Table 1.
The sequence of primers used for qRT-PCR.
Fig 1.
The expression level of miR-93 in gastric cancer clinical specimens and cell lines.
(A) qRT-PCR result for the expression of miR-93 in gastric cancer tissues and matched adjacent normal tissues. (B) Relative expression level of miR-93 in patients at different clinical stages. (C) Relative expression level of miR-93 in patients with metastasis or without metastasis. (D) Relative expression level of miR-93 in gastric cancer cell lines relative to the normal human gastric epithelial cell line GES-1. *P<0.05, **P<0.01.
Table 2.
Correlation between clinicopathological characteristics and miR-93 expression.
Fig 2.
Effect of miR-93 upregulation on cell viability, cell cycle and apoptosis rate of BGC-823 cells.
(A) miR-93 mimics significantly enhanced the expression of miR-93 in BGC-823 cells. (B) Graphical representation of MTT assay showed in BGC-823 cells transfected with miR-93 mimics or control. (C and D) Cell cycle analysis and apoptosis assay of BGC-823 cells transfected with miR-93 mimics or control. *P<0.05, **P<0.01.
Fig 3.
Effect of inhibition of miR-93 on cell proliferation, cell cycle distribution and apoptosis rate of SGC-7901 cells.
(A) miR-93 inhibitors significantly decreased the expression of miR-93 in SGC-7901 cells. (B) Representative graphs of MTT assay showed in SGC-7901 cells transfected with miR-93 inhibitors or control. (C and D) Cell cycle analysis and apoptosis assay of SGC-7901 cells transfected with miR-93 inhibitors or control. *P<0.05, **P<0.01.
Fig 4.
MiR-93 promoted cell migration, invasion and metastasis in vitro.
(A and B) Ectopic expression of miR-93 in BGC-823 cells promoted cell migration and invasion, moreover, knockdown of miR-93 in SGC-7901 cells inhibited cell migration and invasion. (C) Levels of MMP-2, MMP-9 proteins in gastric cancer cells measured by western blot analysis, GAPDH was used as control. *P<0.05, **P<0.01.
Fig 5.
Antagomir-93 inhibits tumor growth in vivo.
After transfection of SGC-7901 cells with antagomir-93 or antagomir-NC for 24h, cells were subcutaneously injected into the Left subaxillary of nude mice. (E) Representative images of excised xenograft tumors from nude mice. (A) The growth curve of tumors in nude mice. (B) Tumor weight. (C) Tumor levels of miR-93 measured by Quantitative RT-PCR. (D and F) TIMP2 miRNA expression and proteins levels measured in tumor tissues. (G) TIMP2 proteins levels in xenograft tumors determined by immunohistochemical staining. *P<0.05, **P<0.01, (magnification × 200).
Fig 6.
The correlation between miR-93 and epithelial-mesenchymal markers in gastric cancer.
(A) The western blot assay showed that the EMT markers including N-cadherin and Vimentin expression were up-regulated after transfection of BGC-823 cells with miR-93 mimics. In contrast, N-cadherin and vimentin protein were suppressed after transfection of SGC-7901 cells with miR-93 inhibitors. (B and C) qRT-PCR analysis of E-cadherin, N-cadherin and Vimentin in BGC-823 and SGC-7901 cell lines. (D) qRT-PCR analysis of E-cadherin, N-cadherin and Vimentin in gastric cancer tissues.*P<0.05, **P<0.01.
Fig 7.
TIMP2 is a direct target of miR-93.
(A) The predicted complementary sequence interaction between human miR-93 3'-UTR and TIMP2. (B) Up-regulation of miR-93 significantly inhibited while miR-93 inhibition promoted the luciferase activity of wild-type rather than mutant 3'-UTR of TIMP2. (C and D) Ectopic expression of miR-93 in BGC-823 significantly reduced the mRNA level and protein of TIMP2. Knockdown of miR-93 in SGC-7901 cells increased TIMP2 mRNA and protein level. (E) Relative expression level of TIMP2 in gastric cancer tissues and corresponding normal tissues. (F) The correlation between TIMP2 and miR-93 mRNA expression in 70 cases of gastric cancer specimens was evaluated using Spearman’s correlation analysis (P<0.01, R2 = 0.33). *P<0.05, **P<0.01.
Fig 8.
TIMP2 mediates the effects of miR-93 in gastric cancer cells.
(A) TIMP2 protein expression was inhibited by siRNA. (B) MTT assays of SGC-7901 cells transfected with miR-93 mimics, NC, si-TIMP2 or Scramble. (C) The expression of MMP-2, MMP-9, E-cadherin, N-cadherin and Vimentin was evaluated by Western blot analysis. (D) Migration and invasion assays were performed in SGC-7901 cells transfected with miR-93 mimics, NC, siTIMP2 or Scramble. *P<0.05, **P<0.01.