Expression of ZNF281 in colorectal cancer correlates with response to radiotherapy and survival

Figures & data

Figure 1. ZNF281 promotes CRC cell radioresistance. (A) Positive ZNF281 expression in a representative biopsy specimen from a patient who exhibited resistance to PRT (×200). (B) Negative ZNF281 expression in a representative biopsy specimen from a patient who exhibited sensitivity to PRT (×200). (C) Positive ZNF281 expression in a representative biopsy specimen with poor tumor regression grade (TRG ≤ 2) (×200). (D) Negative ZNF281 expression in a representative biopsy specimen with good tumor regression grade (TRG ≥ 3) (×200). (E) Kaplan–Meier curves of DFS in our cohort. (F) Kaplan–Meier curves of OS in our cohort. (G) ZNF281 levels in different subgroups (responder, respond to radiation; nonresponder, no response to radiation). (H) Kaplan–Meier curves of OS in GSE133057 dataset. (*p < 0.05; **p < 0.01).

Table 1. Chi-square test of clinicopathologic characteristics and ZNF281 mRNA expression in our cohort.

Characteristics		ZNF281 expression
		High (n = 91)	Low (n = 35)	χ2	P value
Age	<60	24	9	0.006	0.940
	>60	67	26
Gender	Female	26	14	1.524	0.217
	Male	65	21
T stage after pCRT	T0-2	44	18	0.096	0.757
	T3-4	47	17
N stage after pCRT	N0	62	30	3.966	0.046
	N1-2	29	5
TNM stage after pCRT	0-II	61	30	4.397	0.036
	III	30	5
Tumor downstage after pCRT	0-1	62	23	0.067	0.795
	2-3	29	12
Tumor regression grade after pCRT	0-2	74	18	11.462	0.001
	3-4	17	17

pCRT: preoperative chemoradiotherapy.

Table 2. Univariate and multivariate Cox regression analysis of clinical pathologic features according to our cohort.

Parameters	OS
	Univariate		Multivariate
	HR (95% CI)	P	HR (95% CI)	P
ZNF281	5.494 (1.574-19.173)	0.008	11.419 (1.694-76.961)	0.012
(high vs low)
Age	0.484 (0.186-1.258)	0.136	–	–
(<60 vs > 60)
Gender	1.880 (0.663-5.333)	0.236	–	–
(female vs male)
T stage after pCRT	6.980 (2.000-24.364)	0.002	3.422 (0.696-16.826)	0.130
(T0-2 vs T3-4)
N stage after pCRT	3.374 (1.304-8.733)	0.012	0.139 (0.015-1.297)	0.083
(N0 vs N1-2)
TNM stage after pCRT	4.190 (1.623-10.816)	0.003	9.679 (1.041-89.971)	0.046
(0- II vs III)
Tumor downstage after pCRT	0.093 (0.012-0.697)	0.021	0.163 (0.011-2.320)	0.181
(0-I vs II-III)
Tumor regression grade after pCRT	0.020 (0.000-1.086)	0.055	–	–
(0-2 vs 3-4)

HR: hazard ratio; CI: confidence interval; pCRT: preoperative chemoradiotherapy.

Figure 2. ZNF281 was identified as an anti-radiation factor that improved the survival of CRC cells irradiated by X-rays. (A) Verification of ShRNA efficiency for ZNF281 knockdown by Western blot analysis. (B) Verification of ShRNA efficiency for ZNF281 knockdown by RT-PCR. (C) Colony formation assay was used to assess the survival of CRC cells after 8 days of exposure to X-rays (Vector, lentiviral vector; ShZNF281, ZNF281 after stable knockdown).

Figure 3. ZNF281 promotes radiation resistance by promoting DSB repair in CRC cells. (A) Immunofluorescence images of γH2AX levels and time course of lesion changes in X-ray-treated CRC cells (×400). (B) The foci number in CRC cells (mean ± SD). (C) The effects of ZNF281 on γH2AX levels in CRC cells treated with X-rays (loading control: GAPDH. Vector, lentiviral vector; ShZNF281, ZNF281 after stable knockdown; ***, p < 0.01).

Figure 4. ZNF281 promotes the resistance of CRC cells in a nude mouse model (A) HCT116 cells transfected with ZNF281 low expression (ShZNF281) and vector (vector) to establish transplanted xenografts were treated with X-rays (2 Gy/day) for 14 days. (B) Tumour volumes measured at different time points. (C) Images of IHC staining of Ki67 and γH2AX in xenografts (×200). (***, p < 0.001; vector, lentiviral vector; ShZNF281, ZNF281 after stable knockdown).

Availability of data and materials

The data used to support the findings of this study are included within the article.