High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis

Abstract

Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients.

Keywords: Ku70; XRCC6; osteosarcoma; proliferation; β-catenin/Wnt signaling pathway.

Figure 1

XRCC6 expression is up-regulated in osteosarcoma (OS) clinical samples and cell lines. (A–C) The mRNA and protein expression level of XRCC6 were measured by TaqMan real-time PCR and Western blot assays in OS cell lines (including MNNG/HOS, MG63 and U2OS) and human osteoblastic cell line (hFOB); (D,E) relative expression of XRCC6 was detected using qRT-PCR in 20 pairs of OS samples and their corresponding noncancerous samples. The expression of XRCC6 was overexpressed in OS tissues compared with the noncancerous tissues. Statistical analysis was performed using paired t test (D).

Figure 2

Knockdown of XRCC6 inhibited cell proliferation through G2/M phase arrest. (A,B) The expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect the proliferation of MNNG/HOS cells and U2OS cells after transfection with targeted siRNA. Diagrams showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were inhibited by downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; (F,H) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of U2OS transfected with si-XRCC6 or si-NC. The data are representative of three independent experiments. Error bars represent SD (Standard Deviation). * p < 0.05, ** p < 0.01 by Student’s t test.

Figure 3

Decreased XRCC6 impaired OS cell colony-forming capacity. (A,C) Colony formation assay of MNNG/HOS cells and U2OS cells transfected with targeted siRNA or si-NC. After two weeks, cells in each well were fixed and counted. Representative photo micrographs of MNNG/HOS cells (A); and U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy in MNNG/HOS cells (B), and U2OS cells (D). Following XRCC6 knockdown. Data are expressed as mean ± SD. of three independent experiments. ** p < 0.01, by student’s t test. (Magnification: 1×).

Figure 4

XRCC6 regulates the β-catenin/Wnt signaling pathway. (A) Western blot analysis of β-catenin and the downstream protein level of this pathway including c-MYC, Cyclin D1 in both MNNG/HOS and U2OS cells transfected with targeted siRNA or si-NC. Knockdown of XRCC6 expression by targeted siRNA led to reduced expression of β-catenin and the downstream protein level of this pathway in both MNNG/HOS and U2OS cells; (B) a densitometric analysis of the Western blotting bands was performed. * p < 0.05 by Student’s t test.

Figure 5

High expression of XRCC6 correlates to OS clinical stage and tumor size. Representative images of histological inspection of an OS tissue or noncancerous tissue micrographs were shown as labeled. XRCC6 was overexpressed in OS tissues compared with corresponding noncancerous tissues.