Additional file 1 of Bmi-1-induced miR-27a and miR-155 promote tumor metastasis and chemoresistance by targeting RKIP in gastric cancer

Additional file 1: Fig. S1 The association between clinical data and Bmi-1 and RKIP. A. qRT-PCR analysis of Bmi-1 and RKIP RNA expression in 15 paired GC tissues (T) and adjacent normal tissue samples (N). B. Western blotting analysis of Bmi-1 and RKIP in 15 paired GC tissues. The definitions of T and N were the same as mentioned in A. C. Kaplan-Meier analysis of the 3-year overall survival of patients with intestinal-type or diffuse-type GC from TCGA. D. Bmi-1, miR-27a and miR-155 were upregulated, while RKIP was downregulated significantly in GC tissues from the TCGA database. *P < 0.05, **P < 0.01. Fig. S2 Bmi-1 does not upregulate RKIP at the mRNA level nor induce RKIP protein degradation. A. Bmi-1 and RKIP mRNA expression in GES-1 cells overexpressing Bmi-1. *P < 0.05 vs. GES-1-Vector. B. GES-1-Bmi-1 cells and GES-1-Vector cells were subjected to the protein synthesis inhibitor cycloheximide for the indicated period of time. The half-life of RKIP protein in Bmi-1-transduced cells was comparable to that in the control cells, which indicated that Bmi-1 did not induce RKIP protein degradation. Fig. S3 Quantification of Western blotting assays as well as invasion and migration assays. A. The densitometry analysis of bands from the Western blotting assays in Fig. 2f. *P < 0.05 vs. NC mimic/NC inhibitor. B. The densitometry analysis of bands from the Western blotting assays in Fig. 3a. *P < 0.05 vs. Vector-Ctrl/siNC. C. Analysis of the quantities of invading cells in migration and invasion assays. *P < 0.05 vs. shcon, **P < 0.01 vs. shcon/Vector-Ctrl, ##P < 0.01 vs. NC mimic/NC inhibitor. Fig. S4 miR-27a inhibitor and miR-155 inhibitor weakened the effects of Bmi-1 overexpression in functional experiments. A. Bmi-1 upregulation induced gastric cancer cell migration and invasion, which were decreased by the miR-155 inhibitor or miR-27a inhibitor (100 × magnification). B. The reduced ability of cell proliferation due to the transient transfection of the miR-155 inhibitor or miR-27a inhibitor was improved by Bmi-1 overexpression. C. Colony formation assays either in soft agar or on plates showed that the Bmi-1 overexpression group generated more colonies than any other group, and the effect could be reversed by miR-155 inhibitor or miR-27a inhibitor. D. The IC50 values of cells treated with 5-Fu or oxaliplatin were detected by CCK8 reagent. The increase in Bmi-1 reduced chemosensitivity, while the miR-155 inhibitor and miR-27a inhibitor lowered the IC50. *P < 0.05 vs. Vector-Ctrl, #P < 0.05 vs. NC inhibitor. Fig. S5 Immunohistochemistry of tumors for the detection of Bmi-1, RKIP, Vimentin, Bax and Bcl-2. A. Image from immunohistochemistry of isolated tumors from animals. The animals were subcutaneously implanted with cells stably overexpressing miR-155 or miR-27a and then subjected to intraperitoneal injection of 5-Fu. B. Immunostained sections of different tumors from animals that were implanted with cells stably transfected with shRNA or cotransfected with shRNA and miRNA. Magnification: 200 × . Supplementary Table S1. Sequences of primers. Supplementary Table S2. All differentially expressed miRNAs.