MiRNA-145 Induces Apoptosis in a Gallbladder Carcinoma Cell Line by Targeting DFF45

Abstract

Bakcground: We measured expression of miRNA-145 in gallbladder carcinoma and its influence on propagation, invasion, and apoptosis of gallbladder carcinoma cells in vitro.

Methods: miRNA-145 expression was compared between normal gallbladder epithelial cells and GBS-SD (gallbladder series) cells using miRNA chip technology. Propagation, apoptosis, and invasion properties of each cell group were tested using MTT, a clone-formation assay, flow cytometry, Western blot, and Transwell assays.

Results: Expression of miRNA-145 was observed to be down-regulated and GBC-SD cell clones transiently transfected with hsa-miRNA-145 were substantially reduced compared with controls (p<0.01). We observed that GBC-SD cells transfected with hsa-miRNA-145 and double-positive (Annexin V and PI) for apoptosis were more numerous than controls. Moreover, GBC-SD cells over-expressing miRNA-145 had significantly greater expression of apoptosis-related protein, caspase-3. A Transwell assay confirmed that GBC-SD cells over-expressing miRNA-145 that migrated to the lower chamber were fewer compared with controls. Post-transcriptional regulation of gene expression was measured using dualluciferase reporter assays and data show that miRNA-145 facilitates the inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45.

Conclusion: Thus, we speculate that miRNA-145 facilitates inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45; however, miRNA-145 does not directly affect the GBC-SD cell cycle.

Keywords: DFF-45; MTT; clone formation assay; flow cytometry; miRNA-145.

Figure 1

miRNA-145 expression in adjacent gallbladder tissues (A) and gallbladder carcinoma tissues (B).

Figure 2

GBC-SD cells stably transfected with hsa-miRNA-145 have more hsa-miRNA-145 expression. A: hsa-miRNA-145 expression in normal non-transfected GBC-SD cells; B: GBC-SD cells transfected with empty plasmid C: cells over-expressing hsa-miRNA-145 (ns: non-significant differences; ^*: p<0.05, significant difference).

Figure 3

Effect of hsa-miRNA-145 on gallbladder carcinoma cell growth according to MTT assay. normal control : normal non-transfected GBC-SD cells; vect: GBC-SD cells transfected with empty plasmids; miR-145: GBC-SD cells transfected with hsa-miRNA-145 over-expression plasmids; p<0.05: significant difference.

Figure 4

Cell cycle distribution for all phases of GBC-SD cells according to flow cytometry. A:normal non-transfected GBC-SD cells, B: GBC-SD cells transfected with hsa-miRNA-145 over-expression plasmids.

Figure 5

Flow cytometry indicating apoptosis of GBC-SD cells (Annexin V-FITC). A:represents normal non-transfected GBC-SD cells; B: represents hsa-miRNA-145 transfection group.

Figure 6

Expression of caspase-3 protein in GBC-SD cells according to Western blot.

Figure 7

Hsa-miRNA-145 inhibits GBC-SD clone formation. A: cells over-expressing hsa-miRNA-145; B: GBC-SD cells transfected with empty plasmids; C: normal non-transfected GBC-SD cells.

Figure 8

Western blot quantification of DFF45 protein expression in controls, transfected empty plasmid group and transfected miRNA-145 cell lines. Normal control: normal GBC-SDE cells not transfected; Vect: GBC-SD cells transfected with empty plasmids; MiRNA-145 GBC-SD cells transfected with over-expressed mlRNA-145 plasmids.

Figure 9

hsa-miRNA-145 targets DFF45-854 to regulate transcriptional activity. A, bioinformatics predicted target region and sequence of hsa-miRNA-145 in DFF45-854 and hsa-miRNA-145 target sequence mutants. B, hsa-miRNA-145 suppressed activity of normal DFF45-854; C, hsa-miRNA-145 had no suppressive effects related to activity of DFF45-854 mutants.