N6-methyladenosine (m6A) RNA methylation may play an important role in the process of malignant transformation of cells induced by environmental carcinogens. However, the specific roles and mechanisms need to be further explored.

To explore the role and mechanism of m6A binding protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the malignant transformation of human gastric mucosal epithelial cells GES-1 induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Based on the GES-1 malignant transformation cells MC-30, a stable knockdown IGF2BP3 MC-30 cell line (MC30-shIGF2BP3, abbreviated as MC30-shI3) was constructed by lentiviral transfection technology, and a negative control group (MC30-NC) was also prepared. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect the mRNA expression and protein levels of IGF2BP3. RNA binding protein immunoprecipitation (RIP-qPCR) was used to examine the combination between IGF2BP3 protein and MYC mRNA in malignant cells MC-30. Furthermore, the stability of MYC mRNA was detected by actinomycin D assay. CCK-8 and Transwell respectively were employed to detect cell proliferation, migration, and invasion. Western blotting was applied to detect the expression of EMT markers (N-cadherin, Vimentin, α-SMA, and Snail). The role of the downstream target gene MYC was further elucidated by a rescue assay in MC30-shI3 cells transfected with a plasmid overexpressing MYC to observe changes in cellular phenotypes (proliferation, migration, invasion) and expression of key EMT proteins.

Compared with the control group, the expression of IGF2BP3 mRNA was up-regulated after 5, 10, 20, and 40 μmol·L⁻¹ MNNG infection of GES-1 cells (P<0.05). After 20 μmol·L⁻¹ MNNG infection, the expression level of IGF2BP3 mRNA increased with prolongation of exposure time (P<0.05). Compared with the control group, the mRNA and protein expression levels of IGF2BP3 were up-regulated in the 10th, 20th, and 30th generations of 5 μmol·L⁻¹ MNNG malignant transformation (P<0.05). The results of qRT-PCR and Western blotting showed that, compared with the MC30-NC group, the IGF2BP3 and MYC mRNA expression and protein expression decreased in the MC30-shI3 group (P<0.01). The CCK8 and transwell assay results showed that, compared with the MC30-NC group, the cell proliferation, migration, and invasion abilities significantly reduced in the MC30-shI3 group (P<0.01). The results of the Western blotting showed that, compared with the MC30-NC group, the protein levels of EMT markers N-cadherin, Vimentin, α-SMA, and Snail decreased in the MC30-shI3 group (P<0.01). The results of RIP-qPCR showed that, compared with the IgG group, the mRNA level was higher for the enriched MYC in the IGF2BP3 group (P<0.01); the results of the actinomycin D assay showed that, compared with the MC30-NC group, the stability of MYC mRNA significantly reduced in the MC30-shI3 group (P<0.01). While the rescue experiment showed that, compared with the IGF2BP3 knock-down+vector group, the MYC protein level significantly increased in the IGF2BP3 knock-down + MYC over-expression group (P<0.01), the proliferation, migration, and invasion abilities significantly enhanced (P<0.01), and the EMT key proteins (N-cadherin, Vimentin, α-SMA, Snail) increased in the MC30-shI3+MYC group (P<0.01).

Exposure to MNNG could result in up-regulation of IGF2BP3 expression in GES-1 cells. IGF2BP3 may enhance the proliferation, migration, and invasion of malignantly transformed human gastric epithelial cells by binding to MYC mRNA and increasing its stability and expression level and thus promoting the EMT process, which in turn affects the progression of malignant transformation.