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.
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.