Fig 2: FAS knockdown promotes lung cancer cell proliferation and cell cycle.a qRT-PCR measure FAS expression in FAS knockdown A549 and H292 lung cancer cells. The expression was normalized by GAPDH. Results are represented as means ± SD (n = 3). b Western blot analyses of FAS protein in FAS knockdown A549 and H292 lung cancer cells. The bands were quantified using Image J software, and relative values were obtained by normalizing to the value of each corresponding GAPDH. c Cell proliferation assay for A549 and H292 cells after transfection with siFAS for 4 days. Results are represented as means ± SD (n = 3). d The percentage of cell cycle G1 phase cells in FAS knockdown A549 lung cancer cells and control cells. Results are represented as means ± SD (n = 3). e Western blot analyses for cell cycle G1 phase-related proteins in FAS knockdown A549 lung cancer cells and control cells. The bands were quantified using Image J software, and relative values were obtained by normalizing to the value of each corresponding Vinculin. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: miR-196b-5p plays an oncogenic function in lung cancer cells and is negatively correlated with FAS in non-small cell lung cancer (NSCLC).a miR-196b-5p expression data were obtained from the TCGA miR-seq dataset. Of evaluable 666 NSCLC patients, 86 patients had matched normal adjacent tissues. b qRT-PCR examines miR-196b-5p expression in miR-196b-5p overexpressing H460 cells and control cells. Results are represented as means ± SD (n = 3). c Cell proliferation assay for miR-196b-5p overexpressing H460 cells and control cells. Results are represented as means ± SD (n = 3). d Colony-formation assay for miR-196b-5p overexpressing H460 cells and control cells. Colony-forming areas were measured by Image J software, and relative colony-forming areas were calculated by comparing with corresponding controls. Results are represented as means ± SD (n = 3). e, f FAS expression from TCGA RNA-seq data and miR-196b-5p expression from miR-seq data were used to examine the correlation between miR-196b-5p and FAS expressions in lung ADC dataset (n = 469) (e) and lung squamous cell carcinoma (SCC) dataset (n = 378) (f). ***P < 0.001, ****P < 0.0001.

Fig 4: FAS knockdown-mediated p65 activation promotes IL6 expression.a qRT-PCR measure IL6 expression in p65 overexpressing A549 cells and control cells. The expression was normalized by GAPDH. Results are represented as means ± SD (n = 3). b ELISA measures secreted IL6 protein levels in conditioned media from p65 overexpressing A549 cells and control cells. c Western blot analysis for p-STAT3 (Y705) expression in A549 cells treated with conditioned mediums from p65 overexpressing lung cancer cells or control cells. The p-STAT3 bands were quantified using Image J software, and relative values were obtained by normalizing to the value of the corresponding STAT3. d Cell proliferation assay of A549 cells treated with conditioned mediums from p65 overexpressing lung cancer cells or control cells. Results are represented as means ± SD (n = 3). e Western blot analyses for p-STAT3 (Y705), STAT3, p-Jak2, and Jak2 in miR-196b-5p overexpressing lung cancer cells and control cells. f Western blot analyses for p65 protein in miR-196b-5p overexpressing A549 cells and control cells. Both nuclear and cytosol proteins were extracted and subjected to western blot analysis. **P < 0.01, ****P < 0.0001.

Fig 5: c-Myb restrains the activity of T cells, and enhances the immune escape of FLO-1 cell cells by stimulating the expression of miR-145-5p. (A) The expression of c-Myb in each group detected using Western blot analysis. (B) Expression of c-Myb and miR-145-5p in each group measured using qPCR. (C) Proliferation of T cells cocultured with different groups of FLO-1 cells examined by CFSE dye and CD3 antibody. (D) Apoptosis of T cells cocultured with different groups of FLO-1 cells detected using flow cytometry combining with CD3 antibody. (E) The effect of invasion and migration-related protein expression in FLO-1 cells of each group detected by Western blot analysis. (F) Statistics of the number of invasive and migratory cells in each group. (G) The expression of Fas and FasL in FLO-1 cells of each group detected using Western blot analysis. (H) Apoptosis of FLO-1 cells in different groups assessed by flow cytometry. (I) The cytokine levels secreted by T cells in each treatment group detected by ELISA. *P < .05. Measurement data were expressed as mean ± standard deviation. Data in panel C were analyzed by two-way ANOVA, and in other panels were analyzed by one-way ANOVA and Tukey's post-hoc test