PYGB siRNA inhibits the cell proliferation of human osteosarcoma cell lines

Abstract

Osteosarcoma is the most common malignant bone carcinoma that primarily occurs between childhood to adolescence. It was suggested by recent research that the Brain type glycogen phosphorylase (PYGB) gene may serve an important role in various types of cancer. In the present study, the PYGB gene was knocked down in order to evaluate the cell viability, invasion and migration of the human osteosarcoma cell lines MG63 and HOS. The expression levels of PYGB in osteosarcoma and bone cyst tissue samples, as well as in the osteosarcoma cell lines were identified using reverse transcription‑quantitative polymerase chain reaction and western blot assay. Subsequently, a Cell Counting kit 8 assay was employed to evaluate cell proliferation. Cell apoptosis rate and cell cycle distribution were measured by flow cytometry. In addition, cell invasion and migration were evaluated through a Transwell assay. The expression levels of the cell apoptosis and tumor metastasis associated proteins B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein, E‑cadherin, Twist, matrix metalloproteinase (MMP)‑9 and MMP2 were measured via western blotting. PYGB exhibited a higher expression level in the osteosarcoma tissue samples, particularly in the human osteosarcoma cell lines MG63 and HOS. Knockdown of PYGB resulted in a decline in cell proliferation, invasion and migration, which was coupled with induced cell apoptosis and cell cycle arrest in MG63 and HOS cells. Furthermore, alterations in the expression of apoptosis and metastasis associated proteins indicated that small interfering (si)PYGB may have regulated cell viability by targeting the Bcl/Caspase and cyclin dependent kinase (CDK)‑1 signaling pathway. In conclusion, PYGB siRNA exerted an inhibitory effect on the cell viability of the human osteosarcoma cells MG63 and HOS by blocking the Caspase/Bcl and CDK1 signaling pathway, highlighting novel potential therapeutic methods for treating osteosarcoma.

Figure 1.

Screening of osteosarcoma cell lines with high expression level of PYGB. (A) RT-qPCR was used to measure the mRNA expression levels of PYGB in 15 bone cyst samples and 35 osteosarcoma samples. **P<0.01, as indicated. (B and C) The protein expression levels of PYGB in the human osteosarcoma cell lines U-20S, HOS, MG63, SaoS-2 and SW1353 were detected by western blot analysis. ***P<0.001 vs. U-2OS, SaoS-2 and SW1353. (D) Western blotting was also used to measure the PYGB protein expression in (E) MG63 and HOS cells following the transfection of PYGB siRNA at 3 interference sites. (F) In addition, the mRNA expression level of PYGB was also measured using RT-qPCR. ***P<0.001 vs. the NC group. Data are expressed as the mean ± standard deviation (n=3). PYGB, Brain type glycogen phosphorylase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; siRNA, small interfering RNA.

Figure 2.

Effect of PYGB siRNA on the cell viability of the osteosarcoma cell lines MG63 and HOS. Following transfection with PYGB siRNA, the effect of PYGB siRNA on (A) the cell viability of MG63 cells and (B) the cell proliferation of HOS cells was evaluated by a Cell Counting kit-8 assay for 0, 24, 48 and 72 h. Data are expressed as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the NC group. Data are expressed as the mean ± standard deviation (n=3). PYGB, Brain type glycogen phosphorylase; NC, negative control; siRNA, small interfering RNA.

Figure 3.

Effect of PYGB siRNA on cell apoptosis and the cell cycle in the osteosarcoma cell lines MG63 and HOS. (A) Cell apoptosis in MG63 cells was evaluated using flow cytometry following the transfection of PYGB siRNA for 48 h. Early apoptotic cells were identified using Annexin V/propidium iodide double staining and are shown in the lower right quadrant. (B) Cell apoptosis induced by PYGB siRNA in HOS cells was also identified by flow cytometry. (C) The cell cycle distribution of MG63 cells following 48 h post-transfection with PYGB siRNA was detected using flow cytometry. (D) The cell cycle distribution of HOS cells was also evaluated using flow cytometry. *P<0.05 and ***P<0.001 vs. the NC group. Data are expressed as the mean ± standard deviation (n=3). PYGB, Brain type glycogen phosphorylase; NC, negative control; siRNA, small interfering RNA.

Figure 4.

Effect of PYGB siRNA on the invasion and migration of osteosarcoma cell lines MG63 and HOS. (A and B) The invasive ability of MG63 and HOS cells was evaluated using a Transwell assay following 48 h post-transfection with PYGB siRNA. A smaller number of invaded MG63 and HOS cells were observed and counted in the PYGB siRNA group when compared with the control and NC groups. (C and D) Migrated MG63 and HOS cells were also identified by Transwell assay (magnification, ×200). **P<0.01 and ***P<0.001 vs. the NC group. Data are expressed as the mean ± standard deviation (n=3). PYGB, Brain type glycogen phosphorylase; NC, negative control; siRNA, small interfering RNA.

Figure 5.

Effect of PYGB siRNA on the expression of E-cadherin, Twist, MMP9, MMP2, Bax and Bcl2 in the osteosarcoma cell lines MG63 and HOS. (A) The blots show the results of western blot analysis. The protein expression levels in (B) MG63 and (C) HOS cells were measured by western blot analysis 48 h following treatment with PYGB siRNA. *P<0.05, **P<0.01 and ***P<0.001 vs. the NC group. Data are expressed as the mean ± standard deviation (n=3). PYGB, Brain type glycogen phosphorylase; NC, negative control; siRNA, small interfering RNA; MMP, matrix metalloproteinase; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.