Foxj2 overexpression is associated with poor prognosis, progression, and metastasis in nasopharyngeal carcinoma

Figures & data

Table 1 Sequences of siRNAs targeting Foxj2

siRNA names	Sequences (5′-3′)
Foxj2_siR1	Sense: CGAACAACUACUACAUGUAdTdT
	Antisense: UACAUGUAGUAGUUGUUCGdTdT
Foxj2_siR2	Sense: CCUUCUGACUGGUGCUCUAdTdT
	Antisense: UAGAGCACCAGUCAGAAGGdTdT
Foxj2_siR3	Sense: GGCACAACCUUUCUCUCAAdTdT
	Antisense: UUGAGAGAAAGGUUGUGCCdTdT
Foxj2_siR4	Sense: GGUCCUUCCGCAACCUCUAdTdT
	Antisense: UAGAGGUUGCGGAAGGACCdTdT

Abbreviations: siRNA, small interfering RNA; siR, siRNA.

Figure 1 Expression pattern and prognosis role of Foxj2 in NPC tissues.

Notes: (A) Immunohistochemistry analysis (1:50, ab22857) of Foxj2 expression in NPC tissues. Original magnifications: ×200 (a–c); ×400 (d–f) (B) Protein levels of Foxj2 in NPC (ie, tumor tissue T) and noncancerous nasopharyngeal tissues (labeled “N”) by Western blotting. (C) Quantitative results of Western blotting. β-actin was used as a loading control. The same experiment was repeated at least 3 times. *P<0.05. Data are presented as mean ± SD.
Abbreviation: NPC, nasopharyngeal carcinoma.

Table 2 The association between Foxj2 expression and clinicopathological features in 57 NPC patients

Clinicopathological parameters	Total	Foxj2 expression, n		P-value
		Low	High
Gender
Male	41	17	24	1.000
Female	16	6	10
Age, years
<50	20	8	12	1.000
≥50	37	15	22
T-classification
T1–T2	37	19	18	0.026*
T3–T4	20	4	16
Lymph node metastasis
N0–N1	38	13	25	0.253
N2–N3	19	10	9
Distant metastasis
No	43	22	21	0.004*
Yes	14	1	13
TNM clinical stage
I–II	24	14	10	0.029*
III–IV	33	9	24

Notes: Statistical analyses were performed by the Pearson’s χ² test.

* P<0.05 was considered significant.

Abbreviations: NPC, nasopharyngeal carcinoma; TNM, tumor–node–metastasis.

Figure 2 Prognosis role of Foxj2 in NPC.

Notes: Kaplan–Meier survival curves of NPC patients based on Foxj2 expression status; P<0.05, log-rank test.
Abbreviation: NPC, nasopharyngeal carcinoma.

Table 3 Survival status and clinicopathological parameters in 57 human NPC tissues

Clinicopathological parameters	Total	Survival status, n		P-value
		Alive	Dead
Gender
Male	41	29	12	0.545
Female	16	10	6
Age, years
<50	20	12	8	0.377
≥50	37	27	10
T-classification
T1–T2	37	29	8	0.039*
T3–T4	20	10	10
Lymph node metastasis
N0–N1	38	23	15	0.081
N2–N3	19	16	3
Distant metastasis
No	43	36	7	0.000*
Yes	14	3	11
TNM clinical stage
I–II	24	19	5	0.161
III–IV	33	20	13
Foxj2 expression
Low	23	22	1	0.000*
High	34	17	17

Notes: Statistical analyses were performed by the Pearson’s χ² test.

* P<0.05 was considered significant.

Abbreviations: NPC, nasopharyngeal carcinoma; TNM, tumor–node–metastasis.

Table 4 Contribution of various potential prognostic factors to survival, determined by Cox regression analysis, in 57 human NPC tissues

Prognostic factors	Hazard ratio	P-value	95% confidence interval
T-classification	2.455	0.165	0.691–8.721
I–II versus III–IV
Distant metastasis	6.689	0.002*	2.021–22.143
M0 versus M1
TNM clinical stage	0.275	0.123	0.053–1.420
I–II versus III–IV
Foxj2 expression	0.099	0.031*	0.012–0.806
Low versus high

Notes: Statistical analyses were performed by Cox regression analysis.

* P<0.05 was considered significant.

Abbreviations: NPC, nasopharyngeal carcinoma; TNM, tumor–node–metastasis.

Figure 3 Expression of Foxj2 in NPC cells.

Notes: (A, B) Western blot analysis of Foxj2 expression in 4 kinds of NPC cell lines (CNE-1, CNE-2, 5-8F, and 6-10B) and in NP-69 cell line. (C, D) The expression of Foxj2 was suppressed using siRNAs, and interference efficiency was detected by Western blotting. β-actin was used as control. *P<0.05. Data are presented as mean ± SD.
Abbreviations: NPC, nasopharyngeal carcinoma; siRNA, small interfering RNA.

Figure 4 Knockdown of Foxj2 reduces CNE-2 cell proliferation.

Notes: (A) IHC analysis (1:50, ab22857) of Foxj2 expression in T1–T2 and T3–T4 NPC tissues. Original magnifications: ×200 (a–b); ×400 (c–d); (e) percentage expression of Foxj2. (B, C) CCK-8 assay was used to determine cell proliferation of CNE-2 cells treated with Foxj2 siRNA for the indicated time. (D) Cell cycle distribution analyzed by flow cytometry in CNE-2 cells after Foxj2 downregulation. The data are presented as mean value ± SEM (compared with the control). The same experiment was repeated at least 3 times. *P<0.05; ***P<0.001. Data are presented as mean ± SD.
Abbreviations: CCK-8, cell counting kit 8; IHC, immunohistochemistry; NPC, nasopharyngeal carcinoma; OD, optical density; siRNA, small interfering RNA.

Figure 5 Knockdown of Foxj2 decreases the migration of CNE-2 cells.

Notes: (A) IHC analysis (1:50, ab22857) of Foxj2 expression in metastatic and nonmetastatic NPC tissues. Original magnifications: ×200 (a–b); ×400 (c–d); (e) percentage expression of Foxj2. (B) CNE-2 cells transfected with Foxj2 siRNAs showed a slower migration rate than those transfected with the control siRNA. Migration of cells to the wound was visualized at 0 and 48 h with a microscope (original magnification: ×200). (C) The histogram shows the relative migration distance of (B). (D–E) Knockdown of Foxj2 in CNE-2 cells inhibited cell migration, as determined by transwell assays. Number of cells migrating through the membrane was counted in 10 fields under ×20 objective lens. Error bars, SD. n=3; *P<0.05, **P<0.01, ***P<0.001. The same experiment was repeated at least 3 times.
Abbreviations: IHC, immunohistochemistry; NPC, nasopharyngeal carcinoma; siRNA, small interfering RNA.