DNA hypermethylated status and gene expression of PAX1/SOX1 in patients with colorectal carcinoma

Figures & data

Figure 1 DNA methylation of PAX1 and SOX1 analyzed in CRC using TCGA dataset.

Notes: (A) Comparison of DNA methylation status of SOX1 and PAX1 in 166 CRC tissues and 37 normal tissues. P<0.05 was considered significant. (B) DNA methylation status of SOX1 and PAX1 in 37 CRC tissues and paired normal tissues. (C and D) Kaplan–Meier estimation of overall survival according to PAX1 and SOX1 methylation status in 166 patients. Median methylation status of each gene was defined as cutoff. *P<0.05 was calculated by independent sample t-test. **P<0.05 was calculated by Wilcoxon signed-rank test. AUC data shown as value (specificity sensitivity).
Abbreviations: CRC, colorectal cancer; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; TCGA, The Cancer Genome Atlas.

Figure 2 DNA methylation status of PAX1 and SOX1 genes in CRC tissues and paired normal tissues from 41 patients with CRC.

Notes: Comparison of DNA methylation status log (meth-index) of PAX1 (A) and SOX1 (B) genes in 41 CRC tissues and paired normal tissues. The AUC of the ROC curve was calculated to diagnose the CRC and normal tissues for PAX1 (C) and SOX1 (D). P-value was calculated by Wilcoxon signed-rank test.
Abbreviations: AUC, areas under the curve; CRC, colorectal cancer; PAX1, paired boxed gene 1; ROC, receiver-operating characteristic; SOX1, sex-determining region Y-related high-mobility group box 1.

Table 1 Association of the frequency of methylation occurrence of PAX1 and SOX1 in cancer tissues to the clinical characteristics of 41 patients with CRC

Characteristics	N	SOX1				PAX1
		M	U	Methylation percentage	P-value*	M	U	Methylation percentage	P-value*
Age, years					0.460				0.460
Mean (SD) [range]	56	(12.65)	(27–84)
<60	25	16	9	64.00		16	9	64.00
>60	16	12	4	75.00		12	4	75.00
Gender					0.658				0.025
Male	20	13	7	65.00		17	3	85.00
Female	21	15	6	71.43		11	10	52.38
Tumor location					0.987				0.987
Colon	19	13	6	68.42		13	6	68.42
Rectum	22	15	7	68.18		15	7	68.18
LN metastasis					0.203				0.072
N0	29	19	10	65.52		18	11	62.07
N1	11	9	2	81.82		10	1	90.91
N2	1	0	1	0.00		0	1	0.00
Tumor invasion					0.548				0.661
T1	3	3	0	100.00		3	0	100.00
T2	12	8	4	66.67		8	4	66.67
T3	5	4	1	80.00		3	2	60.00
T4	21	13	8	61.90		14	7	66.67
TNM stage					0.666				0.646
I	9	7	2	77.78		7	2	77.78
II	18	9	9	50.00		9	9	50.00
III	12	10	2	83.33		10	2	83.33
IV	2	2	0	100.00		2	0	100.00
Tumor HG					0.432				0.432
Poor	7	6	1	85.71		6	1	85.71
Moderate	20	12	8	60.00		12	8	60.00
Well	14	10	4	71.43		10	4	71.43
Tumor size					0.063				0.128
<3 cm	3	0	3	0.00		1	2	33.33
3–5 cm	9	6	3	66.67		4	5	44.44
>5 cm	5	4	1	80.00		4	1	80.00
NA	24	18	6	75.00		19	5	79.17

Note:

* Calculated by χ² test.

Abbreviations: CRC, colorectal cancer; HG, histological grade; LN, lymph node; M, methylated; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; U, unmethylated.

Table 2 Sensitivities, specificities, and AUC of PAX1 and SOX1 methylation in detecting CRC tumor (n=41)

Detection modality	Cancer		Paracancer		Sensitivity %	Specificity %	AUC	95% CI	P-value*
	U	M	U	M
PAX1	13	28	36	5	68.3	87.8	0.743	0.627–0.858	<0.001
SOX1	13	28	41	0	68.3	100.0	0.798	0.689–0.907	<0.001

Note:

* Calculated by χ² test.

Abbreviations: AUC, areas under the curve; CRC, colorectal cancer; M, methylated; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; U, unmethylated.

Table 4 Correlation between methylation levels of PAX1 and SOX1 in the cancer tissues and clinical characteristics of 41 patients with CRC

Characteristics	PAX1 methylation level		SOX1 methylation level
	Mean ± SD	P-value*	Mean ± SD	P-value*
Age, years		0.621		0.779
<60	1.59±3.10		1.94±2.83
>60	2.37±2.53		2.46±2.69
Gender		0.050		0.531
Male	2.92±2.07		2.00±2.79
Female	0.92±3.24		2.28±2.78
Tumor location		0.296		0.143
Colon	2.17±2.77		2.33±2.66
Rectum	1.66±3.02		1.98±2.89
TNM stage		0.007		0.046
I/II	1.26±2.94		1.77±2.88
III/IV	3.11±2.43		2.87±2.43
Tumor size		0.399		0.792
<5 cm	0.19±3.37		1.09±3.14
>5 cm	2.60±2.90		2.57±2.84
Tumor HG		0.273		0.080
W/M	1.52±2.99		1.86±2.82
Poor	3.39±1.93		3.21±2.41
Tumor invasion		0.665		0.807
T1/2	1.92±2.85		2.41±2.70
T3/4	1.89±2.96		1.99±2.83
LN metastasis		0.071		0.192
N0	1.46±2.93		1.94±2.85
N1/2/3	2.93±2.60		2.65±2.56

Notes:

* Association analysis for methylation status by Mann–Whitney U-test. Significant values <0.05 were shown in bold font.

Abbreviations: CRC, colorectal carcinoma; LN, lymph node; HG, histological grade; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; W/M, well/moderate.

Table 3 Sensitivities, specificities, and AUC of PAX1 and SOX1 methylation in detecting low (n=27) and high (n=14) TNM stages

Detection modality	Sensitivity %	Specificity %	AUC	95% CI	P-value*
PAX1	0.714	0.704	0.759	0.588–0.930	0.007
SOX1	0.714	0.630	0.692	0.515–0.869	0.046

Note:

* Calculated by χ² test.

Abbreviations: AUC, areas under the curve; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1.

Figure 3 Methylation distribution of CRC tissues at various TNM stages.

Notes: (A) Dot plots illustrate the log (meth-index) distributions of PAX1 gene at various TNM stages. (B) The AUC of ROC curve was calculated to diagnose the TNM stage I/II and stage III/IV for PAX1. (C) Dot plots illustrate the log (meth-index) distributions of SOX1 gene at various TNM stages. (D) The AUC of ROC curve was calculated to diagnose the TNM stage I/II and stage III/IV for SOX1 gene. P<0.05 was determined by Mann–Whitney U-test. Arrows show the best specificity and sensitivity.
Abbreviations: AUC, areas under the curve; CRC, colorectal cancer; PAX1, paired boxed gene 1; ROC, receiver-operating characteristic; SOX1, sex-determining region Y-related high-mobility group box 1.

Figure 4 mRNA expression of SOX1/PAX1 genes in CRC tissues of 41 patients with CRC.

Notes: (A) Relative SOX1 and (B) PAX1 mRNA expression levels ± SEM in CRC tissues and paired normal tissues. Pearson’s correlation was used to measure the linear relationship between mRNA expression (x-axis) and methylation level log (meth-index) (y-axis) of (C) SOX1 and (D) PAX1 genes in 41 CRC tissues. P-values were performed by t-test.
Abbreviations: CRC, colorectal cancer; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1.

Figure 5 Representative expression of PAX1 and SOX1 protein in colorectal tissues compared with paired normal tissues.

Notes: (A) IHC staining of SOX1 protein expression in normal tissue samples. (B) IHC staining of SOX1 protein expression in CRC tissue samples. (C) IHC staining of PAX1 protein expression in normal tissue samples. (D) IHC staining of PAX1 protein expression in CRC tissue samples (×200).
Abbreviations: CRC, colorectal cancer; IHC, immunohistochemical; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1.

Figure S1 Correlation between the methylation status of SOX1 and PAX1 in the same CRC samples.

Notes: Pearson’s correlation was used to measure the linear relationship between methylation log (meth-index) of PAX1 (x-axis) and SOX1 (y-axis) in 41 CRC tissues (A) and 166 CRCs downloaded from TCGA dataset (B). P-values were performed by independent sample t-test.

Abbreviations: CRC, colorectal cancer; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; TCGA, The Cancer Genome Atlas.

Table S1 Association of the frequency of methylation occurrence of cg20907471 (PAX1) and average of cg22303211 and cg06675478 (SOX1) in cancer tissues to the clinical characteristics of 166 patients with CRC using TCGA datasets (n=166)

Characteristics	N	SOX1				PAX1
		M	U	Methylated percentage	P-value	M	U	Methylated percentage	P-value*
Age, years					0.239				0.560
Mean (SD) [range]	70	(11.9)	(36–90)
<50	15	7	8	46.67		3	12	20.00
50–70	61	42	19	68.85		21	40	34.43
>70	90	54	36	60.00		29	61	32.22
Gender					0.631				0.617
Male	83	53	30	63.86		28	55	33.73
Female	83	50	33	60.24		25	58	30.12
M					0.576				0.191
M0	142	88	54	61.97		43	99	30.28
M1	24	15	9	62.50		19	14	79.17
N					0.329				0.209
N0	99	65	34	65.66		27	72	27.27
N1	32	20	12	62.50		14	18	43.75
N2	34	18	17	52.94		12	23	35.29
T					0.310				0.834
T1	4	1	3	25.00		2	2	50.00
T2	35	24	11	68.57		10	25	28.57
T3	110	69	41	62.73		36	74	32.73
T4	17	9	8	52.94		5	12	29.41
Stage					0.666				0.646
I	32	22	10	68.75		9	23	28.13
II	64	41	23	64.06		18	46	28.13
III	45	25	20	55.56		16	29	35.56
IV	24	15	10	62.50		10	15	41.67
CEA					0.338				0.887
<9.7	78	46	32	58.97		26	52	33.33
>9.7	24	13	11	54.17		8	16	33.33
NA	64	44	20	68.75		19	45	29.69

Note:

* Calculated χ² test.

Abbreviations: CEA, carcinoembryonic antigen; CRC, colorectal cancer; M, methylated; PAX1, paired boxed gene 1; SOX1, sex-determining region Y-related high-mobility group box 1; TCGA, The Cancer Genome Atlas; U, unmethylated.