Fig 1.
Bag-1 is a direct target of miR-138.
(A) A schematic representation showing the putative target site of Bag-1 and mutated target site for miR-138 with the seed region and base substitutions underlined. (B and C) Luciferase reporter assay in HEK293T cells with co-transfection of Bag-1-UTR-WT or Bag-1-UTR-MUT at indicated times. (D and E) The mRNA and protein expression levels of Bag-1 in OCUG-1 and NOZ cells transfected with miR-138 mimic or the control. β-Actin was used as an internal quantitative control. Error bars represent the SD from three independent trials. **P<0.01.
Fig 2.
Expression of miR-138 and Bag-1 in gallbladder carcinoma specimens.
(A) The expression of miR-138 was determined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (B) Relative expression of Bag-1 at mRNA level was examined in gallbladder carcinoma tissues compared with matched normal adjacent gallbladder tissues using qRT-PCR. (C) Inverse correlation between miR-138 and Bag-1 expression in gallbladder carcinoma tissues using Pearson’s correlation coefficient. The expression of miR-138 was normalized to that of U6, and the expression of Bag-1 mRNA was normalized to that of β-actin in each sample.
Fig 3.
Effect of miR-138 on gallbladder carcinoma cell proliferation and apoptosis.
(A) The qRT-PCR analysis confirmed that the expression of miR-138 was clearly increased in cells transduced with miR-138 compared with the control vector. (B and C) Effect of miR-138 on cell proliferation was measured using MTT assay in OCUG-1 and NOZ cells transduced with miR-138 or the control vector. (D) Flow cytometric analysis of the effect of miR-138 on apoptosis of OCUG-1 and NOZ cells. Error bars represented the SD from three independent trials. **P<0.01.
Fig 4.
Silencing the expression of Bag-1 could inhibit the proliferation of gallbladder carcinoma cells.
(A) Immunoblots were performed to analyze Bag-1, BCL-2, Bax, and cleavage of caspase-3 expression in Bag-1 siRNA-transfected OCUG-1 and NOZ cells. (B and C) MTT assay was used to measure cell proliferative capacity in gallbladder carcinoma cells treated with the control siRNA or Bag-1 siRNA. (D) Gallbladder carcinoma cells transduced with miR-138 compared with the control vector were subjected to Western blot analysis with the indicated antibodies. Error bars represented the SD from three independent trials. *P<0.05; **P<0.01.
Fig 5.
Effects of overexpression of Bag-1 on cell proliferation and apoptosis.
(A) Expression of Bag-1 protein in OCUG-1 and NOZ cells stably expressing miR-138 transfected with pcDNA3.1 vector as a control or Bag-1 vector was detected using Western blot at 48 h after transfection. (B and C) Cell proliferation assay was measured in pcDNA3.1 vector or Bag-1-transfected OCUG-1 and NOZ cells stably expressing miR-138. (D) Apoptosis assay was performed after transfection of pcDNA3.1 vector or Bag-1 in gallbladder carcinoma cells. Error bars represented the SD from three independent trials. **P<0.01.
Fig 6.
miR-138 inhibits the growth of tumor in vivo.
(A) Twelve nude mice were used to establish the gallbladder carcinoma xenograft models. Representative image of tumors formed at the fifth week after injection. Experiments were repeated at least three times. (B) Growth curve was drawn by measuring tumor volumes at the indicated times. **P<0.01.