Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion, migration, and angiogenesis in colorectal cancer

Table 1 Gene sequences used for transfection

Gene	Sequences
STAT3-siRNA	siRNA-S1-a	5’-CCG GGC TGA CCA ACA ATC CCA AGA ACT CGA GTT CTT GGG ATT GTT GGT CAG CTT TTT G-3’
	siRNA-S1-b	5’-AAT TCA AAA AGC TGA CCA ACA ATC CCA AGA ACT CGA GTT CTT GGG ATT GTT GGT CAG C-3’
	siRNA-S2-a	5’-CCG GGC ACA ATC TAC GAA GAA TCA ACT CGA GTT GAT TCT TCG TAG ATT GTG CTT TTT G-3’
	siRNA-S2-b	5’- AAT TCA AAA AGC ACA ATC TAC GAA GAA TCA ACT CGA GTT GAT TCT TCG TAG ATT GTG C-3’
	siRNA-S3-a	5’- CCG GGC TGA AAT CAT CAT GGG CTA TCT CGA GAT AGC CCA TGA TGA TTT CAG CTT TTT G-3’
	siRNA-S3-b	5’- AAT TCA AAA AGC TGA AAT CAT CAT GGG CTA TCT CGA GAT AGC CCA TGA TGA TTT CAG C-3’
VASH1-siRNA	siRNA-V1-a	5’-CCG GCT GCC AAT CAA ATG CCT GGA ACT CGA GTT CCA GGC ATT TGA TTG GCA GTT TTT G -3’
	siRNA-V1-b	5’-AAT TCA AAA ACT GCC AAT CAA ATG CCT GGA ACT CGA GTT CCA GGC ATT TGA TTG GCA G -3’
	siRNA-V2-a	5’-CCG GGA GCT GCA GTA CAA TCA CAC ACT CGA GTG TGT GAT TGT ACT GCA GCT CTT TTT G-3’
	siRNA-V2-b	5’-AAT TCA AAA AGA GCT GCA GTA CAA TCA CAC ACT CGA GTG TGT GAT TGT ACT GCA GCT C-3’
	siRNA-V3-a	5’-CCG GCA CAG GAC ATA GTG GTG CTT TCT CGA GAA AGC ACC ACT ATG TCC TGT GTT TTT G-3’
	siRNA-V3-b	5’-AAT TCA AAA ACA CAG GAC ATA GTG GTG CTT TCT CGA GAA AGC ACC ACT ATG TCC TGT G -3’

STAT3: Signal transducer and activator of transcription 3; VASH1: Vasohibin 1.

Table 2 Baseline characteristics of patients with advanced colorectal cancer and the correlation with immunohistochemical expression of ELL-associated factor 2 and vasohibin 1

Clinicopathologic characteristics	Cases, n (%)	EAF2		P value	VASH1		P value
		Low (n = 100)	High (n = 20)		Low (n = 36)	High (n = 84)
Age (yr)				0.569			0.248
< 61 (median)	44.17	43	10		13	40
≥ 61	55.83	57	10		23	44
Gender				0.935			0.843
Male	54.17	54	11		20	45
Female	45.83	46	9		16	39
Tumor site				0.657			0.515
Colon	29.17	30	5		9	26
Rectum	70.83	70	15		27	58
Size of the tumor, cm				0.250			0.689
≤ 4	41.67	44	6		16	34
> 4	58.33	56	14		20	50
Histological type				0.695			0.566
Mucinous	21.67	21	5		9	17
Non-mucinous	78.33	79	15		27	67
Degree of differentiation				0.013			0.042
Well/moderately differentiated	64.17	69	8		28	49
Poor/mucinous differentiated	35.83	31	12		8	35
Angiolymphatic and/or perineural invasion				0.049			0.006
Absent	45.00	41	13		23	31
Present	55.00	59	7		13	53
TNM				0.034			0.068
I-II	48.33	44	14		22	36
III-IV	51.67	56	6		14	48
Tumor invasion depth				0.219			0.084
T2-T3	57.50	55	14		25	44
T4	42.50	45	6		11	40
Lymph node status				0.073			0.095
N0	46.67	43	13		21	35
N1-N2	53.33	57	7		15	49
Distant metastasis				0.007			0.037
Absent	70.00	65	19		30	54
Present	30.00	35	1		6	30
CEA				0.021			0.874
Normal	56.67	52	16		20	48
High	43.33	48	4		16	36
CA19-9				0.321			0.809
Normal	40.00	38	10		15	33
High	60.00	62	10		21	51
P53				0.219			0.905
Normal	42.50	40	11		15	36
High	57.50	60	9		21	48
CDX2				0.224			0.921
Normal	20.00	18	6		7	17
High	80.00	82	14		29	67

EAF2: ELL protein-associated factor 2; TNM: Tumor-node-metastasis; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; VASH1: Vasohibin 1.

Table 3 Univariate and multivariate survival analysis of overall survival for colorectal cancer by Cox regression.

Variables	Univariate analysis			Multivariate analysis
	HR	95%CI	P value	HR	95%CI	P value
Age (yr) (≥ 61/< 61)	1.703	0.969-2.991	0.064	1.413	0.764-2.613	0.270
Gender (female/male)	0.962	0.561-1.651	0.890
Tumor site (colon/rectum)	1.410	0.798-2.492	0.237	1.211	0.638-2.298	0.558
Size of the tumor, cm (> 4/≤ 4)	0.777	0.446-1.356	0.375
Histological type (mucinous/non-mucinous)	1.218	0.612-2.424	0.574
Degree of differentiation (poor/mucinous, well/moderate)	0.917	0.523-1.608	0.761
Angiolymphatic and/or perineural invasion (present/absent)	0.209	0.107-0.408	< 0.001	0.255	0.072-0.898	0.033
Tumor stage (III-IV/I-II)	0.240	0.128-0.450	< 0.001	1.455	0.339-6.256	0.614
Tumor invasion depth (T4/T2-T3)	0.353	0.202-0.614	< 0.001	0.547	0.225-1.331	0.184
Lymph node status (N1-N2/N0)	0.288	0.155-0.532	< 0.001	2.150	0.511-9.038	0.296
Distant metastasis (present/absent)	0.161	0.091-0.284	< 0.001	0.200	0.090-0.444	< 0.001
EAF2 (low/high)	3.244	1.170-8.999	0.024	2.049	0.698-6.014	0.191
VASH1 (high/low)	0.518	0.266-1.007	0.052	0.785	0.389-1.584	0.499
CEA (high/low)	1.590	0.906-2.790	0.106	2.506	1.336-4.702	0.004
CA19-9 (high/low)	0.817	0.466-1.433	0.481
P53 (high/low)	0.558	0.313-0.994	0.048	0.545	0.299-0.995	0.048
CDX2 (high/low)	0.705	0.332-1.495	0.362

HR: Hazard ratio; CI: Confidence interval; EAF2: ELL protein-associated factor 2; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9.