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Association of calcium/calmodulin-dependent protein kinase kinase1 rs7214723 polymorphism with lung cancer risk in a

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Chinese population Da Chen1, Fangming Zhong1, Ye Chen2* 1

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Huanchengdong Road, Hangzhou, Zhejiang, China. 2

Department of Pneumology, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China

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*Correspondence

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+86-0571-85632119; Fax: +86-0571-86574316

to:

Ye

Chen;

E-mail:

[email protected];

Tel:

Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press ( http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).

ACCEPTED MANUSCRIPT

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The Cardiothoracic Surgery Department, Hang Zhou Red Cross Hospital, 208

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Abstract

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Calcium/calmodulin-dependent protein kinase kinase1 (CAMKK1) could specially

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recognize and activate Calcium/calmodulin-dependent protein kinase (CAMK) I and

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IV. Furthermore, The activation of CAMK showed positively correlation with

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proliferation of lung cancer (LC). In addition, A genome-wide association study has

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identified rs7214723 (E375G) in the CAMKK1 gene as a susceptibility locus for LC

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in the UK population. Therefore, we conducted a case-control study involving 320 LC

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patients and 320 controls to validate this conclusion in a Chinese population.

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Genotyping was performed using a custom-by-design 48-Plex single nucleotide

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polymorphism (SNP) Scan™ Kit. Our results indicate that the individuals with CC

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genotype of rs7214723 polymorphism had the higher risk of LC than those who

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carried TT genotype. Moreover, CAMKK1 rs7214723 polymorphism showed

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positively correlation with the elevated risk of LC in the allelic model and recessive

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model, but not in the dominant model. Stratified analysis further confirmed this

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significant association in male groups and smokers. In conclusion, CAMKK1

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rs7214723 polymorphism may be associated with the increased risk of LC. However,

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larger studies with more diverse ethnic populations are needed to confirm these

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results.

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Keywords: CAMKK1, polymorphism, lung cancer, molecular epidemiology

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Introduction

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Lung cancer (LC) is the leading cause of cancer death with high incidence rate

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[1]. Due to complex biological characteristics, lung cancer has extremely difficulty in

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diagnosis and treatment at early stage [2]. In 2007, new 222,500 LC cases and

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155,870 LC deaths are expected to occur in United States [3]. Tobacco using, air

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pollution, exposure to carcinogens, genetic factors and other factors can cause LC [4,

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5]. In addition, genetic factors play an important role in modifying an individual’s risk

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for LC.

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The calcium/calmodulin dependent protein kinase kinase (CAMKK) gene is

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located on chromosome 17p13.2 and has 19 exons. CAMKKs phosphorylate and

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activate specific downstream protein kinases, including calcium/calmodulin

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dependent protein kinase (CAMK) I, CAMK IV, and 5'-AMP-activated protein kinase

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(AMPK), which mediates a variety of Ca2+ signaling cascades [6]. CAMK I was

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demonstrated to be associated with the proliferation of LC cell lines [7]. Moreover,

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Williams et al. demonstrated that the activation of CAMK (CAMK Ⅱ and CAMK IV)

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inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells [8].

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Furthermore, Shao et al. revealed that the activation of MAPK contributes to growth

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inhibition and apoptosis in human LC cell [9]. There is a wide consensus that

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CAMKK have been shown to undergo autophosphorylation and contains two

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isoforms (CAMKK1 and CAMKK2) [10]. Therefore, we guessed that CAMMK1, a

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member of CAMKK, may play a role in the development of LC indirectly.

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Rs7214723, a T to C transition, leads to a glutamate (E) to glycine (G)

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substitution at the amino acid position 375. A genome-wide association study (GWAS)

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has identified rs7214723 (E375G) polymorphism of CAMKK1 gene as a

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susceptibility locus for LC in UK Caucasians [11]. Subsequently, Truong et al. tended 3

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to replicate this finding, but failed to find significant association between this single

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nuclear polymorphism (SNP) and LC risk among Caucasians and Asians [12]. In 2013,

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Zhang et al. found CAMKK1 rs7214723 polymorphism contribute to LC risk in a

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Chinese population and the T allele of rs7214723 could be viewed as a risk allele for

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LC [13]. Notably, there were two contradictory findings in the Asian populations.

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Therefore, we conducted a hospital-based study with 320 cases and 320 controls to

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validate the role of CAMKK1 rs7214723 polymorphism in modifying the risk of LC.

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Patients and methods

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Study subjects

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A total of 320 patients diagnosed with lung cancer were consecutively recruited

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from the Second Affiliated Hospital of Zhejiang Chinese Medical University, between

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September 2014 and October 2016. The subjects with family history of cancer and

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biochemical abnormalities did not conform to our inclusion criteria. The healthy

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controls were free of LC and recruited from the same institutions during the same

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time period. They were frequency matched (1:1) to the LC cases based on sex and age

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(± 5 years), A detailed questionnaire related to smoking habits was completed for each

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patient and control by a trained interviewer. Informed consent was obtained from all

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patients and controls prior to their participation. The protocol for this study was

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approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang

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Chinese Medical University (Hangzhou, Zhejiang, China).

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DNA extraction and Genotyping To investigate the polymorphism of CAMKK1, all study participants provided 2 4

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mL of peripheral blood in ethylenediaminetetraacetic acid (EDTA) tubes and stored at

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-80 ℃ until use. DNA was extracted by using the QIAamp DNA Blood Mini Kit

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(Qiagen,

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custom-by-design 48-Plex SNP scanTM Kit (Genesky Biotechnologies Inc., Shanghai,

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China), which was described in previous case-control studies [14, 15]. This kit was

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developed according to patented SNP genotyping technology by Genesky

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Biotechnologies Inc., which was based on double ligation and multiplex fluorescence

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PCR. For quality control, repeated analyses were done for 4% of randomly selected

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samples with high DNA quality.

Hilden,

Germany).

SNP

genotyping

was

performed

using

a

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Statistical analysis

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Hardy–Weinberg equilibrium (HWE) for CAMKK1 genotype distributions in

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controls was tested by a goodness-of-fit chi-squared test. The demographic and

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clinical characteristics of study participants were evaluated by using the chi-squared

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test. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the

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association between CAMKK1 gene polymorphisms and risk of LC by logistic

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regression analyses. The most common homozygote was seen as a reference group.

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All statistical analyses were performed using the SPSS ver22.0 software package. P
A polymorphism was associated with a decreased risk of

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esophageal cancer in a Chinese population. Clin Biochem. 46, 1469-1473.

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Witczak, C.A., N. Fujii, M.F. Hirshman, and L.J. Goodyear. (2007)

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Ca2+/calmodulin-dependent protein kinase kinase-alpha regulates skeletal

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muscle glucose uptake independent of AMP-activated protein kinase and Akt

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activation. Diabetes. 56, 1403-1409.

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Mizuno, K., L. Ris, A. Sanchez-Capelo, E. Godaux, and K.P. Giese. (2006)

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Ca2+/calmodulin kinase kinase alpha is dispensable for brain development but

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is required for distinct memories in male, though not in female, mice. Mol Cell

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Biol. 26, 9094-9104.

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Okuno, S., T. Kitani, and H. Fujisawa. (1997) Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha. J 11

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Biochem. 122, 337-343.

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Table 1 Patient demographics and risk factors in lung cancer Variable Cases (n=320) Age (years) ≤50 122 >50 198 Sex Female 70(21.9%) Male 250(78.1%) Smoking status Nonsmoker 65(20.3%) Smoke 255(79.7%) Histology Squamous cell carcinoma 180(56.3%) Adenocarcinoma 94(29.4%) others 46(14.4%)

Controls (n=320)

P

102 218

0.369

75(23.4%) 245(76.6%)

0.495

78(24.4%) 242(75.6%)

0.217

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Table 2 Logistic regression analysis of associations between CAMKK1 rs7214723 polymorphism and risk of lung cancer Genotype Cases*(n=320) Controls*(n=320) OR (95% CI) n % n % TC vs. TT 122/133 38.1/41.6 130/149 40.6/46.6 1.05 (0.75–1.48) CC vs. TT 61/133 19.1/41.6 38/149 11.9/46.6 1.80 (1.13-2.87) CC vs. TC vs. TT TC+CC vs. TT 183/133 57.2/41.6 168/149 52.5/46.6 1.22 (0.89-1.67) CC vs. TC+TT 61/255 19.1/79.9 38/279 11.9/87.2 1.76 (1.13-2.73) C vs. T 244/388 38.1/60.6 206/428 32.2/66.9 1.31(1.04-1.65) *The genotyping was successful in 316 cases and 317 controls. Bold values are statistically significant (P

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