Functional polymorphisms in NFκB1/IκBα predict risks of chronic obstructive pulmonary disease and lung cancer in Chinese

Abstract

Lung inflammation is the major pathogenetic feature for both chronic obstructive pulmonary disease (COPD) and lung cancer. The nuclear factor-kappa B (NFκB) and its inhibitor (IκB) play crucial roles in inflammatory. Here, we tested the hypothesis that single nucleotide polymorphisms (SNPs) in NFκB/IκB confer consistent risks for COPD and lung cancer. Four putative functional SNPs (NFκB1: -94del>insATTG; NFκB2: -2966G>A; IκBα: -826C>T, 2758G>A) were analyzed in southern and validated in eastern Chineses to test their associations with COPD risk in 1,511 COPD patients and 1,677 normal lung function controls, as well as lung cancer risk in 1,559 lung cancer cases and 1,679 cancer-free controls. We found that the -94ins ATTG variants (ins/del + ins/ins) in NFκB1 conferred an increased risk of COPD (OR 1.27, 95 % CI 1.06-1.52) and promoted COPD progression by accelerating annual FEV1 decline (P = 0.015). The 2758AA variant in IκBα had an increased risk of lung cancer (OR 1.53, 95 % CI 1.30-1.80) by decreasing IκBα expression due to the modulation of microRNA hsa-miR-449a but not hsa-miR-34b. Furthermore, both adverse genotypes exerted effect on increasing lung cancer risk in individuals with pre-existing COPD, while the -94del>insATTG did not in those without pre-existing COPD. However, no significant association with COPD or lung cancer was observed for -2966G>A and -826C>T. Our data suggested a common susceptible mechanism of inflammation in lung induced by genetic variants in NFκB1 (-94del>ins ATTG) or IκBα (2758G>A) to predict risk of COPD or lung cancer.

Full text

1 23
Human Genetics
ISSN 0340-6717
Volume 132
Number 4
Hum Genet (2013) 132:451-460
DOI 10.1007/s00439-013-1264-9
Functional polymorphisms in NFκB1/IκBα
predict risks of chronic obstructive
pulmonary disease and lung cancer in
Chinese
Dongsheng Huang, Lei Yang, Yehua
Liu, Yumin Zhou, Yuan Guo, Mingan
Pan, Yunnan Wang, Yigang Tan, Haibo
Zhong, Min Hu, Wenju Lu, et al.

1 23
Your article is protected by copyright and
all rights are held exclusively by Springer-
Verlag Berlin Heidelberg. This e-offprint is
for personal use only and shall not be self-
archived in electronic repositories. If you
wish to self-archive your work, please use the
accepted author’s version for posting to your
own website or your institution’s repository.
You may further deposit the accepted author’s
version on a funder’s repository at a funder’s
request, provided it is not made publicly
available until 12 months after publication.

ORIGINAL INVESTIGATION
Functional polymorphisms in NFjB1/IjBapredict risks
of chronic obstructive pulmonary disease and lung cancer
in Chinese
Dongsheng Huang •Lei Yang •Yehua Liu •Yumin Zhou •Yuan Guo •Mingan Pan •
Yunnan Wang •Yigang Tan •Haibo Zhong •Min Hu •Wenju Lu •Weidong Ji •
Jian Wang •Pixin Ran •Nanshan Zhong •Yifeng Zhou •Jiachun Lu
Received: 20 September 2012 / Accepted: 3 January 2013 / Published online: 16 January 2013
ÓSpringer-Verlag Berlin Heidelberg 2013
Abstract Lung inflammation is the major pathogenetic
feature for both chronic obstructive pulmonary disease
(COPD) and lung cancer. The nuclear factor-kappa B
(NFjB) and its inhibitor (IjB) play crucial roles in
inflammatory. Here, we tested the hypothesis that single
nucleotide polymorphisms (SNPs) in NFjB/IjBconfer
consistent risks for COPD and lung cancer. Four putative
functional SNPs (NFjB1:-94del[insATTG; NFjB2:
-2966G[A; IjBa:-826C[T, 2758G[A) were analyzed
in southern and validated in eastern Chineses to test their
associations with COPD risk in 1,511 COPD patients and
1,677 normal lung function controls, as well as lung cancer
risk in 1,559 lung cancer cases and 1,679 cancer-free
controls. We found that the -94ins ATTG variants (ins/
del ?ins/ins) in NFjB1 conferred an increased risk of
COPD (OR 1.27, 95 % CI 1.06–1.52) and promoted COPD
progression by accelerating annual FEV1 decline
(P=0.015). The 2758AA variant in IjBahad an increased
risk of lung cancer (OR 1.53, 95 % CI 1.30–1.80) by
decreasing IjBaexpression due to the modulation of
microRNA hsa-miR-449a but not hsa-miR-34b. Further-
more, both adverse genotypes exerted effect on increasing
lung cancer risk in individuals with pre-existing COPD,
while the -94del[insATTG did not in those without pre-
existing COPD. However, no significant association with
COPD or lung cancer was observed for -2966G[A and
-826C[T. Our data suggested a common susceptible
mechanism of inflammation in lung induced by genetic
variants in NFjB1 (-94del[ins ATTG) or IjBa
(2758G[A) to predict risk of COPD or lung cancer.
D. Huang, L. Yang and Y. Liu contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00439-013-1264-9) contains supplementary
material, which is available to authorized users.
D. Huang L. Yang Y. Liu W. Ji J. Lu (&)
School of Public Health, The Institute for Chemical
Carcinogenesis, The State Key Lab of Respiratory Disease,
Guangzhou Medical University, 195 Dongfengxi Road,
Guangzhou 510182, China
e-mail: jcLu@gzhmc.edu.cn
D. Huang Y. Wang Y. Tan
Department of Respiratory Medicine, Guangzhou Chest
Hospital, Guangzhou, Guangdong, China
Y. Zhou W. Lu J. Wang P. Ran N. Zhong
Guangzhou Institute of Respiratory Diseases, The First Affiliated
Hospital, The State Key Lab of Respiratory Disease, Guangzhou
Medical University, Guangzhou, Guangdong, China
Y. Guo
The Third Affiliated Hospital of Guangzhou Medical University,
Guangzhou, Guangdong, China
M. Pan
Department of Respiratory Medicine, The Third Affiliated
Hospital of Sun Yat-sen University, Guangzhou,
Guangdong, China
H. Zhong
Department of Respiratory Medicine, Guangzhou Red Cross
Hospital, Guangzhou, Guangdong, China
M. Hu Y. Zhou
Soochow University Laboratory of Cancer Molecular Genetics,
Medical College of Soochow University, Suzhou 215123, China
123
Hum Genet (2013) 132:451–460
DOI 10.1007/s00439-013-1264-9
Author's personal copy

Introduction
Chronic obstructive pulmonary disease (COPD) and lung
cancer, two major smoking-related diseases, are the leading
causes of morbidity and mortality in China and worldwide
(Ferlay et al. 2010). Tobacco smoking is the most important
risk factor of COPD and lung cancer (Zhang and Cai 2003;
Zhong et al. 2007). Smoking can induce inflammatory
response in the airways, which play decisive roles in the
developments of both COPD and lung cancer (Grivennikov
et al. 2010; Lee et al. 2012). Moreover, patients with COPD
develop a greater degree of inflammation and thus provide
protumorigenic effects in lung (Hogg et al. 2004), and it has
been reported that COPD patients would develop lung cancer
with a high incidence rate of 16.7 cases per 1,000 person-
years (de Torres et al. 2011). COPD and lung cancer are both
inheritable (Chen 1999; Lichtenstein et al. 2000), therefore,
to reveal the common genetic susceptible factors of them
would be useful for prevention of both diseases, especially
for preventing the COPD patients to develop lung cancer.
As a crucial inflammatory mediator, nuclear factor
kappa-B (NFjB) and its endogenous inhibitors NFjBI (IjB)
are recognized as molecular link between COPD and lung
cancer (Garcia-Rio et al. 2010; Petrescu et al. 2010; Tanni
et al. 2010). Many signal transduction pathways, originating
from a wide variety of cellular stimuli, converge on the
NFjB/IjB complex in the bronchial epithelium, inflamma-
tory cells, premalignant lesions of the bronchial epithelium
and neoplastic cells (Karin 2009); and subsequent activation
of NFjB might cause chronic inflammation in the lower
airways that further promote the developments of COPD and
lung cancer. Furthermore, NFjB mediates tumor-promoting
inflammation during every stages of carcinogenesis, includ-
ing initiation, promotion, malignant conversion, invasion and
metastasis (Chaturvedi et al. 2011).
NFjB1 and NFjB2 are two major forms of NFjB
family in human (Chen et al. 1999), they can be inactivated
by the most common protein of IjB family-NFkB inhibitor
a(IjBa) (Hayden et al. 2006). Previous studies have
identified several single nucleotide polymorphisms (SNPs)
in NFjB1/NFjB2 and IjBato be associated with various
diseases including inflammatory disorders and cancer (He
et al. 2009; Karban et al. 2004; Song et al. 2011). However,
the pathophysiological effects of these SNPs on chronic
pulmonary diseases are unclear. We hypothesized that the
SNPs in NFjB/IjBagenes may influence the development
of COPD and lung cancer.
In this study, we tested the associations between four
putative functional SNPs (-94del[ins ATTG in NFjB1;
-2966G[AinNFjB2;-826C[T and 2758G[AinIjBa)
and COPD risk in a total of 1,511 COPD patients and 1,677
normal lung function controls, as well as lung cancer risk
with totally 1,559 lung cancer cases and 1,679 cancer-free
controls. We further performed a series of biological assays
to identify the biological effects of these polymorphisms.
Methods
Study subjects
Retrospective case–control studies were conducted for
COPD and lung cancer in two stages. The discovery set
included 1,025 COPD patients and 1,061 normal lung
function controls (Yang et al. 2012b), as well as 1,056 lung
cancer cases and 1,056 cancer-free controls of southern
Chinese (Liu et al. 2012; Lu et al. 2011; Yang et al. 2012a).
The validation set comprised 486 COPD patients and 616
normal controls, as well as 503 lung cancer cases and 623
healthy controls (Liu et al. 2012; Lu et al. 2011; Yang et al.
2012a). The detailed information of subjects’ recruitment
was presented in Electronic Supplementary Material.
Definition of COPD and its severity stage were according
to the global initiative for chronic obstructive lung disease
(Rabe et al. 2007). Individual’s demographic characters
and surrounding variables were obtained during an inter-
view after a written informed consent was signed. The
definitions of variables such as pre-existing COPD were
described in Electronic Supplementary Material and
elsewheres (Liu et al. 2012; Lu et al. 2011; Yang et al.
2012a). Demographic and selected variables of study sub-
jects were presented in Supplementary Table S1, S2. Fur-
thermore, all subjects that we recruited in previous studies
(Buist et al. 2007; Zhong et al. 2007; Zhou et al. 2010) with
at least 4 years spirometric follow-up data between 2002
and 2010 were selected for further phenotype analysis (i.e.,
116 COPD patients and 357 controls). The study was
approved by the institutional review boards of Guangzhou
Medical University and Soochow University.
SNP selection
Several SNPs located in NFjBor IjBagene have been
identified in previous studies. Among them, four polymor-
phisms [i.e., -94del[ins ATTG (rs28362491) of NFjB1;
-2966G[A (rs12769316) of NFjB2;-826C[T (rs2233406)
and 2758G[A(rs696)ofIjBa] were putatively functional and
reported to be associated with various human diseases (He
et al. 2009;Hungetal.2010; Marco s et al. 2009; Sampath et al.
2011;Songetal.2011; Zhang et al. 2009). Therefore, we
selected these four SNPs in our study.
Genotype and phenotype detection
The Taqman allelic discrimination assay was used to detect
the genotypes of -94del[ins ATTG (rs28362491) of
452 Hum Genet (2013) 132:451–460
123
Author's personal copy

NFjB1;-2966G[A (rs12769316) of NFjB2;-826C[T
(rs2233406) and 2758G[A (rs696) of IjBa. Primers and
probes were designed by using Primer Express 3.0
(Applied Biosystems, Foster City, CA, USA) and synthe-
sized by Shanghai GeneCore Biotechnologies (Shanghai,
China) as shown in Supplementary Table S3. The PCR was
performed in the ABI PRISM 7900 Sequence Detection
Systems (Applied Biosystems) and the genotypes were
automatically determined by Sequence Detection Systems
software 2.0.1 (Applied Biosystems; Supplementary Figure
S1). To confirm the genotyping results, we randomly
selected 10 % samples to repeat by Taqman assay and 60
samples to re-sequence, as expected, the results were all
100 % concordant (Supplementary Figure S1).
Because the biological effect of the -94del[ins ATTG
variants has been identified (Karban et al. 2004), we
focused on assaying the effect of 2758G[A polymorphism.
The IjBaexpression level was detected in mRNA level by
real-time PCR, in protein level by western blotting like
described previously (Lu et al. 2011). We also performed
immunohistochemistry to detect the IjBaexpression
in situ. The detail protocols were described in Electronic
Supplementary Material.
RNA interference and luciferase assays
The protocol for construction of two luciferase reporter
genes comprising the 30-UTR of IjBawith different
2758G or A allele was presented in Electronic Supple-
mentary Material. The IjBain vitro luciferase assays
were performed first without any microRNA treatment
(Lu et al. 2011). Because the bioinformatics analysis
showed that the 2758G[A would change the binding of
the microRNA miR-449a and miR-34b, we added the
RNA interference assay to show their effect interacted
with the SNP. The mimics and inhibitors of miR-449a and
miR-34b synthesized by GenePharma Co. (Shanghai,
China) were co-transferred with the luciferase reporters to
show the effect of microRNA on IjBareporter genes
in vitro. A549 (2758GG genotype) and NCI-520 (2758AG
genotype) were seeded into 24-well plates at
1910
5
cells/well and cultured at 37 °Cin5%CO
2
for
24 h. The cells were then transiently transfected with
1.5 lg of reporter plasmids (G or A allele) alone or co-
transfected with or without microRNA mimics or inhibi-
tors using Lipofectamine 2000 according to the protocol
(Invitrogen, Carlsbad, CA, USA). The activities of
reporter genes with renilla luciferase and the internal
standard firefly luciferase were quantified by a Dual-
Luciferase Reporter Assay System (Promega, Madison,
WI, USA). Independent triplicate experiments were done
for each plasmid construct. We further detect the effect of
the operant microRNA on IjBaexpression with real-time
PCR analysis in A549 (2758GG genotype) and NCI-520
(2758AG genotype).
Statistical analysis
Differences in the distributions of demographic character-
istics and genotypes between cases and controls were
evaluated using the Chi-square test or the Student’s ttest.
The association between each SNP and diseases risk was
estimated using an unconditional logistic regression model
with adjustments for surrounding factors. A multiplicative
interaction was suggested to detect the possible gene–
environment interaction (Lu et al. 2011). Homogeneity test
was performed with Breslow–Day test. The statistical
power was calculated by the PS Software (Dupont and
Plummer 1990). The One-way ANOVA test, Student’s
ttest and linear regression analysis were used to assay the
deviation of annual decline of pre-bronchodilator FEV1,
which refers to the volume exhaled during the first second
of a forced expiratory maneuver started from the level of
total lung capacity and IjBaexpression in groups with
different genotypes. The Student’s ttest was also used to
examine the difference in levels of luciferase reporter
genes expression between different constructs. All tests
were two-sided by using the SAS software (version 9.3;
SAS Institute, Cary, NC, USA). P\0.05 was considered
statistically significant.
Results
NFjB/IjBagenotypes and COPD or lung cancer risk
As summarized in Table 1, the observed genotype fre-
quencies of all SNPs were in agreement with the Hardy–
Weinberg equilibrium in controls (P[0.05 for all). In
discovery set, according to criteria of the smallest AIC
value, the -94ins variant genotypes (ins/ins ?ins/del) of
NFjB1 conferred a 1.23-fold increased risk of COPD
compared with del/del genotype (OR 1.23; 95 % CI
1.03–1.54; P=0.037) in a dominant genetic model. And
the 2758AA genotype of IjBaconferred a 1.49-fold
increased risk of lung cancer compared with 2758G
(GG ?GA) genotypes (OR 1.49; 95 % CI 1.22–1.82;
P=9.5 910
-5
) under a recessive genetic model. How-
ever, no other significant association between these poly-
morphisms and the risk of either COPD or lung cancer was
observed. The results in validation set confirmed the above
findings, the -94ins variant genotypes conferred a 1.33-
fold increased risk of COPD compared with del/del geno-
type (OR 1.33; 95 % CI 1.04–1.81; P=0.047). Mean-
while, the 2758AA adverse genotype had a 1.61-fold risk of
lung cancer compared to 2758G genotypes (OR 1.61; 95 %
Hum Genet (2013) 132:451–460 453
123
Author's personal copy

Table 1 Associations between genotypes in NFjB1/NFjB2 and IjBagenes and COPD as well as lung cancer risk
Genotypes COPD case–control study Lung cancer case–control study
Discovery set (Southern Chinese) Validation set (Eastern Chinese) Discovery set (Southern Chinese) Validation set (Eastern Chinese)
Cases n(%) Controls n(%) Adjusted OR (95 % CI)
a
Cases n(%) Controls n(%) OR (95 % CI)
a
Cases n(%) Controls n(%) Adjusted OR (95 % CI)
a
Cases n(%) Controls n(%) OR (95 % CI)
a
Total no. of subjects 1,025 1,061 486 616 1,056 1,056 503 623
NFjB1 -94del [insATTG
del/del 170 (16.6) 214 (20.2) 1.00 (ref) 82 (16.9) 133 (21.6) 1.00 (ref) 225 (21.3) 210 (19.9) 1.00 (ref) 104 (20.7) 145 (23.3) 1.00 (ref)
ins/del 454 (44.3) 496 (46.7) 1.13 (0.89–1.44) 214 (44.0) 261 (42.4) 1.33 (0.96–1.86) 459 (43.4) 491 (46.5) 0.87 (0.69–1.10) 230 (45.7) 289 (46.4) 1.08
(0.79–1.47)
ins/ins 401 (39.1) 351 (33.1) 1.38 (1.07–1.77) 190 (39.1) 222 (36.0) 1.39 (1.00–1.95) 372 (35.5) 355 (33.6) 0.97 (0.77–1.24) 169 (33.6) 189 (30.3) 1.25
(0.90–1.73)
Trend test Pvalue 0.006 0.095 0.982 0.173
ins/del ?ins/ins 855 (83.4) 847 (79.8) 1.23 (1.03–1.54) 404 (83.1) 483 (78.4) 1.33 (1.04–1.81) 831 (78.7) 846 (80.1) 0.92 (0.74–1.13) 404 (83.1) 483 (78.4) 1.14
(0.86–1.52)
NFjB2-2966G[A
GG 412 (40.2) 451 (42.5) 1.00 (ref) 440 (41.7) 459 (43.5) 1.00 (ref)
AG 480 (46.8) 475 (44.8) 1.11 (0.91–1.34) 494 (46.8) 481 (45.5) 1.11 (0.91–1.34)
AA 133 (13.0) 135 (12.7) 1.08 (0.79–1.48) 122 (11.5) 116 (11.0) 1.08 (0.79–1.48)
Trend test Pvalue 0.687 0.798
IjBa-826T[C
TT 788 (76.9) 822 (77.5) 1.00 (ref) 815 (77.2) 808 (76.5) 1.00 (ref)
TC 210 (20.5) 225 (21.2) 0.99 (0.80–1.23) 217 (20.5) 229 (21.7) 0.95 (0.77–1.17)
CC 27 (2.6) 14 (1.32) 1.76 (0.90–3.47) 24 (2.3) 19 (1.8) 1.26 (0.69–2.33)
Trend test Pvalue 0.227 0.976
IjBa2758G[A
GG 311 (30.3) 305 (28.8) 1.00 (ref) 150 (30.9) 171 (27.8) 1.00 (ref.) (ref.) 294 (27.9) 293 (27.8) 1.00 (ref) 132 (26.2) 206 (33.1) 1.00 (ref.) (ref.)
AG 503 (49.1) 532 (50.1) 0.93 (0.76–1.14) 244 (50.2) 324 (52.6) 0.88 (0.66–1.16) 466 (44.1) 544 (51.5) 0.86 (0.70–1.06) 226 (44.9) 290 (46.5) 1.23
(0.93–1.63)
AA 211 (20.6) 224 (21.1) 0.91 (0.71–1.17) 92 (18.9) 121 (19.6) 0.87 (0.61–1.24) 296 (28.0) 219 (20.7) 1.36 (1.07–1.72) 145 (28.8) 127 (20.4) 1.82 (1.31-2.53)
Trend test Pvalue 0.448 0.350 0.018 4.0 310
25
GG ?AG 814 (79.4) 837 (78.9) 1.00 (ref) 394 (81.1) 495 (80.4) 1.00 (ref) 760 (72.0) 837 (79.3) 1.00 (ref) 358 (71.2) 496 (79.6) 1.00 (ref)
AA 211 (20.6) 224 (21.1) 0.96 (0.77–1.19) 92 (18.9) 121 (19.6) 0.95 (0.70–1.29) 296 (28.0) 219 (20.7) 1.49 (1.22–1.82) 145 (28.8) 127 (20.4) 1.61
(1.22–2.12)
Significant statistics with tested Pvalues less than 0.05 were presented in bold
a
Adjusted in a logistic regression model that included age, sex, smoking status and drinking status
454 Hum Genet (2013) 132:451–460
123
Author's personal copy

CI 1.22–2.12; P=0.001). Because the associations of
above adverse genotypes in the two datasets were homo-
geneous (P=0.734 for -94del[ins ATTG, P=0.726 for
2758G[A), we then merged the two sets, the -94ins variant
genotypes of NFjB1 conferred a 1.27-fold increased risk of
COPD (OR 1.27; 95 % CI 1.06–1.52; P=0.009; Supple-
mentary Figure S2), while the 2758AA genotype had a
1.53-fold increased risk of lung cancer (OR 1.53, 95 % CI
1.30–1.80; P=3.0 910
-4
; Supplementary Figure S3).
We further analyze the effect of pre-existing COPD
condition on the risk genotypes for lung cancer because
COPD promotes inflammation and thus provide protu-
morigenic effects (Hogg et al. 2004). Interestingly, both the
adverse genotypes were significant in those subjects with
pre-existing COPD (P\0.05 for all), while the -94ins
variant genotypes was not in those without pre-existing
COPD (Table 2). When combined the number of risk
genotypes of these two SNPs, we found that the subjects
carrying two risk genotypes with pre-existing COPD had a
higher risk of lung cancer (OR 3.15, 95 % CI 1.48–6.70)
than those without pre-existing COPD (OR 1.43, 95 % CI
1.12–1.48) on a borderline statistically significance (Bre-
slow–Day test: P=0.087, Table 2). In the stratification
analysis, we did not observe any significant differences for
associations between the -94del[ins ATTG or 2758G[A
polymorphism and risk of COPD or lung cancer in each
stratum (Supplementary Figure S2, S3; homogeneity test
P[0.05 for all), and no significant interaction between
these risk genotypes and surrounding factors on both dis-
eases risk. In addition, there was no significant association
between the risk genotypes and smoking status as well as
pack years smoked (P[0.05, data not shown).
NFjB1 genotypes and pulmonary functions
Only the effect of -94del[ins ATTG on pulmonary function
was presented because no significant association was
observed in other SNPs for pulmonary function. In subjects
with pulmonary function follow-up data, there was a signi-
ficant decreasing trend of annual average decline of pre-
bronchodilator FEV1 in COPD patients according to -94ins
ATTG allele-dependent manner (-94ins/ins ATTG: n=62,
-0.126 ±0.113 L, -94ins/del ATTG: n=42, -0.091 ±
0.083 L, -94del/del ATTG: n=12, -0.040 ±0.036 L,
ANOVA test P=0.015, linear regression test P=0.017),
but not in controls (Fig. 1). And the -94del[insATTG
genotypes were significantly correlated with COPD Gold
stages (P=0.048). Furthermore, as shown in Supplemen-
tary Table S4, sex, smoking status and pack-year smoked
were correlated with the annual decline of pre-bronchodilator
FEV1 in both COPD cases and controls.
IjBagenotypes and its expression
As shown in Fig. 2, the mRNA levels of IjBawere much
lower in lung cancer tissues compared to their adjacent
Table 2 Effect of NFjB1/IjBapolymorphisms on risk of lung cancer with or without pre-existing COPD
Genotypes Non pre-existing COPD individuals Pre-existing COPD individuals Homogeneity test
Pvalue
Lung cancers n(%) Controls n(%) OR (95 % CI)
a
Lung cancers n(%) Controls n(%) OR (95 % CI)
a
Total no. of subjects 1,342 1,510 217 169
NFjB1 -94del[insATTG
del/del 289 (21.6) 309 (20.5) 1.00 (ref.) 40 (18.4) 46 (27.2) 1.00 (ref.)
ins/del 599 (44.6) 653 (43.2) 1.05 (0.91–1.22) 90 (41.5) 77 (45.6) 0.86 (0.57–1.30) 0.267
ins/ins 454 (33.8) 548 (36.3) 0.90 (0.77–1.05) 87 (40.1) 46 (27.2) 1.80 (1.15–2.80) 0.003
del/del 289 (21.6) 309 (20.5) 1.00 (ref.) 40 (18.4) 46 (27.2) 1.00 (ref.)
ins/del ?ins/ins 1,053 (78.5) 1,201 (79.5) 0.93 (0.78–1.12) 177 (81.6) 123 (72.8) 1.69 (1.03–2.78) 0.030
IjBa2758G[A
GG?AG 968 (72.1) 1,202 (79.6) 1.00 (ref.) 150 (69.1) 131 (77.5) 1.00 (ref.)
AA 374 (27.9) 308 (20.4) 1.52 (1.28–1.80) 67 (30.9) 38 (22.5) 1.60 (1.00–2.58) 0.934
No. of the combined risk genotypes
b
0 210 (15.6) 251 (16.6) 1.00 (ref.) 26 (12.0) 33 (19.5) 1.00 (ref.)
1 837 (62.4) 1,009 (66.8) 0.98 (0.80–1.21) 138 (63.6) 111 (65.7) 1.60 (0.89–2.89) 0.132
2 295 (22.0) 250 (16.6) 1.43 (1.12–1.84) 53 (24.4) 25 (14.8) 3.15 (1.48-6.70) 0.087
Significant statistics with tested Pvalues less than 0.05 were presented in bold
a
Adjusted in a logistic regression model that included age, sex, smoking status and drinking status
b
Genotype combinations of the two polymorphisms in the NFjB1 and IjBa: ins variant genotypes (ins/del ?ins/ins) and 2758AA genotype are defined as risk
genotypes: i.e., the carriers of del/del and 2758GG/AG have zero risk genotype, the carriers of ins/del (ins/ins) and 2758GG/AG, or del/del and 2758AA have one
risk genotype; and the ins/del (ins/ins) and 2758AA carriers have two risk genotypes
Hum Genet (2013) 132:451–460 455
123
Author's personal copy

normal tissues (P=0.032), and they were significantly
lower in cases carrying 2758AA genotype than in cases
with 2758G genotypes of both normal and cancer tissues
(P\0.05 for all). The IjBaprotein expression confirmed
the above findings as they were significantly decreased in
carriers of 2758AA genotype compared with those of
2758G genotypes (P\0.05 for all). Furthermore, the
immunohistochemical stain also shown that 2758AA
genotype exerted a significantly lower IjBaexpression
in situ than 2758G genotypes (Supplementary Figure S4;
P=0.045).
Luciferase activity
As shown in Fig. 3b, the transcription activity of the
reporter gene integrated the IjBa30-UTR with 2758A
allele was significantly lower than that with G allele both in
A549 cell and NCI-520 cell (Pvalue is 0.022, 0.036,
respectively). The miR-449a mimics could further reduce
the reporter genes’ activity with 2758A allele (P\0.05),
and the miR-449a inhibitor reversed and up-regulated
reporter genes’ activity (P\0.05). However, the miR-34b
failed to exert any effect on the reporter genes either with
2758A allele or G allele (P[0.05 for all). Furthermore,
the expressions of miR-449a were observed in A549 and
NCI-520 (Fig. 3c). As shown in Fig. 3d, the mimics of
miR-449a suppressed the mRNA expression of IjBaand
its inhibitors could reverse and up-regulate IjBa’s
expression only in NCI-520 with 2758AG genotype
(P\0.01). However, in A549 with 2758GG genotype, the
miR-449a mimics could significantly suppress IjBa, but
the miR-449a inhibitors did not significantly reverse the
IjBaexpression.
Fig. 1 Association between the NFjB1 -94del[ins ATTG poly-
morphism and the annual average decline of pre-bronchodilator
FEV1. aThe annual average decline of pre-bronchodilator FEV1 by
-94del[ins ATTG genotypes in COPD patients. bThe annual
average decline of pre-bronchodilator FEV1 by -94del[ins ATTG
genotypes in healthy controls. A significant decreased trend of annual
average decline of pre-bronchodilator FEV1 in COPD patients
according to -94ins ATTG allele-dependent manner (ANOVA test
P=0.015) but not in controls (ANOVA test P=0.139) was
observed
Fig. 2 Association between the 2758G[A genotypes and IjBa
expressions. aRelative mRNA levels of the IjBaexpression in lung
tissues by 2758G[A genotypes. bThe western blotting assay on
detecting IjBaprotein with b-actin as an internal reference. cThe
IjBaprotein expression by 2758G[A genotypes in lung tissues. Both
the mRNA and protein expressions of IjBawere significantly
decreased in carriers of 2758AA genotype than those of 2758G
genotypes in lung cancer tissues (ANOVA test P\0.05 for all) as
well as in adjacent normal tissues (ANOVA test P\0.05 for all)
456 Hum Genet (2013) 132:451–460
123
Author's personal copy

Discussion
In this study, we found that the -94ins ATTG variants of
NFjB1 conferred an increased risk of COPD and accel-
erated COPD progression by making carriers more rapid
reduction in pulmonary function; and the 2758AA variant
of IjBacontributed an increased risk of lung cancer by
decreasing IjBaexpression in lung tissues under the
regulation of hsa-miR-449a but not hsa-miR-34b. Espe-
cially in individuals with pre-existing COPD, both
adverse genotypes exerted effect on increasing lung can-
cer risk while the -94del[insATTG did not in those
without pre-existing COPD. All these supported a com-
mon susceptible mechanism of inflammation in lung
induced by genetic variants in NFjB1 or IjBafor COPD
and lung cancer.
The NFjB1 and IjBaplay critical roles in multiple
human diseases by responding to the environmental stim-
ulus and regulating inflammatory responses, cell growth
and apoptosis (Escarcega 2010). Up expression or over
activation of NFjB1 that promotes exacerbation of
inflammation has been found in both COPD and lung
cancer (Brown et al. 2009; Tang et al. 2006), and NFjB1
also possibly predisposes those COPD patients to further
develop lung cancer. Meanwhile, IjBa, which functions to
suppress the effect of NFjB1, has been reported be inactive
or down-regulated during various stimuli induced NFjB
activation progresses (Abe et al. 2011; Kim et al. 2000),
and in consequence loss its protective role in the devel-
opment of human disease. Previous study has demonstrated
that the -94ins ATTG allele has an effect on increasing
expression of NFjB1 and in turn promoting inflammation
Fig. 3 Effects of the 2758G[A polymorphism and treatment with
microRNAs on reporter gene’s activity and IjBain different cell
lines. aSchematic of the reporter gene construct with a 296 bp 30-
UTR of IjBa(?1ntto?296 nt downstream to the translation stop
site TGA) including 2758G[A polymorphism and a putative target
site of miR-449a and miR-34b highly conserved in the IjBamRNA
30-UTR. bLuciferase expression of the two constructs in lung cancer
cells (A549 and NCI-520). The renilla luciferase activity of each
construct was normalized against the internal control of firefly
luciferase. cThe gene expressions of has-miR-449a in lung cell lines.
dThe differences of IjBamRNA level in A549 and NCI-520 with
different 2758G[A genotypes after the treatment of mimics or
inhibitors of miR-449a. Columns, mean from three independent
experiments; bars, SD; and Student’s ttest was used to test the
differences in the expression levels of different constructs as well as
IjBamRNA levels. The transcription activity of the reporter gene
integrated the IjBa30-UTR with 2758A allele was significantly lower
than that with G allele with a modulation by miR-449a
Hum Genet (2013) 132:451–460 457
123
Author's personal copy

(Karban et al. 2004), so, it is conceivable that the -94ins
ATTG variant genotypes conferred an increased risk of
COPD and decreased pulmonary functions. Correspond-
ingly, the 2758G[A polymorphism exerted an effect on
decreasing IjBaexpression in lung tissues and might loss
its suppression on NFjB dependent inflammation, there-
fore, the 2758AA variants shared a consistent pro-inflam-
matory role with the -94ins ATTG variants, thus
contributed high risk to lung cancer. The in vitro assays
further revealed an adverse effect of 2758A allele on
IjBa’s expression, and the miR-449a could specially reg-
ulate the activities of reporter genes with 2758A but not G
allele. The stronger effect of miR-449a on modulating IjBa
in NCI-520 with 2758AG genotype than that of A549 with
2758AA genotype confirmed this finding. Interestingly,
high miR-449a levels have been found in lung cancer tis-
sues and cancer cells (Lize et al. 2010), further suggesting a
physical and endogenous function of the miR-449a on
regulating the effect of IjBa. Two reports of colorectal
cancer have showed significant associations of the
2758G[A polymorphism in southern Chinese and Swedish
population (Song et al. 2011; Zou et al. 2011). They were
consistent with our finding that the 2758G[A polymor-
phism increased the risk of lung cancer. Taken these
together, the genetic variations -94del[ins ATTG of
NFjB1 or 2758G[AofIjBathat involved in a common
mechanism as pro-inflammation may predispose a sus-
ceptibility to COPD or lung cancer. Additionally, the
genetic effect of above two risk genotypes was more evi-
dent in subjects with pre-existing COPD, indicating a
possibly high risk of lung cancer in COPD cases owing to
over-activated inflammation.
Many studies have reported the -94ins ATTG variant
genotypes conferred an increased risk of inflammatory
diseases (He et al. 2009; Karban et al. 2004). Here, we
consistently found -94ins ATTG variants were associated
with COPD risk and correlated with COPD stages by
causing more rapid reduction in pre-bronchodilator FEV1
in COPD cases. The reduction in FEV1 is a marker of
airflow obstruction and the FEV1 is used to assess the
severity of the airflow obstruction (Ferrer et al. 1997), so
the patients with -94ins ATTG genotypes got more serious
COPD stages. Our findings of the -94ins ATTG variants
significantly associated with FEV1 decline in COPD cases
but not in controls implies that the ATTG polymorphism
increases the inflammation may involve in the development
of COPD.
As a hospital-based case–control study, there were some
limitations in current study such as information bias.
However, with the fairly large sample size and two study
populations, we have achieved high statistical powers
(88.0 % for COPD, 99.7 % for lung cancer) and the
functional assays also confirmed the associations. In
addition, recent genome-wide association studies (GWAS)
identified several SNPs in Chromosome 4q24 to be sus-
ceptible loci for COPD or lung function (Castaldi et al.
2011; Repapi et al. 2010). The NFjB1 gene is also located
on 4q24. However, the GWAS did not report any loci in
NFjB1, this may be due to that the SNP (rs28362491) was
not included in the Affymetrix 6.0 genechip assay, and
there is no LD between the SNP and other SNPs of NFjB1
comprised in the gene chip. For 2758G[A (rs696), the
results from Chinese GWAS that we previously partici-
pated in (Hu et al. 2011) showed that the SNP is significant
associated with lung cancer risk (P=0.016) although it
does not meet the GWAS significant criteria (P\10
-7
).
Therefore, it appears that our finding is unlikely by chance.
In conclusion, in current study, we found significant
associations between NFjB1 and IjBapolymorphisms and
risk of COPD or lung cancer in Chinese. The findings were
consistent in COPD and lung cancer, because the -94ins
variants carriers with reduced expression of NF-jB had an
increased risk of COPD, and the carriers of 2758AA
genotype with decreased expression of IjB and conse-
quently less suppression of NF-jB were associated with an
increased risk of lung cancer. Our results suggested that the
genetic variation in genes encoding NF-jB/IjB may con-
tribute to the developments of both COPD and lung cancer
in Chinese people.
Acknowledgments This study was supported by the National Nat-
ural Scientific Foundation of China grants 30671813, 30872178,
81072366, 81273149 (Dr. J. Lu), and partly by 81170043 (Dr. P.Ran),
30872142 (Dr. W. Ji) and 81001278, 81171895 (Dr. Y. Zhou);
Guangdong Provincial High Level Experts Grants 2010-79 (Dr.
J. Lu); Guangdong Provincial Science and Technology Planning
Project Grant 2011B031800378 (Dr. D. Huang), Guangdong Provin-
cial Medical Scientific Research Grants A2012520 (Dr. D. Huang);
Guangzhou civic Science and Technology grant 2012-Y2-00029 (Dr.
B. Liu); Changjiang Scholars and Innovative Research Team in
University grant IRT0961 (Dr. J. Wang), Guangdong natural science
foundation team grant 10351012003000000 (Dr. W. Lu). We thank
Dr. Bohang Zeng, Dr. Yunnan Wang, Dr. Zhanhong Xie and Ms.
Wanmin Zeng for their assistance in recruiting the subjects; Hongjun
Zhao, Xiaoxuan Ling and Lin Liu for their laboratory assistance.
Conflict of interest The authors have declared no conflicts of
interest.
References
Abe H, Hayes CN, Ochi H, Tsuge M, Miki D, Hiraga N, Imamura M,
Takahashi S, Kubo M, Nakamura Y, Kamatani N, Chayama K
(2011) Inverse association of IL28B genotype and liver mRNA
expression of genes promoting or suppressing antiviral state.
J Med Virol 83:1597–1607. doi:10.1002/jmv.22158
Brown V, Elborn JS, Bradley J, Ennis M (2009) Dysregulated
apoptosis and NFkappaB expression in COPD subjects. Respir
Res 10:24. doi:10.1186/1465-9921-10-24
458 Hum Genet (2013) 132:451–460
123
Author's personal copy

Buist AS, McBurnie MA, Vollmer WM, Gillespie S, Burney P,
Mannino DM, Menezes AM, Sullivan SD, Lee TA, Weiss KB,
Jensen RL, Marks GB, Gulsvik A, Nizankowska-Mogilnicka E
(2007) International variation in the prevalence of COPD (the
BOLD Study): a population-based prevalence study. Lancet
370:741–750. doi:10.1016/S0140-6736(07)61377-4
Castaldi PJ, Cho MH, Litonjua AA, Bakke P, Gulsvik A, Lomas DA,
Anderson W, Beaty TH, Hokanson JE, Crapo JD, Laird N,
Silverman EK (2011) The association of genome-wide signifi-
cant spirometric loci with chronic obstructive pulmonary disease
susceptibility. Am J Respir Cell Mol Biol 45:1147–1153. doi:
10.1165/rcmb.2011-0055OC
Chaturvedi MM, Sung B, Yadav VR, Kannappan R, Aggarwal BB
(2011) NF-kappaB addiction and its role in cancer: ‘one size does
not fit all’. Oncogene 30:1615–1630. doi:10.1038/onc.2010.566
Chen Y (1999) Genetics and pulmonary medicine. 10: genetic
epidemiology of pulmonary function. Thorax 54:818–824
Chen F, Castranova V, Shi X, Demers LM (1999) New insights into
the role of nuclear factor-kappaB, a ubiquitous transcription
factor in the initiation of diseases. Clin Chem 45:7–17
de Torres JP, Marin JM, Casanova C, Cote C, Carrizo S, Cordoba-
Lanus E, Baz-Davila R, Zulueta JJ, Aguirre-Jaime A, Saetta M,
Cosio MG, Celli BR (2011) Lung cancer in patients with chronic
obstructive pulmonary disease–incidence and predicting factors.
Am J Respir Crit Care Med 184:913–919. doi:10.1164/rccm.201
103-0430OC
Dupont WD, Plummer WD (1990) Power and sample size calcula-
tions: a review and computer program. Control Clin Trials
11:116–128
Escarcega RO (2010) The transcription factor NF-kappaB in human
diseases. Rev Med Inst Mex Seguro Soc 48:55–60
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM (2010)
Estimates of worldwide burden of cancer in 2008: gLOBOCAN
2008. Int J Cancer. doi:10.1002/ijc.25516
Ferrer M, Alonso J, Morera J, Marrades RM, Khalaf A, Aguar MC,
Plaza V, Prieto L, Anto JM (1997) Chronic obstructive
pulmonary disease stage and health-related quality of life. The
Quality of Life of Chronic Obstructive Pulmonary Disease Study
Group. Ann Intern Med 127:1072–1079
Garcia-Rio F, Miravitlles M, Soriano JB, Munoz L, Duran-Tauleria E,
Sanchez G, Sobradillo V, Ancochea J (2010) Systemic inflam-
mation in chronic obstructive pulmonary disease: a population-
based study. Respir Res 11:63. doi:10.1186/1465-9921-11-63
Grivennikov SI, Greten FR, Karin M (2010) Immunity, inflammation,
and cancer. Cell 140:883–899. doi:10.1016/j.cell.2010.01.025
Hayden MS, West AP, Ghosh S (2006) SnapShot: NF-kappaB
signaling pathways. Cell 127:1286–1287. doi:10.1016/j.cell.
2006.12.005
He Y, Zhang H, Yin J, Xie J, Tan X, Liu S, Zhang Q, Li C, Zhao J,
Wang H, Cao G (2009) IkappaBalpha gene promoter polymor-
phisms are associated with hepatocarcinogenesis in patients
infected with hepatitis B virus genotype C. Carcinogenesis
30:1916–1922. doi:10.1093/carcin/bgp226
Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L,
Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD
(2004) The nature of small-airway obstruction in chronic
obstructive pulmonary disease. N Engl J Med 350:2645–2653.
doi:10.1056/NEJMoa032158
Hu Z, Wu C, Shi Y, Guo H, Zhao X, Yin Z, Yang L, Dai J, Hu L, Tan
W, Li Z, Deng Q, Wang J, Wu W, Jin G, Jiang Y, Yu D, Zhou G,
Chen H, Guan P, Chen Y, Shu Y, Xu L, Liu X, Liu L, Xu P, Han
B, Bai C, Zhao Y, Zhang H, Yan Y, Ma H, Chen J, Chu M, Lu F,
Zhang Z, Chen F, Wang X, Jin L, Lu J, Zhou B, Lu D, Wu T, Lin
D, Shen H (2011) A genome-wide association study identifies
two new lung cancer susceptibility loci at 13q12.12 and 22q12.2
in Han Chinese. Nat Genet 43:792–796. doi:10.1038/ng.875
Hung YH, Wu CC, Ou TT, Lin CH, Li RN, Lin YC, Tsai WC, Liu
HW, Yen JH (2010) IkappaBalpha promoter polymorphisms in
patients with Behcet’s disease. Dis Markers 28:55–62. doi:
10.3233/DMA-2010-0684
Karban AS, Okazaki T, Panhuysen CI, Gallegos T, Potter JJ, Bailey-
Wilson JE, Silverberg MS, Duerr RH, Cho JH, Gregersen PK,
Wu Y, Achkar JP, Dassopoulos T, Mezey E, Bayless TM,
Nouvet FJ, Brant SR (2004) Functional annotation of a novel
NFKB1 promoter polymorphism that increases risk for ulcera-
tive colitis. Hum Mol Genet 13:35–45. doi:10.1093/hmg/ddh008
Karin M (2009) NF-kappaB as a critical link between inflammation
and cancer. Cold Spring Harb Perspect Biol 1:a000141. doi:
10.1101/cshperspect.a000141
Kim HJ, Kim KW, Yu BP, Chung HY (2000) The effect of age on
cyclooxygenase-2 gene expression: NF-kappaB activation and
IkappaBalpha degradation. Free Radic Biol Med 28:683–692
Lee J, Taneja V, Vassallo R (2012) Cigarette smoking and
inflammation: cellular and molecular mechanisms. J Dent Res
91(2):142–149. doi:10.1177/0022034511421200
Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J,
Koskenvuo M, Pukkala E, Skytthe A, Hemminki K (2000)
Environmental and heritable factors in the causation of cancer—
analyses of cohorts of twins from Sweden, Denmark, and
Finland. N Engl J Med 343:78–85. doi:10.1056/NEJM20
0007133430201
Liu B, Yang L, Huang B, Cheng M, Wang H, Li Y, Huang D, Zheng
J, Li Q, Zhang X, Ji W, Zhou Y, Lu J (2012) A functional copy-
number variation in MAPKAPK2 predicts risk and prognosis of
lung cancer. Am J Hum Genet 91:384–390. doi:10.1016/j.ajhg.
2012.07.003
Lize M, Pilarski S, Dobbelstein M (2010) E2F1-inducible microRNA
449a/b suppresses cell proliferation and promotes apoptosis. Cell
Death Differ 17:452–458. doi:10.1038/cdd.2009.188
Lu J, Yang L, Zhao H, Liu B, Li Y, Wu H, Li Q, Zeng B, Wang Y, Ji
W, Zhou Y (2011) The polymorphism and haplotypes of PIN1
gene are associated with the risk of lung cancer in Southern and
Eastern Chinese populations. Hum Mutat 32:1299–1308. doi:
10.1002/humu.21574
Marcos M, Pastor I, Gonzalez-Sarmiento R, Laso FJ (2009) A
functional polymorphism of the NFKB1 gene increases the risk
for alcoholic liver cirrhosis in patients with alcohol dependence.
Alcohol Clin Exp Res 33:1857–1862. doi:10.1111/j.1530-0277.
2009.01023.x
Petrescu F, Voican SC, Silosi I (2010) Tumor necrosis factor-alpha
serum levels in healthy smokers and nonsmokers. Int J Chron
Obstruct Pulmon Dis 5:217–222
Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P,
Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C,
Zielinski J (2007) Global strategy for the diagnosis, manage-
ment, and prevention of chronic obstructive pulmonary disease:
GOLD executive summary. Am J Respir Crit Care Med
176:532–555. doi:10.1164/rccm.200703-456SO
Repapi E, Sayers I, Wain LV, Burton PR, Johnson T, Obeidat M, Zhao
JH, Ramasamy A, Zhai G, Vitart V, Huffman JE, Igl W, Albrecht
E, Deloukas P, Henderson J, Granell R, McArdle WL, Rudnicka
AR, Barroso I, Loos RJ, Wareham NJ, Mustelin L, Rantanen T,
Surakka I, Imboden M, Wichmann HE, Grkovic I, Jankovic S,
Zgaga L, Hartikainen AL, Peltonen L, Gyllensten U, Johansson
A, Zaboli G, Campbell H, Wild SH, Wilson JF, Glaser S, Homuth
G, Volzke H, Mangino M, Soranzo N, Spector TD, Polasek O,
Rudan I, Wright AF, Heliovaara M, Ripatti S, Pouta A, Naluai
AT, Olin AC, Toren K, Cooper MN, James AL, Palmer LJ,
Hingorani AD, Wannamethee SG, Whincup PH, Smith GD,
Ebrahim S, McKeever TM, Pavord ID, MacLeod AK, Morris
AD, Porteous DJ, Cooper C, Dennison E, Shaheen S, Karrasch S,
Schnabel E, Schulz H, Grallert H, Bouatia-Naji N, Delplanque J,
Hum Genet (2013) 132:451–460 459
123
Author's personal copy

Froguel P, Blakey JD, Britton JR, Morris RW, Holloway JW,
Lawlor DA, Hui J, Nyberg F, Jarvelin MR, Jackson C, Kahonen
M, Kaprio J, Probst-Hensch NM, Koch B, Hayward C, Evans
DM, Elliott P, Strachan DP, Hall IP, Tobin MD (2010) Genome-
wide association study identifies five loci associated with lung
function. Nat Genet 42:36–44. doi:10.1038/ng.501
Sampath V, Le M, Lane L, Patel AL, Cohen JD, Simpson PM,
Garland JS, Hines RN (2011) The NFKB1 (g.-24519delATTG)
variant is associated with necrotizing enterocolitis (NEC) in
premature infants. J Surg Res 169:e51–e57. doi:10.1016/j.jss.
2011.03.017
Song S, Chen D, Lu J, Liao J, Luo Y, Yang Z, Fu X, Fan X, Wei Y,
Yang L, Wang L, Wang J (2011) NFkappaB1 and NFkappaBIA
polymorphisms are associated with increased risk for sporadic
colorectal cancer in a southern Chinese population. PLoS ONE
6:e21726. doi:10.1371/journal.pone.0021726
Tang X, Liu D, Shishodia S, Ozburn N, Behrens C, Lee JJ, Hong WK,
Aggarwal BB, Wistuba II (2006) Nuclear factor-kappaB (NF-
kappaB) is frequently expressed in lung cancer and preneoplastic
lesions. Cancer 107:2637–2646. doi:10.1002/cncr.22315
Tanni SE, Pelegrino NR, Angeleli AY, Correa C, Godoy I (2010)
Smoking status and tumor necrosis factor-alpha mediated
systemic inflammation in COPD patients. J Inflamm (Lond)
7:29. doi:10.1186/1476-9255-7-29
Yang L, Li Y, Cheng M, Huang D, Zheng J, Liu B, Ling X, Li Q,
Zhang X, Ji W, Zhou Y, Lu J (2012a) A functional polymor-
phism at microRNA-629-binding site in the 30-untranslated
region of NBS1 gene confers an increased risk of lung cancer in
Southern and Eastern Chinese population. Carcinogenesis
33:338–347. doi:10.1093/carcin/bgr272
Yang L, Qiu F, Lu X, Huang D, Ma G, Guo Y, Hu M, Zhou Y, Pan M,
Tan Y, Zhong H, Ji W, Wei Q, Ran P, Zhong N, Lu J (2012b)
Functional polymorphisms of CHRNA3 predict risks of chronic
obstructive pulmonary disease and lung cancer in Chinese. PLoS
ONE 7:e46071. doi:10.1371/journal.pone.0046071
Zhang H, Cai B (2003) The impact of tobacco on lung health in
China. Respirology 8:17–21
Zhang P, Wei Q, Li X, Wang K, Zeng H, Bu H, Li H (2009) A
functional insertion/deletion polymorphism in the promoter region
of the NFKB1 gene increases susceptibility for prostate cancer.
Cancer Genet Cytogenet 191:73–77. doi:10.1016/j.cancergencyto.
2009.01.017
Zhong N, Wang C, Yao W, Chen P, Kang J, Huang S, Chen B, Ni D,
Zhou Y, Liu S, Wang X, Wang D, Lu J, Zheng J, Ran P (2007)
Prevalence of chronic obstructive pulmonary disease in China: a
large, population-based survey. Am J Respir Crit Care Med
176:753–760. doi:10.1164/rccm.200612-1749OC
Zhou Y, Hu G, Wang D, Wang S, Wang Y, Liu Z, Hu J, Shi Z, Peng
G, Liu S, Lu J, Zheng J, Wang J, Zhong N, Ran P (2010)
Community based integrated intervention for prevention and
management of chronic obstructive pulmonary disease (COPD)
in Guangdong, China: cluster randomised controlled trial. BMJ
341:c6387. doi:10.1136/bmj.c6387
Zou YF, Wang F, Feng XL, Tao JH, Zhu JM, Pan FM, Su H (2011)
Association of NFKB1 -94ins/delATTG promoter polymorphism
with susceptibility to autoimmune and inflammatory diseases: a
meta-analysis. TissueAntigens 77:9–17. doi:10.1111/j.1399-0039.
2010.01559.x
460 Hum Genet (2013) 132:451–460
123
Author's personal copy