Role of the Na+/K+/2Cl- cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma

Table 1 Correlations between clinicopathological parameters and Na⁺/K⁺/2Cl^- cotransporter 1 expression

Variable		NKCC1 expression		P value
		Low grade	High grade
Age (yr)	< 60	12	10	0.1874
	≥ 60	16	30
Gender	Male	25	32	0.5049
	Female	3	8
Location of tumor	Ce/Ut	4	3	0.4346
	Mt/Lt/Ae	24	37
Tumor size (mm)	< 50	18	30	0.4206
	≥ 50	10	10
Histological type	Differentiated type SCC	25	21	0.0015a
	Poorly differentiated type SCC	3	19
pT	pT1	10	21	0.2191
	pT2-3	18	19
pN	negative	13	20	0.8095
	positive	15	20
pStage	I	6	16	0.1231
	II-III	22	24
Ki-67 labeling index		28.7 ± 2.3	29.9 ± 2.0	0.6834

Ce: Cervical esophagus; Ut: Upper thoracic esophagus; Mt: Middle thoracic esophagus; Lt: Lower thoracic esophagus; Ae: Abdominal esophagus; pT: Pathological T stage; pN: Pathological N stage; pStage: Pathological stage; SCC: Squamous cell carcinoma;

^aP < 0.05 vs control, Fisher’s exact test.

Table 2 Twenty genes displaying the highest change in expression levels in Na⁺/K⁺/2Cl^- cotransporter 1 depleted KYSE170 cells

Gene Symbol	Gene ID	Gene Name	Fold Change
Upregulated Genes
C18orf34	NM_001105528	Chromosome 18 open reading frame 34	155.49
KCNA6	NM_002235	Potassium voltage-gated channel, shaker-related subfamily, member 6	140.4
CCDC147	NM_001008723	Coiled-coil domain containing 147	105.98
C20orf202	NM_001009612	Chromosome 20 open reading frame 202	86.17
A1CF	NM_138933	APOBEC1 complementation factor	70.98
SH3GL2	NM_003026	SH3-domain GRB2-like 2	70.93
PTGFR	NM_001039585	Prostaglandin F receptor (FP)	66.99
NDN	NM_002487	Necdin homolog (mouse)	66.45
INPP5D	NM_001017915	Inositol polyphosphate-5-phosphatase, 145 kDa	52.83
CYP2E1	NM_000773	Cytochrome P450, family 2, subfamily E, polypeptide 1	52.44
AGBL3	NM_178563	ATP/GTP binding protein-like 3	50.88
UBTFL1	NM_001143975	Upstream binding transcription factor, RNA polymerase I-like 1	47.88
PADI2	NM_007365	Peptidyl arginine deiminase, type II	46.83
CCR1	NM_001295	Chemokine (C-C motif) receptor 1	44.86
ARC	NM_015193	Activity-regulated cytoskeleton-associated protein	44.41
COLEC10	NM_006438	Homo sapiens collectin sub-family member 10 (C-type lectin)	44.28
DNAH6	NM_001370	Dynein, axonemal, heavy chain 6	41.96
BOLL	NM_033030	Bol, boule-like (Drosophila)	41.31
CORO2B	NM_006091	Coronin, actin binding protein, 2B	41.04
MUC7	NM_152291	Mucin 7, secreted	36.97
Downregulated Genes
NPFFR1	NM_022146	Neuropeptide FF receptor 1	-54.97
LRRFIP1	NM_001137550	Leucine rich repeat (in FLII) interacting protein 1	-44.72
PPIL6	NM_173672	Peptidylprolyl isomerase (cyclophilin)-like 6	-44.46
CRHR2	NM_001883	Corticotropin releasing hormone receptor 2	-39.78
CMTM2	NM_144673	CKLF-like MARVEL transmembrane domain containing 2	-39.62
C5	NM_001735	Complement component 5	-39.13
KCNMA1	NM_001014797	Potassium large conductance calcium-activated channel, subfamily M, alpha member 1	-38.59
HESX1	NM_003865	HESX homeobox 1	-33.03
SLC22A2	NM_003058	Solute carrier family 22 (organic cation transporter), member 2	-32.49
WNT8B	NM_003393	Wingless-type MMTV integration site family, member 8B	-32.17
GRIA1	NM_000827	Glutamate receptor, ionotropic, AMPA 1	-31.27
ZNF367	NM_153695	Zinc finger protein 367	-30.04
GPR128	NM_032787	G protein-coupled receptor 128	-29.88
SLC12A2	NM_001046	Solute carrier family 12 (sodium/potassium/chloride transporters), member 2	-28.92
KCNG2	NM_012283	Potassium voltage-gated channel, subfamily G, member 2	-28.3
ECT2L	NM_001077706	Epithelial cell transforming sequence 2 oncogene-like	-27
ERMN	NM_020711	Ermin, ERM-like protein	-26.61
DPP10	NM_020868	Dipeptidyl-peptidase 10 (non-functional)	-26.58
TSPAN7	NM_004615	Tetraspanin 7	-25.54
APOA1	NM_000039	Apolipoprotein A-I	-25.21

Table 3 Top biological functions, canonical pathways, and networks of Na⁺/K⁺/2Cl^- cotransporter 1 according to Ingenuity Pathway Analysis

Top Biological Functions
Diseases and disorders
Name	P value	Number of molecules
Cancer	2.08E-12 - 1.59E-02	277
Gastrointestinal disease	8.03E-12 - 1.60E-02	149
Reproductive system disease	2.25E-09 - 1.57E-02	138
Hematological disease	1.72E-06 - 1.22E-02	70
Hereditary disorder	2.10E-06 - 1.57E-02	120
Molecular and cellular functions
Name	P value	Number of molecules
Cell cycle	1.06E-20 - 1.60E-02	158
Cellular assembly and organization	1.06E-20 - 1.36E-02	111
DNA replication, recombination, and repair	1.06E-20 - 1.16E-02	133
Cellular movement	8.36E-11 - 1.51E-02	82
Cell death	2.98E-06 - 1.56E-02	216
Top canonical pathways
Name	P value	Ratio
Role of BRCA1 in the DNA damage response	9.79E-6	12/65 (0.185)
Mitotic roles of Polo-Like kinase	7.37E-5	11/69 (0.159)
Estrogen-mediated S-phase entry	4.91E-4	6/28 (0.214)
Cell Cycle: G2/M DNA damage checkpoint regulation	5.07E-4	8/49 (0.163)
Role of CHK proteins in cell cycle checkpoint control	5.37E-4	9/56 (0.161)
Top networks
Associated network functions	Score
Cellular assembly and organization; DNA replication, recombination, and repair; Cell cycle	47
Cellular assembly and organization, Cell cycle, DNA replication, recombination, and repair	43
Cell cycle; DNA replication, recombination, and repair; Cancer	37
Digestive system development and function, organismal injury and abnormalities, cellular function and maintenance	37
Cellular assembly and organization; DNA replication, recombination, and repair; Cardiovascular disease	35

Table 4 Cell growth-related genes with expression levels in KYSE170 cells that were changed by the depletion of Na⁺/K⁺/2Cl^- cotransporter 1

Gene symbol	Gene ID	Biological functions		Fold change
		Cell growth and proliferation	Cell cycle
Upregulated genes
NDN	NM_002487	●		66.45
INPP5D	NM_001017915	●	●	52.83
CCR1	NM_001295	●		44.86
COL1A2	NM_000089	●		36.72
EDAR	NM_022336		●	35.86
RBP4	NM_006744	●		30.40
DCLK1	NM_004734		●	28.95
RARRES1	NM_002888	●		22.92
FMOD	NM_002023	●		22.85
BARX1	NM_021570	●		21.47
MAPK10	NM_138980	●		20.00
ADORA2A	NM_000675	●		18.78
CHRNA7	NM_001190455	●	●	18.47
SOX10	NM_006941	●		18.03
FGF20	NM_019851	●		16.73
FAM5C	NM_199051	●	●	14.35
MBD2	NM_015832	●		12.13
EGF	NM_001963	●	●	11.50
TLR5	NM_003268	●		11.35
TNN	NM_022093	●		10.98
SLC1A2	NM_004171	●		10.74
CD36	NM_001001547	●		10.43
CD52	NM_001803	●		10.34
NR2E3	NM_016346	●		10.31
PLCB1	NM_182734		●	10.26
MYCN	NM_005378	●	●	10.23
ZNF365	NM_199451		●	10.12
ERG	NM_004449	●		10.04
MSH4	NM_002440		●	10.03
DMRT1	NM_021951		●	9.61
RNF128	NM_194463	●		9.37
CD69	NM_001781	●		8.89
PDE3A	NM_000921	●	●	8.58
ACVR1C	NM_145259	●		8.57
SPI1	NM_001080547	●	●	8.55
SH2D3C	NM_170600	●		8.53
IFNG	NM_000619	●	●	8.22
MRAS	NM_012219	●		8.20
MCF2L	NM_024979	●		7.43
RRAD	NM_004165	●	●	7.42
E2F5	NM_001951	●	●	7.32
BGN	NM_001711	●		7.13
KIFC1	NM_002263		●	7.02
ABCC6	NM_001171			6.98
SERPINE1	NM_000602	●		6.74
CIITA	NM_000246	●		6.74
GJB6	NM_006783	●		6.45
TP53INP1	NM_033285	●	●	6.38
GHRL	NM_016362	●		6.30
CCNG2	NM_004354	●	●	6.29
RORC	NM_005060	●		6.24
NCF1	NM_000265	●		6.24
NFATC4	NM_001136022	●		6.15
CHRM5	NM_012125	●		6.01
HMOX1	NM_002133	●	●	6.00
IL18RAP	NM_003853	●		5.98
C8orf4	NM_020130	●		5.95
L1CAM	NM_024003	●		5.87
TNFSF8	NM_001244	●		5.85
MSMB	NM_002443	●		5.77
ITPR1	NM_002222	●		5.77
ITGAL	NM_002209	●	●	5.73
INHBA	NM_002192	●	●	5.53
HEYL	NM_014571	●		5.38
JAK3	NM_000215	●	●	5.30
MMP13	NM_002427	●		5.23
NNMT	NM_006169	●		5.06
BNIPL	NM_138278	●		4.86
LTC4S	NM_145867		●	4.74
MMP24	NM_006690	●		4.72
MMP1	NM_002421	●		4.66
CD19	NM_001770	●	●	4.52
ADC	NM_052998	●		4.46
TGFBR1	NM_004612	●	●	4.33
RHOB	NM_004040	●		4.32
CDKN1C	NM_000076	●	●	4.30
HOXB13	NM_006361	●	●	4.20
IPMK	NM_152230	●		4.07
BMF	NM_001003940	●		4.06
VTCN1	NM_024626	●	●	4.05
CEACAM1	NM_001712	●		2.97
TSSK3	NM_052841		●	2.00
Downregulated genes
CRHR2	NM_001883	●		-39.78
C5	NM_001735	●	●	-39.13
KCNMA1	NM_001014797	●		-38.59
GRIA1	NM_000827	●		-31.27
SLC12A2	NM_001046	●		-28.92
APOA1	NM_000039	●		-25.21
PRKAR2B	NM_002736	●	●	-24.25
TF	NM_001063	●	●	-22.85
BRCA2	NM_000059	●	●	-22.04
AURKC	NM_001015878		●	-21.69
PLXNA4	NM_181775	●		-20.54
TYR	NM_000372	●		-20.39
BACH2	NM_021813	●		-20.01
KIF14	NM_014875		●	-18.06
HEY2	NM_012259	●		-18.01
TMPO	NM_003276	●	●	-15.97
FCGR3A	NM_000569	●		-15.18
ARF6	NM_001663			-15.09
MYBL1	NM_001080416	●	●	-14.60
CCNA1	NM_003914	●	●	-14.29
ESCO2	NM_001017420		●	-14.05
TOP2A	NM_001067	●	●	-13.91
CENPI	NM_006733		●	-13.87
ATAD2	NM_014109	●		-13.41
POSTN	NM_006475	●		-12.91
MKI67	NM_002417	●	●	-12.41
ABCB1	NM_000927	●	●	-12.15
KIF20B	NM_016195	●	●	-11.53
SPN	NM_001030288	●		-11.41
MAD2L1	NM_002358	●	●	-11.16
HLA-DPB1	NM_002121	●		-11.12
SGOL1	NM_001012410		●	-10.91
RRM2	NM_001034	●		-10.71
FANCD2	NM_033084	●	●	-10.55
FANCA	NM_001018112	●	●	-10.31
HDAC2	NM_001527	●	●	-9.94
NUF2	NM_145697		●	-9.76
CLSPN	NM_022111	●	●	-9.57
RAD54L	NM_003579		●	-9.47
KLHL13	NM_033495		●	-9.32
CCNA2	NM_001237	●	●	-9.13
MCM10	NM_182751	●	●	-9.11
MCTS1	NM_014060	●		-9.02
ANLN	NM_018685		●	-9.00
HMGB2	NM_002129	●	●	-8.86
VPREB1	NM_007128	●		-8.81
KIF4A	NM_012310		●	-8.78
SPC25	NM_020675		●	-8.75
ALOX5	NM_000698	●	●	-8.55
PBK	NM_018492	●		-8.20
TNFRSF11B	NM_002546	●		-8.20
CIT	NM_001206999		●	-8.17
HELLS	NM_018063	●	●	-8.1
CDC45	NM_003504	●	●	-8.07
DTL	NM_016448	●	●	-8.00
RGS3	NM_017790			-7.93
TYMS	NM_001071	●	●	-7.87
NDC80	NM_006101		●	-7.86
ERCC6L	NM_017669		●	-7.82
CENPE	NM_001813		●	-7.75
TTK	NM_003318	●	●	-7.74
SIM2	NM_009586		●	-7.61
KRT4	NM_002272	●		-7.55
RAD51AP1	NM_006479		●	-7.55
LTA	NM_000595	●		-7.51
PAK2	NM_002577	●	●	-7.50
SLC5A8	NM_145913	●		-7.41
BLM	NM_000057	●	●	-7.40
NUSAP1	NM_016359		●	-7.36
JDP2	NM_130469	●	●	-7.19
CASP3	NM_004346	●	●	-7.17
NEIL3	NM_018248	●		-7.17
POLH	NM_006502	●	●	-7.11
KIF20A	NM_005733	●	●	-7.08
MYO7A	NM_000260			-6.93
NRGN	NM_006176	●		-6.82
NCAPG	NM_022346	●	●	-6.78
CDCA8	NM_018101	●	●	-6.72
CEP55	NM_018131		●	-6.65
DLGAP5	NM_014750	●	●	-6.60
CDC25C	NM_001790	●	●	-6.59
ARL2BP	NM_012106	●		-6.58
IL12A	NM_000882	●	●	-6.53
MYH14	NM_001077186	●		-6.52
SKA1	NM_001039535		●	-6.46
CASC1	NM_018272		●	-6.44
HJURP	NM_018410		●	-6.42
TACC3	NM_006342	●	●	-6.33
ENPP3	NM_005021	●		-6.30
STIL	NM_001048166	●	●	-6.27
KNTC1	NM_014708		●	-6.26
NR1I2	NM_003889	●	●	-6.24
AKR1B10	NM_020299	●		-6.22
E2F2	NM_004091	●	●	-6.20
USP47	NM_017944	●		-6.14
KIF11	NM_004523	●	●	-6.09
E2F8	NM_024680	●	●	-6.05
PLK1	NM_005030	●	●	-6.02
CCDC6	NM_005436	●		-6.00
ORC6	NM_014321		●	-6.00
EXO1	NM_003686		●	-5.95
GPC5	NM_004466	●		-5.94
GSG2	NM_031965		●	-5.93
PRC1	NM_003981		●	-5.89
RAD51	NM_002875	●	●	-5.78
KIF2C	NM_006845	●	●	-5.71
TNFRSF13C	NM_052945	●		-5.70
BLZF1	NM_003666	●		-5.63
FEN1	NM_004111	●		-5.51
PLK4	NM_014264	●	●	-5.49
HAS2	NM_005328	●	●	-5.44
PKMYT1	NM_182687		●	-5.40
BUB1	NM_001211	●	●	-5.34
BUB1B	NM_001211	●	●	-5.34
NEK2	NM_002497	●	●	-5.33
IQGAP3	NM_178229		●	-5.27
SKA3	NM_145061		●	-5.23
PNN	NM_002687	●	●	-5.20
NTRK3	NM_001007156	●	●	-5.17
IL25	NM_022789	●		-5.09
UBE2C	NM_181803	●	●	-5.09
AURKB	NM_004217	●	●	-5.08
CDC6	NM_001254	●	●	-5.08
CDKN2C	NM_078626	●	●	-5.06
EDN2	NM_001956	●		-5.06
CDC20	NM_001255	●	●	-5.05
RRM1	NM_001033	●	●	-5.05
APC	NM_000038	●	●	-5.04
KIF15	NM_020242	●	●	-5.03
LMNB1	NM_005573	●		-5.02
NCAPG2	NM_017760		●	-4.96
CCNE2	NM_057749	●	●	-4.94
HMMR	NM_012484	●		-4.93
BRIP1	NM_032043	●		-4.90
ECT2	NM_018098	●	●	-4.89
CDT1	NM_030928	●	●	-4.87
MCAM	NM_006500	●		-4.82
LAG3	NM_002286	●	●	-4.78
ZWINT	NM_032997		●	-4.73
DCLK2	NM_001040260		●	-4.72
TRAIP	NM_005879	●		-4.71
SSTR2	NM_001050	●	●	-4.69
TXK	NM_003328	●		-4.65
TBC1D9	NM_015130	●		-4.63
IL1RN	NM_173843	●		-4.61
CDCA7	NM_031942	●		-4.56
STK38	NM_007271	●	●	-4.56
CDCA5	NM_080668	●	●	-4.54
E2F7	NM_203394	●		-4.54
FIGNL1	NM_001042762	●		-4.51
SMC4	NM_005496		●	-4.50
CYCS	NM_018947	●		-4.48
FBN1	NM_000138	●		-4.48
NCAPD3	NM_015261		●	-4.46
IL16	NM_172217	●	●	-4.44
PCNA	NM_002592	●	●	-4.42
FBXO5	NM_001142522		●	-4.37
CKAP2	NM_018204		●	-4.34
IL34	NM_152456	●		-4.34
PSRC1	NM_032636	●	●	-4.33
C11orf82	NM_145018		●	-4.32
CHRDL1	NM_145234		●	-4.31
RAD54B	NM_012415		●	-4.31
DIAPH3	NM_001042517		●	-4.29
AKR1C1	NM_001353	●		-4.26
INHBB	NM_002193	●		-4.25
MDM2	NM_002392	●	●	-4.25
PRKAA1	NM_206907	●		-4.25
MASTL	NM_032844		●	-4.23
MCM5	NM_006739	●		-4.21
CD2AP	NM_012120	●	●	-4.20
BRCA1	NM_007300	●	●	-4.18
TPX2	NM_012112	●	●	-4.15
FGFBP1	NM_005130	●		-4.14
EIF4G2	NM_001172705	●	●	-4.12
AURKA	NM_198433	●	●	-4.10
PTTG1	NM_004219	●	●	-4.08
ADRA1B	NM_000679	●	●	-4.07
RECQL4	NM_004260	●		-4.02
GJB2	NM_004004	●		-4.00
BIRC5	NM_001012271	●	●	-3.35
TERF1	NM_017489	●	●	-3.18
LRP1	NM_032832	●		-2.43
CDK1	NM_012395	●	●	-2.21
ARHGEF10	NM_014629		●	-2.08