Fig 2: Suppression of Protein Levels of Target Genes by Bioengineered let-7c Agent in HCC Cell Lines(A) Immunoblot analyses of let-7c targeted ARID3B, c-MYC, LIN28B, and Bcl-xl protein levels and (B) immunofluorescent analysis of HMGA2 in Huh7 and Hep3B cells treated with 15 nM let-7c or control MSA. Immunoblot intensity was normalized to total protein and Lipofectamine 3000/vehicle control for comparison between groups. ap < 0.05 compared with vehicle; bp < 0.05 compared with MSA (one-way ANOVA with Bonferroni’s post hoc test). Immunofluorescent intensity of HMGA2 staining was normalized to total DAPI-positive cells. *p < 0.05 compared with MSA (Student’s t test).

Fig 3: GCMSCs-derived HGF promotes HK2 expression by regulating c-Myc. (A) ELISA analysis of the HGF expression in BMMSC-CM and GCMSC-CM. (B) Immunoblotting of p-MET, total MET in MGC-803 cells treated with GCMSC-CM or GCMSC-CM+SGX-523 (4 nM) for 30 min. (C) Immunoblotting of p-MET, total MET in HGC-27 cells treated with GCMSC-CM or GCMSC-CM+SGX-523 (4 nM) for 30 min. (D) Immunoblotting of HK2 and c-Myc in MGC-803 cells treated with GCMSC-CM, GCMSC-CM+SGX-523 (4 nM), or GCMSC-CM+JQ1 (0.8 μM) for 48 h. (E) Quantitative statistics of the c-Myc and HK2 expression in MGC-803 cells. (F) Immunoblotting of HK2 and c-Myc in HGC-27 cells treated with GCMSC-CM, GCMSC-CM+SGX-523 (4 nM), or GCMSC-CM+JQ1 (0.8 μM) for 48 h. (G) Quantitative statistics of the c-Myc and HK2 expression in HGC-27 cells (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001).

Fig 4: Proposed model for LKB1-mediated m6A modification in KRAS-mutated lung cancer progression.Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2, and MYC are crucial targets of m6A meditated by LKB1 deficiency, and are involved in KRAS mutation lung cancer progression.

Fig 5: Loss of LKB1 clinically associates with the increase of ALKBH5 and decrease of m6A modification on SOX2, SMAD7, and MYC genes.A Representative immunostaining images and quantification of 5mC DNA in lung cancer patients with KW and KM. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. B, C High levels of ALKBH5 DNA methylation and mRNA expression in cases of LKB1 loss compared with that of LKB1 positive expression in KM patients. D–F The correlation of ALKBH5 protein with the global m6A modification (D), m6A levels of SOX2, SMAD7, and MYC genes (E, F) in KM and KW groups. G–I Negative correlations of the m6A enrichment with mRNA expressions of SOX2, SMAD7, and MYC in KM group. J Representative immunostaining images and quantification of SOX2, SMAD7, and MYC in KM group with LKB1 loss or not. K The positive correlations of ALKBH5 with SOX2, SMAD7, and MYC protein expression in KM group. Data as mean ± SD. KRAS mutation (KM). KRAS wild-type (KW). *P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for A, B, and C. Student’s t-test for J.