Figure 4.

miR34a directly regulates GOLPH3 and is responsible for CSC subpopulation enrichment and GC chemoresistance. (A) Small scale miRNA screening using qRT-PCR showed the altered folds of 52 UBC-related miRNAs in T24 GC 3^rd xenograft tissue, compared with xenograft tissue from control group. (B) Sequences of wild type (GOLPH3-3'UTR-WT) and mutated 3'UTR Renilla luciferase reporters (GOLPH3-3'UTR-Mut) were listed. Expression of GOLPH3 was determined by real-time Q-PCR (C) and Western blotting (D) in scramble control and miR-34a overexpressed (miR-34a mimic) in T24-GC 3^rd cell (n=3 per group). (E) Relative luciferase activity was measured in scrambled control miRNA and mimic miR-34a transfected T24-GC 3^rd cells, simultaneously transfected also with GOLPH3-3'UTR-WT or GOLPH3-3'UTR-Mut expression vectors. (F) T24 GC 3^rd cell and 5637 GC 3^rd cell with miR34a elevated expression and control T24 and 5637 cells with miR34a knockdown were treated with various concentrations of cisplatin (0-19.2 µg/mL) and 1.5 µg/mL gemcitabine. (G) Sphere formation assay of T24 GC 3^rd cell and 5637 GC 3^rd cell with miR34a elevated expression and control cells T24 and 5637 with miR34a knockdown. (H) CD44⁺ subpopulation flow cytometry assay of T24 GC 3^rd cell and 5637 GC 3^rd cell with miR34a elevated expression and control cells T24 and 5637 with miR34a knockdown. (I) Western blot and (J) real-time Q-PCR of CSC markers (CD44, ALDH1A1, Sox2, and KLF4) and DNA damage and repair related protein GOLPH3 in T24 GC 3^rd cell and 5637 GC 3^rd cell with miR34a elevated expression and control cells T24 and 5637 with miR34a knockdown. Unpaired 2-tailed Student's t test was used for analysis of statistical significance. All cells were isolated from xenografts. The experiments were done in triplicate. CSC: cancer stem cell; GOLPH3: Golgi phosphoprotein 3; PCR: Polymerase Chain Reaction