Zinc finger protein x-linked (ZFX) contributes to patient prognosis, cell proliferation and apoptosis in human laryngeal squamous cell carcinoma

Abstract

Zinc finger protein, X-linked (ZFX) gene locus on the human X chromosome is structurally similar to the zinc finger protein, Y-linked gene. Its role in human laryngeal squamous cell carcinoma (LSCC) is still not clearly defined. This study was focused on investigating the role of zinc-finger protein X-linked (ZFX) in human LSCC. Expression levels of ZFX were examined in LSCC tissues, corresponding adjacent non-tumoral tissues and vocal leukoplakia tissues by immunohistochemistry (IHC). The association with the expression level of ZFX and LSCC clincopathological parameters was analyzed. The prognostic value of ZFX expression was also analyzed. Lentivirus-mediated RNA interference was applied to silence ZFX expression and the effects of ZFX knockdown on the growth of human LSCC primary cells was investigated. Overexpression of ZFX was found in LSCC tissues. The expression of ZFX was associated with the clinical stage of LSCC. Patients with higher level of ZFX experienced a poorer prognosis compared to those with lower level of ZFX. Knockdown of ZFX inhibited cell proliferation, colony formation and migration of LSCC primary cells. Moreover, ZFX silencing induced cell apoptosis. These results provide the convincing evidence for the first time that ZFX plays an important role in LSCC development and could be a potential therapeutic target or prognostic predictor for LSCC.

Keywords: Laryngeal squamous cell carcinoma (LSCC); apoptosis; prognosis; proliferation; zinc finger protein x-linked (ZFX).

Figure 1

Expression of ZFX proteins in LSCC tissue, adjacent non-tumoral tissue and vocal leukoplakia tissue using immunohistochemistry (IHC) and H&E staining (left 100×, right 400×). T tumor tissue, A adjacent non-tumoral tissue, V vocal leukoplakia tissue.

Figure 2

Expression quantification of ZFX proteins in tissue samples. Quantification of ZFX expression depending on optical density of ZFX positive signals in IHC slices. A. LSCC tissue vs. vocal leukoplakia tissues. B. Early-stage LSCC tissue vs. vocal leukoplakia tissues. C. Corresponding LSCC tissues vs. adjacent non-tumoral tissues. D. Non-tumoral tissues vs. vocal leukoplakia tissues. The asterisk above the bar denotes statistically significant differences from the control group (P<0.05).

Figure 3

Kaplan-Meier graph showing the overall survival for patients with LSCC. Patients with high expression of ZFX had a significant lower overall survival rate than those with low expression of ZFX (P<0.01).

Figure 4

The expression of ZFX was dramatically knocked down by lentivirus mediated shZFX transfection. A. Microscope pictures of GFP-expressing cells after 96 h after pGCSIL-GFP-shZFX or -shNC transfection, including fluorescence microscope images and light microscope images (100×). B. Quantitative analysis of ZFX by qRT-PCR. The asterisk above the bar denotes statistically significant differences from the control group, P<0.05.

Figure 5

The effect of ZFX knockdown on the proliferation of human LSCC primary cells. A. MTT assay of cell proliferation in LSCC primary cells infected with pGCSIL-GFP-shZFX or -shNC. The asterisk under the line denotes statistically significant differences from the control group (P<0.05). B. Knockdown of ZFX decreased colony number of LSCC primary cells. pGCSIL-GFP-shZFX or -shNC infected LSCC primary cells were cultured for 2 weeks and stained with GIEMSA. Quantitative analysis of colony formation was shown. C. A BrdU ELISA was performed to determine proliferation of LSCC primary cells after knockdown of ZFX. The asterisk above the bar denotes statistically significant differences from the control group (P<0.05).

Figure 6

The effect of ZFX-knockdown on cell migration in human LSCC primary cells. A. Transwell assay in LSCC primary cells infected with pGCSIL-GFP-shZFX and -shNC. Migrated cells on the lower surface of the transwell filter were stained by GIEMSA, photographs were taken after 24 h post-migration (200×). B. The average migration rate was measured in the Transwell assay. The average cell migration rate was calculated and represented as OD570/OD490. The asterisk above the bar denotes statistically significant differences from the control group (P<0.05).

Figure 7

Apoptosis induction in LSCC primary cells infected with lentivirus expressing ZFX-specific siRNA. A. Cell apoptosis by annexin-V staining was analyzed with flow cytometry. B. FACS analysis of Annexin V-APC stained control cells or ZFX-knockdown cells 5 days after transient infection. Data shown here is the mean ± SD of cell percentage in apoptosis from three independent experiments. The asterisk above the bar denotes statistically significant differences from the control group (P<0.05).