miR-34 inhibits growth and promotes apoptosis of osteosarcoma in nude mice through targetly regulating TGIF2 expression

Abstract

miR-34 was reported to be involved in multiple tumors occurrence and development. The aim of the present study was to explore the impact of miR-34 on osteosarcoma and related mechanisms. Tumor tissues and non-tumor tissues of 34 patients with osteosarcoma were collected. qRT-PCR detection revealed that miR-34 was significantly down-regulated in tumor tissues (P<0.05). hFOB 1.19 and MG-63 cells were cultured. qRT-PCR detection showed that miR-34 was also significantly down-regulated in MG-63 cells (P<0.05). After transfection by miR-34 mimics, MG-63 cells proliferation in nude mice was significantly impaired (P<0.05), and percentage of apoptosis as well as caspase-3 positive cells proportion of osteosarcoma tissue in nude mice was markly increased (P<0.05). Western blot and immunofluorescence results also demonstrated that TGIF2 relative expression and TGIF2 positive cells proportion were both dramatically decreased (P<0.05). By luciferase reporter assay, we found that TGIF2 was the target gene of miR-34. After transfected by TGIF2 overexpression vector or co-transfected by miR-34 mimics and TGIF2 overexpression vector, we observed that, compared with blank group, tumor volume was significantly increased and apoptotic cells as well as caspase-3 positive cells proportion was obviously decreased in TGIF2 group (P<0.05), while no significant difference was found in these indicators between blank group and TGIF2 + mimics group. We concluded that miR-34 inhibited growth and promoted apoptosis of osteosarcoma in nude mice through targetting regulated TGIF2 expression.

Keywords: Osteosarcoma; TGIF2; apoptosis; miR-34; tumor volume.

Figure 1. miR-34 expression by qRT-PCR

(A) miR-34 expression in tumor tissues and non-tumor tissues of patients with osteosarcoma by qRT-PCR; (B) miR-34 expression in hFOB 1.19 cells and MG-63 cells by qRT-PCR. *P<0.05.

Figure 2. miR-34 inhibited osteosarcoma growth and promoted its apoptosis in nude mice

(A) miR-34 expression of tumor tissues in each group nude mice by qRT-PCR; (B) tumor volume of nude mice in each group; (C) apoptosis of tumor tissues in each group nude mice by flow cytometry; (D) caspase-3 expression by immunohistochemistry. *P<0.05 when compared with blank group or NC group, respectively.

Figure 3. miR-34 regulated TGIF2 expression targetly

(A) TGIF2 protein expression of tumor tissues in each group nude mice by Western blot; (B) TGIF2 positive cells proportion of tumor tissues in each group nude mice by immunofluorescence; (C) prediction of binding sites between TGIF2 and miR-34 by TargetScan; (D) dual luciferase reporter assay. *P<0.05 when compared with blank group or NC group, respectively. ^#P<0.05 when compared with blank group or NC group, respectively.

Figure 4. miR-34 inhibited growth and promoted apoptosis of osteosarcoma in nude mice through targetting regulated TGIF2 expression

(A) TGIF2 mRNA expression of tumor tissues in each group nude mice by qRT-PCR; (B) TGIF2 protein expression of tumor tissues in each group nude mice by Western blot; (C) tumor volume of nude mice in each group; (D) apoptosis of tumor tissues in each group nude mice by flow cytometry; (E) caspase-3 expression by immunohistochemistry. *P<0.05 when compared with blank group.