstream effector.

Conclusion: NOP2 catalyzes the m5C modification of LMNB2 mRNA to facilitate its stability, which contributes to the elevated LMNB2 protein level and CRC progression, suggesting the potential of NOP2 as a therapeutic target in the development of novel CRC treatment.

Keywords: LMNB2; NOP2; colorectal cancer; m5C; methyltransferase.

FIGURE 1

NOP2 is highly expressed in CRC tissues and cell lines. (A) NOP2 expression in different types of cancers and the adjuvant normal tissues. (From GEPIA2 database). (B) Relative expression of NOP2 in COAD and READ tumor tissues compared with adjacent normal tissues was analyzed in the GEPIA2 database. (C) The survival curve of CRC patients with high or low NOP2 expression levels was predicted in the OncLnc database. (D) The relative mRNA expressions and protein levels of NOP2 in four different CRC cell lines and one normal colon cell line (NCM460). (E) Relative expression of NOP2 in CRC tumor tissues and adjuvant normal tissues. Data from the HPA database. (F, G) NOP2 expression in different stages of COAD or READ (from GEPIA2). All data were exhibited with mean ± SD (n = 3). *p < 0.05; **p < 0.01.

FIGURE 2

Silencing of NOP2 inhibited the proliferation, invasion, and migration of CRC cells. (A, B) The migration ability of siNC or siNOP2‐transfected HCT116 and LoVo cells was detected by wound healing assay. Scale bar = 400 μm. (C, D) The invasion ability of HCT116 and LoVo cells was detected by transwell assay with Matrigel in transwell chambers. Scale bar = 50 μm. (E, F) The proliferation of HCT116 or LoVo cells was detected through cell number counting at different time points after transfecting siNC or siNOP2. All data were exhibited with mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Silencing of NOP2 induced the downregulation of LMNB2. (A) HCT116 cells were transfected with siNC or siNOP2, then multi‐omics sequencing was performed. The differentially expressed genes were intersected, and 23 significantly downregulated genes regulated by siNOP2 were selected for further analysis. (B) The 23 selected genes were analyzed in the GEPIA2 database, and 4 genes highly expressed in COAD and READ and positively correlated with NOP2 were selected. (C) The relative expressions of SLC7A5, HMGA1, CCND1, and LMNB2 in COAD and READ tumor tissues compared with adjuvant normal tissues were from the GEPIA2 database. (D) The co‐expression analysis between NOP2 and SLC7A5, HMGA1, CCND1, or LMNB2 in COAD and READ was performed in the GEPIA2 database. (E) After silencing NOP2, the expression of SLC7A5, HMGA1, CCND1, and LMNB2 was measured through qRT‐PCR. (F) NOP2 and LMNB2 protein levels in NOP2‐silenced HCT116 and LoVo cells. All data were exhibited with mean ± SD (n = 3). *p < 0.05; ***p < 0.001.

FIGURE 4

NOP2 promotes the m5C methylation of LMNB2 mRNA. (A) The mRNA expression of LMNB2 in 26 pairs of CRC tumor tissues and adjuvant normal tissues was detected by qRT‐PCR. (B) Co‐expression analysis between NOP2 and LMNB2 in 26 CRC tumor tissues. (C) The relative NOP2 expression in NOP2‐silenced HCT116 and LoVo cells. (D) LMNB2 expression in NOP2‐silenced HCT116 or LoVo cells. (E) Dual‐luciferase assay was used to test the relative luciferase activity of a luciferase reporter with WT (wild‐type) or MUT (mutant) LMNB2 CDS sequence containing the m5C site in NOP2 siRNA‐ or siNC‐transfected HCT116 cells. (F) The LMNB2 mRNA methylation levels in NOP2‐silenced cells were detected by MeRIP‐qPCR. (G) The LMNB2 mRNA stability in HCT116 and LoVo cells with NOP2 knockdown. All data were exhibited with mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

LMNB2 promotes the proliferation, invasion, and migration abilities of HCT116 and LoVo cells. HCT116 and LoVo cells were transfected with siNOP2 or LMNB2 overexpression vectors. (A, B) EdU staining was performed to measure cells' proliferation. Blue immunofluorescence indicated nuclear and red indicated EdU‐positive cells. The EdU‐positive cells in per field were also quantified. Scale bar = 50 μm. (C, D) The invasion ability of cells was measured by transwell assay, and the number of invasive cells in per field was quantified. Scale bar = 50 μm. (E, F) The migration ability of cells was measured by the wound healing assay and the migration rate was quantified. Scale bar = 400 μm. (G, H) Apoptosis level of HCT116 and LoVo cells was measured by flow cytometry and cells apoptosis rate was quantified. All data were exhibited with mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

Silencing of NOP2 inhibited the growth and metastasis of CRC tumors and downregulated the expression of LMNB2 in HCT116‐derived xenografts. (A) The image of dissected tumor tissues. (B) Tumor growth curve during 21 days of treatment. (C) Weight of the dissected tumor tissues. (D, E) IHC images of tumor tissues stained with NOP2, LMNB2, or Ki67, and the relative expression of these genes was quantified by calculating the positive area. (F, G) The images of mice that were injected with HCT116 cells through tail vein injection. The signal intensity of each mouse was quantified. Scale bar = 100 μm. All data were exhibited as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.