Supplementary Figure 1. miR-216a negatively regulates non-small cell lung cancer cell growth. (A) Expression of miR-216a was increased or decreased by transfection of pre-miR-216a or antisense of miR-216a (ASO miR-216a) in NSCLC cell lines, respectively. Indicated cells were transfected with indicated nucleotides. After 72 hrs of transfection, total RNA was isolated from cells and subjected to qRT-PCR analysis. (B) Overexpression of miR-216a suppressed, whereas inhibition of miR-216a stimulated A549 and H23 cell growth. Indicated cells were transfected with indicated oligonucleotides and then subjected to MTT assay. (C) miR-216a negatively regulates NSCLC cell colony formation. Indicated cells were transfected with indicated nucleotides and subjected to colony formation assay. (D) Overexpression of miR-216a arrested, whereas inhibition of miR-216a accelerated A549 and H23 cell proliferation. Indicated cells were transfected with indicated oligonucleotides. After 6 hrs of transfection, cells plated in six-well plates and cell numbers were counted at the indicated time points. *, p<0.05; **, p<0.01; ***, p<0.001 compared to control. Ctrl. Oligo, control oligonucleotide.
Supplementary Figure 2. miR-216a induces apoptosis in vitro and in vivo. (A) miR-216a expression was significantly increased or decreased in stably expressing miR-216a or miR-216a-antisense cells compared to their vector control, respectively. (B) Overexpression of miR-216a induced apoptosis in HOP62 cells, whereas inhibition of miR-216a inhibited apoptosis in H522 cells. Cells were transfected with indicated oligonucleotides. 24 hrs after of incubation, cells were subjected to flow cytometry assay. All in vitro experiments data are presented as the mean ± s.d. from three independent experiments. (C) Cleaved PARP and caspase-3 were increased by overexpression of miR-216a in A549 xenograft samples, whereas decreased by inhibition of miR-216a in H23 xenograft samples. *, p<0.05; **, p<0.01; ***, p<0.001 compared to control. Ctrl. Oligo, control oligonucleotide.
Supplementary Figure 3. miR-216a inhibits EMT in non-small cell lung cancer cell. (A) Western blot and (B) immunofluorescence (IF) show that miR-216a regulates E-cadherin and vimentin expression in vitro. Stably expressing-miR-216a (A549) or miR-216a-antisense cells (H23) were subjected to Western blot and IF analysis.
Supplementary Figure 4. Anti-Argonaute 2 RIP-Chip assay shows that overexpression of miR-216a increased Ago2 complex and ZEB1/eIF4B mRNA interaction. A549 cells were transfected with pre-miR-216a and Ago2-flag plasmid. After 48 h of transfection, cells were harvested and subjected to RIP-Chip assay. ***, p<0.001 compared to control. Ctrl. Oligo, control oligonucleotides. nd: not detected.
Supplementary Figure 5. Correlation of ZEB1/eIF4B expression and miR-216a expression or non-small cell lung cancer (NSCLC) patients' survival. (A) mRNA expression of ZEB1 and eIF4B was measured in high- and low-miR-216a human NSCLC samples. (B) NSCLC patients in the high ZEB1 or eIF4B expression group had a significantly shorter overall survival rate compared to the low ZEB1 or eIF4B expression group, respectively. High expression: mRNA expression level was higher than 2 fold in tumor samples compared to matched normal lung tissue; Low expression: mRNA expression level was lower than 1 fold in tumor samples compared to matched normal lung tissue.
Supplementary Figure 6. Effects of miR-216a on putative target genes expression in A549 and HepG2 cells. (A) Indicated nucleotides were transfected into A549 and HepG2 cells. After 72 h of transfection, indicated proteins were detected by Western blot. (B) Correlation of eIF4B mRNA and miR-216a expression was measured in liver cancer patients (n=21).
Supplementary Figure 7. Expression of miR-216a, ZEB1 and eIF4B. A549 cells were tranfected with indicated plasmid. After 72 hrs of transfection, cells were subjected to qRT-PCR and Western blot analysis.