Functions of Exogenous RUNX2 in Giant Cell Tumor of Bone In Vitro.

OBJECTIVES: This research aimed to investigate the relative level of Runt-related transcription factor 2 (RUNX2) in giant cell tumor of bone (GCTB). Through the histopathological similarities between osteoporosis and GCTB, the biological functions of exogenous RUNXS were demonstrated in GCTB cell lines. This generated awareness of the molecular mechanism of the biogenesis and metastasis of GCTB, as well as showing the pathways and processes involved in this study. This research also expected to provide hints for the clinical treatment of patients with GCTB, to release the tumor burden and reduce the recurrence rate and metastasis of patients with this condition. METHODS: The expression of RUNX2 in the tumors was verified by Western Blot, qRT-PCR and immunohistochemistry, compared with the normal tissues' adjacent tumors. Subsequently, the plasmids expressing RUNX2 were constructed, amplified and transfected into the 0404 cell line through transfection kits (0.4, 0.8, 1.6, 2.4 ng/µl). After that, the proliferation, migration, invasion, cellular viability and apoptosis of 0404 cell lines were examined by EDU assay, wound healing assay, transwell assay, annexin v staining, and CCK8 assay, respectively. RESULTS: The messenger RNA (mRNA) level of RUNX2 in tumors was over 100 folds more than the normal tissues. The protein level of tumors upregulated 8.32(±4.41) folds relatively. After the transfection of RUNX2 overexpressed plasmids into the 0404 cell line, the mRNA level of RUNX2 increased approximately 530.11(±24.87), 1117.96(±77.68), 2835.09(±45.22) and 4781.51(±79.37) folds respectively, and the protein level was upregulated about 4.12(±1.15), 16.73(±1.63), 21.53(±2.41) and 23.39(±0.85) folds respectively. The proliferation of 0404 cells was inhibited by 2.13(±1.02)% of 1.6 ng/µl group and 3.03(±1.76)% of 2.4 ng/µl group. And the migration was inhibited about 45.56(±6.13)%, 50.79(±5.27)%, 63.15(±8.62)% and 93.90(±3.65)% respectively. The invasion was decreased about 14.49(±5.4)%, 37.02(±6.52)%, 42.24(±2.59)% and 48.97(±10.61)% respectively. Meanwhile, FITC Annexin V/PI apoptosis assay demonstrated that RUNX2 plasmids could promote apoptosis rate around 4.15(±0.27)%, 5.07(±0.27)%, 7.61(±0.45)% and 11.32(±1.02)% respectively, and CCK8 proved these plasmids could weaken cellular viability in a concentration-dependent manner with the time passing. CONCLUSIONS: RUNX2 is highly expressed in giant cell tumors of bone. The RUNX2 overexpressed plasmids we constructed could be successfully transfected into 0404 cell line. Far more importantly, the exogenous RUNX2 can seriously block the biological functions of 0404 cell line in a concentration-dependent manner, including proliferation, translocation, invasion, cellular viability, and apoptosis. Meanwhile, the mechanism was hypothesized and discussed in the article.

Expression profiles of miRNAs in giant cell tumor of bone showed miR-187-5p and miR-1323 can regulate biological functions through inhibiting FRS2.

BACKGROUND: Giant cell tumor of bone (GCTB) is considered to be a kind of borderline tumor, which has a tendency to recur and translocate. MicroRNAs are one type of small noncoding RNA, which can inhibit the translation of targeted mRNA through RNA-induced silencing complex. METHODS: Microarray was conducted on three groups of tumor tissues and normal tissues from patients with GCTB, and results showed different expression profiles of miRNAs with Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. The functions of miR-187-5p and miR-1323, which were highly expressed in GCTB, were examined by 5-ethynyl-2'-deoxyuridine (EDU), transwell, and CCK8 assays. RNAhybrid et al. (RNA prediction softwares) predicted that the two microRNAs targeted fibroblast growth factor receptor substrate 2 (FRS2), which was verified by luciferase assay and rescue experiments. RESULTS: miR-187-5p and miR-1323 were highly expressed in tumor tissues. They can jointly regulate the biological functions of GCTB in vitro. Luciferase assay confirmed that the two microRNAs can bind to the 3' untranslated regions (UTR) of mRNA of FRS2. And, rescue experiments verified the relationships between the two microRNAs and FRS2. CONCLUSION: There were some different-expressed microRNAs between GCTB and normal tissues. miR-187-5p and miR-1323 can regulate the biological functions of GCTB through influencing the expression of FRS2.