Cofilin-2 Acts as a Marker for Predicting Radiotherapy Response and Is a Potential Therapeutic Target in Nasopharyngeal Carcinoma

Abstract

BACKGROUND The purpose of this study was to determine whether cofilin-2 could serve as a protein marker for predicting radiotherapy response and as a potential therapeutic target in nasopharyngeal carcinoma (NPC). MATERIAL AND METHODS Cofilin-2 protein levels in serum and tissue samples from patients with NPC were assessed by sandwich ELISA and IHC. In vitro, cofilin-2 levels in CNE-2R cells were significantly higher than those of CNE-2 cells. Meanwhile, CNE-2R cells were silenced for cofilin-2 to obtain a stable cofilin-2-RNAi-LV3 cell line. Then, cell proliferation, radiosensitivity, invasion and migration abilities, cell cycle, and apoptosis were evaluated by Cell Counting Kit 8 assay (CCK-8), flow cytometry (FCM), clone formation assay, and in vitro. RESULTS The secreted levels of the cofilin-2 protein in radioresistant NPC patients were significantly higher than those of radiosensitive cases. After cofilin-2 knockdown in nasopharyngeal carcinoma CNE-2R cells, proliferation was decreased, while apoptosis and radiosensitivity were enhanced; cell cycle distribution was altered, and the transplanted tumors in nude mice grew significantly less. CONCLUSIONS Overall, our findings suggest that cofilin-2 acts as a marker for predicting radiotherapy response and is a potential therapeutic target in nasopharyngeal carcinoma.

Figure 1

Cofilin-2 protein levels in the radioresistance and radiosensitivity groups. The differences showed a statistical significance (P=0.004).

Figure 2

Cofilin-2 expression in NPC tissues. Cofilin-2 levels were assessed by IHC. (A, B) Low expression; (C, D) high expression (200×, HP).

Figure 3

Cofilin-2 levels are reduced by lentiviral cofilin-2-shRNA. (A) Quantitative analysis of cofilin-2 mRNA expression in different groups as assessed by RT-PCR. (B) Western blot analysis showing cofilin-2 protein expression in different groups. GAPDH was used as an internal control. Control – non-transfected group; NC – scrambled shRNA-transfected group.

Figure 4

Cofilin-2 silencing by shRNA results in cell growth inhibition. CCK-8 assay was used to assess cell viability in CNE-2R cells. Survival rates of the control, NC, and cofilin-2-shRNA groups were significantly different at the radiation doses of 2, 4, 6, 8, and 10 Gy (* p<0.001).

Figure 5

ShRNA-mediated knockdown of cofilin-2 promotes G2/M cell cycle arrest in CNE-2R cells. (A) Representative flow-cytograms assessing cell cycle distribution in the control, NC, and cofilin-2-shRNA groups. (B) Quantification of A (* p<0.001).

Figure 6

ShRNA-mediated knockdown of cofilin-2 enhances apoptosis in CNE-2R cells. Cells were submitted to Annexin V APC staining and assessed by flow cytometry. (A) Representative flow-cytograms showing Annexin V APC staining in the control, NC, and cofilin-2-shRNA groups. (B) Quantification of A (* p=0.001).

Figure 7

Cofilin-2 knockdown increases radiosensitivity in CNE-2R cells. (A) Effect of cofilin-2 silencing on clone formation ability of CNE-2R cells at different doses of irradiation. (B) Fit curves were generated with the Graph Pad Prism 6.0 software.

Figure 8

Effects of cofilin-2 silencing in the nude mouse xenograft tumor model. (A) Nude mice and transplanted tumors. (B) Growth curves of the transplanted tumors in nude mice.

Figure 9

Expression of the cofilin-2 protein in nude mouse transplanted tumors. (A) Xenograft tumor morphology after H&E staining; IHC showing cofilin-2 protein levels in nude mice (200×, HP). (B, C) Protein expression of cofilin-2 in nude mice as detected by WB.