Nicotinic cholinergic receptors in esophagus: Early alteration during carcinogenesis and prognostic value

Table 1 Sequences of the oligonucleotides used in reverse transcription-quantitative PCR

Gene	Oligonucleotide sequences (5'-3')	Positive control	Ref.
CHRNA1	Forward: ACCAGGAGTCTAACAATGCG	Glioblastoma cell line, U251	Designed by the authors
	Reverse: ACAAGCATGAAGACTCCGAG
CHRNA3	Forward: AACCTGTGGCTCAAGCAAATCT	Lung cancer cell line, A549	[20]
	Reverse: CATGAACTCTGCCCCACCAT
CHRNA4	Forward: ACACAGACTTCTCGGTGAAG	ESCC cell line, TE1	Designed by the authors
	Reverse: CAGCAGGCAGACGATGATGA
CHRNA5	Forward: AGATGGAACCCTGATGACTATGGT	Lung cancer cell line, A549	[20]
	Reverse: AAACGTCCATCTGCATTATCAAAC
CHRNA7	Forward: GCTGCTCGTGGCTGAGATC	Lung cancer cell line, A549	[20]
	Reverse: TGGCGAAGTACTGGGCTATCA
CHRNA9	Forward: GATGGCCTAGACTCCATCAG	Glioblastoma cell line, U251	Designed by the authors
	Reverse: CTGAAGATTCATCATCAGCCTTG
CHRNA10	Forward: CTACTCCCTGCAGAGTGCCTG	Glioblastoma cell line, U251	Designed by the authors
	Reverse: TCTGGTCTGTGTCTGCCACAG
CHRNB4	Forward: TCACAGCTCATCTCCATCAAGCT	Lung cancer cell line, A549	[20]
	Reverse: CCTGTTTCAGCCAGACATTGGT
GAPDH	Forward: CAACAGCCTCAAGATCATCAGCAA	-	[22]
	Reverse: AGTGATGGCATGGACTGTGGTCAT

ESCC: Esophageal squamous cell carcinoma.

Table 2 Characterization of the healthy individuals and esophageal squamous cell carcinoma patients

Evaluated criteria	Number of individuals (range or %)
	Healthy individuals,n = 44	ESCC patients,n = 28
Age (yr)	56.50 (18-85)	59.50 (46-79)
Sex
Men	13 (29.5)	18 (64.3)
Women	31 (70.5)	10 (35.7)
Smoking
Never	27 (61.4)	1 (3.6)
Ever	17 (38.6)	27 (96.4)
Alcohol consumption
Never	20 (45.5)	2 (7.1)
Ever	24 (54.5)	26 (92.9)
Death
No	NA	8 (28.6)
Yes	NA	20 (71.4)
Tumor localization
Upper third	NA	9 (32.1)
Middle third	NA	15 (53.6)
Lower third	NA	3 (10.7)
Histological grade, differentiation
Poor	NA	6 (21.4)
Moderate	NA	21 (75.0)
Well	NA	1 (3.6)
Tumor stage
1 + 2	NA	7 (25.0)
3 + 4	NA	15 (53.6)
Not determined	NA	6 (21.4)

One patient had the tumor located in the middle/lower third of the esophagus (3.6%). NA: Not applicable.

Table 3 Association of CHRN expression with characteristics of healthy individuals

Variable	CHRNA3		CHRNA5		CHRNA7		CHRNB4
	Expression,	P value	Expression,	P value	Expression,	P value	Expression,	P value
	median (min-max)		median (min-max)		median (min-max)		median (min-max)
Esophageal thirds
Upper	1.563 × 10-5	0.769	8.431 × 10-5	0.945	1.023 × 10-5	0.789	9.027 × 10-6	0.153
	(2.735 × 10-6 - 7.183 × 10-5)		(1.277 × 10-5 - 2.157 × 10-3)		(1.033 × 10-6 - 5.616 × 10-5)		(2.534 × 10-6 - 2.828 × 10-5)
Middle	1.783 × 10-5		8.476 × 10-5		1.346 × 10-5		1.077 × 10-5
	(1.512 × 10-6 - 1.196 × 10-4)		(1.286 × 10-5 - 2.388 × 10-3)		(2.588 × 10-6 - 6.319 × 10-5)		(3.913 × 10-6 - 3.259 × 10-5)
Lower	1.721 × 10-5		8.906 × 10-5		1.263 × 10-5		1.166 × 10-5
	(2.914 × 10-6 - 6.796 × 10-5)		(1.442 × 10-5 - 1.990 × 10-3)		(1.678 × 10-6 - 5.636 × 10-5)		(3.621 × 10-6 - 4.453 × 10-5)
Age, yr	58		56.5		57		57
> median	2.226 × 10-5	0.422	4.982 × 10-5	0.097	1.189 × 10-5	0.272	1.060 × 10-5	0.778
	(1.512 × 10-6 - 1.019 × 10-4)		(1.286 × 10-5 - 2.388 × 10-3)		(2.588 × 10-6 - 4.391 × 10-5)		(3.913 × 10-6 - 3.192 × 10-5)
≤ median	1.274 × 10-5		9.054 × 10-5		1.449 × 10-5		1.094 × 10-5
	(3.977 × 10-6 - 1.196 × 10-4)		(2.657 × 10-5 - 4.635 × 10-4)		(2.931 × 10-6 - 6.319 × 10-5)		(5.010 × 10-6 - 3.259 × 10-5)
Sex
Male	1.344 × 10-5	0.253	1.104 × 10-4	0.243	1.316 × 10-5	0.807	1.772 × 10-5	0.372
	(3.977 × 10-6 - 5.947 × 10-5)		(2.657 × 10-5 - 3.351 × 10-4)		(2.931 × 10-6 - 4.060 × 10-5)		(5.559 × 10-6 - 2.917 × 10-5)
Female	1.812 × 10-5		7.842 × 10-5		1.375 × 10-5		1.034 × 10-5
	(1.512 × 10-6 - 1.196 × 10-4)		(1.286 × 10-5 - 2.388 × 10-3)		(2.588 × 10-6 - 6.319 × 10-5)		(3.913 × 10-6 - 3.259 × 10-5)
Smoking
Never	1.665 × 10-5	0.400	8.531 × 10-5	0.961	1.210 × 10-5	0.582	1.077 × 10-5	0.599
	(1.512 × 10-6 - 1.019 × 10-4)		(2.051 × 10-5 - 2.388 × 10-3)		(2.588 × 10-6 - 6.319 × 10-5)		(3.913 × 10-6 - 3.192 × 10-5)
Ever	1.948 × 10-5		8.405 × 10-5		1.375 × 10-5		1.078 × 10-5
	(5.157 × 10-6 - 1.196 × 10-4)		(1.286 × 10-5 - 3.351 × 10-4)		(2.931 × 10-6 - 4.256 × 10-5)		(5.010 × 10-6 - 3.259 × 10-5)
Alcohol consumption
Never	2.386 × 10-5	0.163	5.955 × 10-5	0.131	1.046 × 10-5	0.412	1.094 × 10-5	0.521
	(1.512 × 10-6 - 1.196 × 10-4)		(2.051 × 10-5 - 3.525 × 10-4)		(2.588 × 10-6 - 4.256 × 10-5)		(3.913 × 10-6 - 3.259 × 10-5)
Ever	1.274 × 10-5		1.018 × 10-4		1.375 × 10-5		1.007 × 10-5
	(3.977 × 10-6 - 8.783 × 10-5)		(1.286 × 10-5 - 2.388 × 10-3)		(2.931 × 10-6 - 6.319 × 10-5)		(5.010 × 10-6 - 2.917 × 10-5)

Statistical analyses were performed using the Mann-Whitney or Kruskal Wallis test and Dunn's post-test as appropriate; no significant differences were observed.

Table 4 Correlation matrices between the mRNA expression of CHRN subunits in healthy esophagus and esophageal squamous cell carcinoma tissues

		CHRNA3	CHRNA5	CHRNA7	CHRNB4
Healthy esophagus	CHRNA3		P = 0.820	P = 0.203	P = 0.007a
	CHRNA5	r = -0.040		P = 0.727	P = 0.672
	CHRNA7	r = 0.220	r = 0.060		P = 0.193
	CHRNB4	r = 0.470	r = -0.079	r = 0.240
Normal surrounding mucosa	CHRNA3		P = 0.486	P = 0.753	P = 0.006a
	CHRNA5	r = 0.170		P = 0.847	P = 0.013a
	CHRNA7	r = -0.077	r = -0.038		P = 0.185
	CHRNB4	r = 0.607	r = 0.556	r = -0.318
Tumor Tissue	CHRNA3		P = 0.450	P = 0.026a	P = 0.016a
	CHRNA5	r = 0.184		P = 0.130	P = 0.071
	CHRNA7	r = 0.511	r = 0.293		P = 0.012a
	CHRNB4	r = 0.544	r = 0.423	r = 0.561

Matrices show the P values and correlation coefficients between the mRNA expression of the different CHRN subunits in the healthy esophagus, in the surrounding mucosa of esophageal squamous cell carcinoma (ESCC) patients and in ESCC.

^aP < 0.05.

Table 5 Association of CHRN expression fold-change and clinicopathological data of esophageal squamous cell carcinoma patients

Clinicopathologicaldata	CHRNA3		CHRNA5		CHRNA7		CHRNB4
	FC,	P value	FC,	P value	FC,	P value	FC,	P value
	median		median		median		median
	(min-max)		(min-max)		(min-max)		(min-max)
Age, yr	60		59.5		59.5		60
	(46-79)		(46-79)		(46-79)		(46-79)
> median	1.1	0.2775	2.166	0.1612	2.867	0.9451	1.984	0.1128
	(0.03136-14.69)		(0.3918-15.07)		(0.2630-20.53)		(0.1586-7.119)
≤ median	0.5844		3.193		1.267		0.7518
	(0.006324-10.62)		(0.8615-11.66)		(0.4813-23.10)		(0.1414-2.051)
Sex
Men	0.9777	0.1053	3.032	0.1719	2.867	0.7192	1.685	0.1053
	(0.006324-14.69)		(0.3918-15.07)		(0.4813-23.10)		(0.2261-7.119)
Women	0.1153		2.102		1.657		0.3153
	(0.03349-1.072 )		(0.5510-3.593)		(0.2630-20.53)		(0.1414-2.809)
Smoking
Never	NA	NA	NA	NA	NA	NA	NA	NA
Ever	0.6941		3.01		1.86		1.229
	(0.006324-14.69)		(0.3918 - 15.07)		(0.2630-23.10)		(0.1414-7.119)
Alcohol consumption
Never	NA	NA	NA	NA	NA	NA	NA	NA
Ever	0.5844		2.971		1.657		0.9571
	(0.006324-14.69)		(0.3918-15.07)		(0.2630-23.10)		(0.1414-7.119)
Tumor localization1
Upper third	0.1153	0.4079	1.589	0.0737	0.8255	0.1525	0.3964	0.3510
	(0.03136-14.69)		(0.3918- 6.284)		(0.2630-7.917)		(0.1414-7.119)
Middle third	0.8265		3.01		2.567		1.5
	(0.006324-10.62)		(0.8615-11.66)		(0.4813-23.10)		(0.2261-2.809)
Lower third	3.904		9.329		4.199		2.211
	(0.07381-7.799)		(3.414-15.07)		(0.7220-13.36)		(1.613-2.552)
Histological grade, differentiation
Well	NA	0.4902	NA	0.9767	NA	0.3661	NA	0.8610
Moderate	0.8039		3.031		1.454		0.9571
	(0.03136-14.69)		(0.3918-15.07)		(0.2630-23.10)		(0.1414-7.119)
Poor	0.2908		2.6		2.514		1.5
	(0.006324-1.626)		(1.826-4.993)		(1.057-18.57)		(0.5035-2.532)
Tumor stage
1 + 2	0.5728	0.9599	2.505	0.778	1.454	0.7780	1.832	0.4457
	(0.03349-10.62)		(1-15.07)		(0.4813-20.53)		(0.1414-2.809)
3 + 4	0.8039		3.01		3.167		0.9571
	(0.006324-7.799)		(0.4633-6.566)		(0.6082-23.10)		(0.2261-2.532)

¹The analyses were performed considering only the upper and middle thirds because the number of individuals with ESCC in the lower third was too small. Statistical analyses were performed using Mann-Whitney or Kruskal Wallis test and Dunn's post-test, as appropriate; no significant differences were observed. NA: Not applicable because the number of individuals was too small.