MicroRNA-645, up-regulated in human adencarcinoma of gastric esophageal junction, inhibits apoptosis by targeting tumor suppressor IFIT2

Abstract

Background: An increasing body of evidence indicates that miRNAs have a critical role in carcinogenesis and cancer progression; however, the role of miRNAs in the tumorigenesis of adencarcinoma of gastric esophageal junction (AGEJ) remains largely unclear.

Methods: The SGC7901 and BGC-823 gastric cancer cell lines were used. The expressions of miR-645 and IFIT2 (Interferon-induced protein with tetratricopeptide repeats 2) were examined by qRT-PCR, The expressions of IFIT2 was examined by western blotting and immunohistochemistry assay. The cell apoptosis was determined by FACS. MiR-645 inhibitor, mimics and plasmid-IFIT2 transfections were performed to study the loss- and gain-function. Caspase-3/7 activity was examined by caspase-3/7 assay.

Results: In the present study, we have reported an increased expression of miR-645 in AGEJ clinical specimens compared with paired non-cancerous tissues. We also observed a significant miR-645 up-regulation in two gastric cancer (GC) cell lines, SGC7901 and BGC-823, which were used as cell models because there was no available AGEJ cell lines established to date. We found that inhibition of miR-645 could sensitize dramatically SGC7901 and BGC-823 cells to both serum starvation- and chemotherapeutic drug-induced apoptosis by up-regulating IFIT2, a mediator of apoptosis via a mitochondrial pathway, with a potential binding site for miR-645 in its mRNA's 3'UTR. Further investigation exhibited that IFIT2 expression decreases in SGC7901 and BGC-823 cells and AGEJ tissues. IFIT2 ectopic expression leads to promotion of cell apoptosis, indicating that IFIT2 may function as a suppressor in the development of AGEJ. Furthermore, inhibition of miR-645 induces up-regulation of IFIT2 and increased caspase-3/7 activity compared with control groups.

Conclusions: Our data suggest that miR-645 functions as an oncogene in human AGEJ by, at least partially through, targeting IFIT2.

Figure 1

Expressions of miR-645 are up-regulated in AGEJ clinical samples. A. expression of miR-645 in 43 human AGEJ clinical samples relative to the adjacent paired normal human gastric cardiac non-cancerous tissues, was measured by quantitative RT-PCR (The values indicate the mean ± SEM, n = 3, t-test,* p < 0.05, ** p < 0.01, *** p < 0.001). B. comparison of relative expression of miR-645 in human AGEJ clinical samples of different tumor size. (Two-tailed t-test,* p < 0.05).

Figure 2

Depletion of miR-645 promotes apoptosis of gastric cancer (GC) cells. A &D. The level of miR-645 was measured by quantitative PCR at designated time (One-way ANOVA analysis, the values indicate the mean ± SD, Figure 2 A, F =426.588, P < 0.001; Figure 2 D, F = 685.026, P < 0.001). B &E. GC cells transfected with miR-645 mimics and inhibitor subjected to MTT assay daily for 6 days (Two-way ANOVA analysis, Figure 2 B, F = 52.602, p < 0.001; Figure 2 E, F = 42.847, p < 0.001). C &F. GC cells cells transfected with miR-645 mimics and inhibitor were collected for FACS analysis after 72 h (The values indicate the mean ± SD, n = 3, One-way ANOVA analysis, Figure 2 C-a, F = 121.600, p <0.001; Figure 2 C-b, F = 250.400, p <0.001; Figure F-a, F = 194.815, p <0.001; Figure 2 F-b, F =412.741, p <0.001).

Figure 3

Validating the predicted binding sites between miR-645 and IFIT2. A. The schematic diagram shows the construct of Luc-IFIT2 3′UTR and Luc-IFIT2 3′Mut UTR. Both Luc-IFIT2 3′UTR and Luc-IFIT2 3′Mut UTR were cloned into a pmirGLO plasmid downstream of the firefly luciferase coding region between the PmeI and XbaI sites. B&C. SGC7901 cells (B) or BGC-823 cells (C) were co-transfected with the psiCHECK-2 constructs containing either IFIT2 3′UTR or IFIT2 3′Mut UTR and either the miR-645 inhibitor or the miR-645 mimics for 48 h. Values indicate the relative luciferase activity after normalization to Renilla luciferase activity (The values indicate the mean ± SD, n = 3, One-way ANOVA analysis, Figure 3 B, F = 283.244, Figure 3 C, F = 143.313. ***p < 0.001).

Figure 4

Expression of miR-645 and IFIT2 negatively correlate in AGEJ clinical samples and IFIT2 was down-regulated in AGEJ tissues compared with paired non-cancerous tissues. A. Expression of IFIT2 and miR-645 in AGEJ clinical samples were analyzed by quantitative PCR. B. comparison of relative expression of IFIT2 in human AGEJ clinical samples of different tumor size. (t-test, *p < 0.05). C. Expression of IFIT2 examined by western blotting (a, b: the values indicate the mean ± SD, normalized to tubulin, n = 3, t-test, ***p <0.001) D. a. Representative images shown are positive immunohistochemical staining of IFIT2 in human AGEJ specimens and matched adjacent normal tissues (magnification 200×). b. Staining scores of IFIT2 (t-test, ***p <0.001). E. Scatter plots showing the negative linear correlation between the mRNA expression of IFIT2 and that of miR-645 in 43 AGEJ clinical samples. F. IFIT2 and IFIT2 expression measured by western blotting in SGC7901(a) and BGC-823 cells (b). (The values indicate the mean ± SD, n = 3, One-way ANOVA analysis, ***p < 0.001).

Figure 5

IFIT2 mediates the function of miR-645 by promoting GC cell apoptosis. A &B. Expression of IFIT2 examined by western blotting (A: SGC7901; B, BGC-823. a, normalized to tubulin, the values indicate the mean ± SD, One-Way ANOVA analysis, for SGC7901, F = 189.307, for BGC-823, F = 85.374; b, The values indicate the mean ± SD, One-Way ANOVA analysis, for SGC7901, F = 85.374, for BGC-823, F = 219.921; ***p < 0.001). C. SGC7901 (a) and BGC-823 (b) cells subjected to MTT assay daily for 6 days (the values indicate the mean ± SD, Two-way ANOVA analysis, for SGC7901, F = 13.768, for BGC-823, F = 16.409, ***p <0.001). D &E. SGC7901 (D) and BGC-823 (E) cells were collected for FACS analysis after 72 h in the presence of ADR (0.05 μg/mL) (the values indicate the mean ± SD, n = 3, One-Way ANOVA analysis; for SGC7901, F = 361.749, for BGC-823, F = 229.952; ***p < 0.001).

Figure 6

Caspase-3/7 activity. Anti-apoptotic ability of SGC7901 cells and BGC-823 cells after exposure to ADR (0.2 μg/mL) or serum starvation was evaluated by caspases-3/7 activity. A &C, SGC7901 cells; B &D, BGC-823 cells. (the values indicate the mean ± SD, n = 3, One-Way ANOVA analysis; for A, F = 183.930, for B, F = 1093.797; for C, F = 1861.50, for D, F = 1483.604, ***p < 0.001).