Figure 5.

MiR-940/Cbl-b/signal transducer and activator of transcription 5A (STAT5A) are involved in 5-fluorouracil (5-FU)-induced upregulation of programmed death-ligand 1 (PD-L1) in gastric cancer cells. (A) Online database Promoter 2.0 Prediction Server and Animal TFDB 3.0 predicted transcriptional factors of PD-L1. (B–D) Correlation analysis of predicted transcriptional factors with PD-L1 using the gene expression profiles of TCGA-STAD database and GSE62254. (E) MKN74 and MGC803 cells were treated with 1.0 μg/ml 5-FU for 6 or 12 h; the phosphorylation of signal transducer and activator of transcription 4 (STAT4) and STAT5A were analyzed by Western blot. (F) After transient knockdown of STAT5A by using small interfering RNA (siRNA) for 72 h, STAT5A and PD-L1 levels were detected by Western blot analysis, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data were representative of three independent experiments. (G) After transient knockdown of Cbl-b by using siRNA for 72 h and treated MGC803 with 1.0 μg/ml 5-FU for 72 h, simultaneously, Cbl-b, p-STAT5, STAT5A, and PD-L1 levels were detected by Western blot analysis. (H) The expression of miR-940 in MGC803 treated with or without 1.0 μg/ml 5-FU for 72 h was analyzed. **P < 0.01. (I) MGC803 was cotransfected with 40 nmol miR-940 mimics and treated with 1.0 μg/ml 5-FU for 72 h. Western blot analysis the expression of Cbl-b, p-STAT5, STAT5A, and PD-L1 in MGC803.