Abstract
Background
A host of studies have explored the potential connection between leptin (LEP) G19A polymorphism and the risk of cancers, but the relationship between gastric cancer (GC) susceptibility and LEP G19A polymorphism was not revealed before. The aim of this study was to investigate this relationship in Chinese Han population.
Methods
Thus, this case–control study with 380 GC cases and 465 controls was designed to unearth the link between LEP G19A polymorphism and GC susceptibility. Genotyping was accomplished by a custom-made 48-Plex SNP scanTM kit. Relative LEP gene expression was detected by real-time reverse transcription-polymerase chain reaction.
Results
LEP G19A polymorphism was shown to relate with a decreased risk of GC. Subgroup analyses uncovered significant connections in the males, nondrinkers, and those at age <60 years. G19A polymorphism was also linked with tumor size and location and pathological type of GC. Last, LEP gene expression in gastric tissues was considerably less than in control tissues.
Conclusion
This study shows that G19A polymorphism of LEP gene is linked with a lower risk of GC in the tested Chinese Han individuals.
Introduction
Gastric cancer (GC) is the 3th dominant cause of cancer mortality and has the 5th highest incidence among cancers worldwide.Citation1 The GC incidence and mortality rates are the highest in East Asia, and about 679,100 new GC patients and 498,000 cancer-associated deaths were reported in 2015 in China.Citation2 GC has two anatomical types: gastro-oesophageal-junction adenocarcinoma and true gastric adenocarcinoma,Citation3 and is histologically separated into diffuse and intestinal types. GC is a multi-step complex disorder due to both environmental and genetic factors.Citation4 Tobacco smoking, alcohol consumption, Helicobacter pylori (HP) infection, and unhealthy dietary habits including low consumption of vegetables and fruits can induce the risk of GC.Citation5–Citation7 Genetic factors are also critical in GC progression. Genome-wide association research has recognized several gene polymorphisms are linked with the risk of GC.Citation8–Citation10
Leptin (LEP), a hormone of energy expenditure, is secreted by white adipose tissues and is associated with endocrinologic metabolism.Citation11 It is expressed in the hypothalamus and regulates appetite and energy expenditure.Citation12 LEP is engaged in energy homeostasis, insulin signaling, immune response, and inflammation.Citation13–Citation15 Reduced sensitivity to LEP may result in the development of metabolic disorders, such as cancers.Citation16 LEP and its receptor are involved in various carcinogenesis-related signal pathways, including MAPK, PI3K, mTOR, and JAK/STAT.Citation17,Citation18 Increased LEP level is involved in the development of many malignancies.Citation19–Citation21 As for GC, LEP plays an important role in GC via stimulating the proliferation of GC cells through activating the ERK1/2 and STAT3 pathways.Citation22 LEP can also enhance GC cell migration by increasing ICAM-1.Citation23 Recombinant human LEP could induce apoptosis and inhibit growth in human GC cell lines.Citation24 Gastric LEP performed diverse functions including nutrient absorption and tumorigenesis in the gastrointestinal system.Citation25 LEP could induce the expression of tumorigenic genes in the gastric mucosa of male rats.Citation26 Dietary fat-accelerating LEP signaling was shown to promote protumorigenic gastric environment in mice.Citation27 Overexpression of LEP was linked with the development of GC in humans and murine.Citation25,Citation28 Serum LEP levels were related with insulin resistance in GC patientsCitation29 and may be a valuable diagnostic indicator.Citation30 LEP is connected with chemotherapy resistance and therapy-independent prognosis of GC.Citation31 Besides, LEP was correlated with the progression and prognosis of GC patients. Citation32
G19A polymorphism, a SNP in the 5ʹ-untranslated region of LEP gene, may impact RNA transcription, translation and steadiness, and change LEP protein expression. This polymorphism was correlated to cancer risk.Citation33–Citation45 However, the existing findings in different types of cancers are discrepant. Furthermore, no researchers have studied the relationship between G19A polymorphism and the risk of GC. To explore such potential relationships, we performed this study to explore this relationship in Chinese Han subjects.
Patients and Methods
Subjects
Totally 380 newly identified GC cases and 465 age- and gender-matched controls were enrolled from Danyang People’s Hospital (Jiangsu, China) from March 2016 to January 2020. All GC patients received surgery treatment. GC tissues and normal tissues were obtained during surgery, which were stored at −80°C after surgery. GC was diagnosed according to pathological examination results. Exclusion criteria for GC patients included: 1) a history of other cancers; 2) patients with gastritis; 3) patients with gastric ulcers; 4) patients not providing enough data and written informed consent. The age and gender-matched control individuals receiving health examinations were recruited from the hospital at the same period. Exclusion criteria for controls were as follows: patients 1) with a history of cancers; 2) with metabolic diseases; 3) with autoimmune diseases. 4) receiving no gastroscopy or having symptoms of gastritis. The demographic characteristics and lifestyle habits of all individuals were collected by a structured questionnaire. Medical records provided the clinical data of GC patients. All subjects completed a C-urea breath test to measure whether they had HP infection. The gastric tissues for RT_qPCR were not fresh because these tissues did not be used immediately after surgery. All individuals signed written informed consent in this study. Approval was obtained from the Ethics Committee of this Hospital (number: 20,200,618), and the Declaration of Helsinki was followed.
Genotyping
The blood samples of participants were stored at −80°C immediately. DNA sample was from peripheral leukocytes using a TIANamp blood DNA kit (Qiagen, Hilden, Germany). LEP G19A polymorphism was genotyped using a custom-by-design 48-Plex SNP scanTM kit (Genesky Biotechnologies Inc., Shanghai, China). Each PCR system (50 μL) contained 5 μL of 10×PCR buffer for KOD-Plus-Neo, 1 μL of upstream and downstream primers, 34 μL of ddH2O, 1 μL of temple, 2 mM dNTPs (5 μL), 1 μL of cDNA, 25 mM MgSO4 (3 μL), and 1 μL of KOD-Plus-Neo (TOYOBO, Japan). Reaction conditions were 95 ºC, 5 min; 94 ºC, 30 s, 50 ºC, 30 s; 72 ºC, 1 min, 35 cycles; extension at 72 ºC, 10 min, cooling to 4 ºC. The PCR products were digested with BglI (New England Biolabs, Beverly, MA) at 37°C for 5 h and then sent to 2% agarose gel electrophoresis. About 10% of the samples were repeatedly genotyped, and the concordance of genotypes was 100%.Citation46,Citation47
Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
The gastric tissues of participants were stored at −80°C. Total RNA was isolated from the gastric tissues from all subjects using the Trizol reagent (Invitrogen, CA, USA) and was reverse-transcribed with a relevant kit (TaKaRa, Shiga, Japan) as instructed by the manufacturers. The SYBR Green PCR master mix (TaKaRa, Otsu, Shiga, Japan) was used for real-time PCR. Forward and reverse primers used for PCR were: 5ʹ-CGTTAAGGGAAGGAACTCTGG-3ʹ, 5ʹ-TTGATGGCTGAAGACCTTGG-3ʹ (LEP); 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ, 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ (β-actin). The PCR condition was: an initial 94°C for 2 min; 35 cycles, 94°C, 30 s, 60°C, 25 s, and 72°C, 30 s; final extension at 72°C, 10 min. The relative expression of LEP gene was calculated using the 2−ΔΔCT method.
Statistical Analysis
Descriptive variables of GC patients and controls were shown as the mean and standard deviations for continuous variables, and frequencies and percentages for categorical variables. The Chi-square test was applied to compare the case–control differences in the distributions of genotypic frequencies and categorical variables. Student’s t-test was adopted for continuous variables. The Hardy–Weinberg equilibrium (HWE) test was examined among the controls for LEP G19A polymorphism. The odds ratios (ORs) and 95% confidence interval (CI) were computed via logistic regression.Citation48 Five genetic models including dominant model, homozygote model, recessive model, heterozygote model, and allele model were utilized in this study. Stratified analyses by sex, age, smoking and alcohol were done. We addressed the association of LEP G19A polymorphism with clinical features of GC patients. P < 0.05 was the significance level. All data analyses were finished on SPSS 22.0 (SPSS Inc., USA).
Results
Population Characteristics
A flowchart of patient enrollment is shown in . Totally 380 GC patients and 465 controls were involved. Clinical data are listed in . Data showed no remarkable discrepancy between cases and controls in age, sex, alcohol, or smoking. The percentage of HP infection was higher in GC patients than the controls. Additionally, LEP gene level in gastric cancer tissues versus normal tissues was significantly lower ().
Figure 1 The flowchart of patient enrollment.
Figure 2 The LEP mRNA expression in gastric tissues and normal tissues. ***The LEP mRNA expression in gastric tissues was significantly lower than those in normal tissues (P < 0.001).
Table 1 Patient Demographics and Risk Factors for Gastric Cancer
Association of LEP G19A Polymorphism with GC Risk
LEP G19A polymorphism was in line with HWE (P > 0.05). Data found that the AA genotype (OR: 0.50; 95% CI: 0.26–0.97; P = 0.040) or A allele (OR: 0.73; 95% CI: 0.58–0.93; P = 0.009) of G19A polymorphism showed a less risk for GC (). These associations hold true even after adjusting for age and sex. Stratified analyses demonstrated that this polymorphism was linked to a lower risk of GC patients among the non-drinkers, males, and those at age <60 years (). No significant associations were obtained in subgroup analysis of smoking and HP status.
Table 2 Genotype Frequencies of LEP G19A Polymorphism in Cases and Controls
Table 3 Stratified Analyses Between LEP G19A Polymorphism and the Risk of Gastric Cancer
Relationship Between LEP G19A Polymorphism and Clinical Features of GC
Last, we addressed the relationship between G19A polymorphism of LEP gene and GC clinical features (). The SNP was related to tumor size, cardia GC and adenocarcinoma, but not to histological grade, R classification, or TNM stage.
Table 4 The Associations Between LEP G19A Polymorphism and Clinical Characteristics of Gastric Cancer
Discussion
In this case–control study, we observed that LEP G19A polymorphism was correlated with less risk for GC in Chinese subjects. Next, this polymorphism can lower the risk of GC patients among non-drinkers, males, and those at age <60 years. In addition, LEP G19A polymorphism showed connection with tumor size, cardia GC and adenocarcinoma. Last, LEP gene level in gastric tissues was remarkably lower than in normal tissues.
Some recent case–control studies have probed into the possible relationship between LEP G19A polymorphism and the risk of various cancers. Tsilidis et al observed no association between this locus and CRC susceptibility in a Caucasian population from the USA.Citation33 Later, a study with 1567 cases and 1965 controls from the USA indicated that G19A polymorphism of LEP gene was related with lower risk for colon cancer.Citation34 Partida-Perez et al suggested that this SNP was associated with colorectal cancer risk in Mexican patients.Citation35 As for esophageal cancer, an Australian study showed LEP G19A polymorphism was inactive for esophageal carcinogenesis,Citation36 which was in line with the findings of a Chinese study.Citation37 However, another Chinese study indicated that LEP G19A polymorphism contributed to less risk for esophagogastric junction adenocarcinoma.Citation38 As for prostate cancer, two studies obtained it was not heavily associated with LEP G19A polymorphism.Citation39,Citation40 Next, several studies addressing the relationship between non-Hodgkin’s lymphoma (NHL) risk and this polymorphism yielded conflicting findings. Willett et al suggested that G19A polymorphism was a protective factor for NHL;Citation41 Skibola et al found this polymorphism increased the risk of NHL.Citation42 Nevertheless, a Chinese study revealed that G19A polymorphism was not associated with susceptibility to NHL.Citation43 In addition, Kim et alCitation44 observed no connection between G19A polymorphism and breast cancer risk and Zhang et al showed this SNP was associated with the risk of hepatocellular carcinoma.Citation45 Obviously, these studies obtained inconsistent results in different cancers regarding LEP G19A polymorphism. Some factors may account for paradoxical results. First, genetic heterogeneity existed in different cancers, and LEP G19A polymorphism may be a specific locus for some cancers. Second, clinical heterogeneity and third, the varying sample sizes may contribute to it. Fourth, different races are neglectable. Fifth, exposure factors and grade malignancy of cancers are different.
We firstly found that LEP G19A polymorphism was connected to decreased risk for GC. Data suggested that AA genotype or A allele carriers decreased the risk of GC. However, another study observed that AA genotype was a risk factor for other cancers.Citation45 We assumed that LEP G19A polymorphism may play diverse roles in different cancers. To our knowledge, this is the first study to uncover the significant link between this SNP and GC susceptibility. Savino et al indicated LEP G19A polymorphism was linked with LEP down-expression.Citation49 In addition, serum LEP downregulation may be a protective factor and predict good prognosis for breast cancer patients.Citation50 Thereby, we assume that lower serum LEP levels related to genotypes of LEP G19A polymorphism may contribute to decreased risk for GC. However, further studies are needed to verify this assumption. Furthermore, subgroup analysis found that LEP G19A polymorphism was linked with a less risk for GC patients among the males, non-drinkers, and those aged <60 years, suggesting that those individuals are less susceptible to GC. On the contrary, the females, drinkers, and those aged >60 years may be prone to GC. From the view of GC management or prevention, those groups need to receive gastroscopy or other early examinations, which could help with early intervention on the occurrence of GC. Analyses concerning the relationship between LEP G19A polymorphism and clinical characteristics of GC patients found that this SNP was linked with the tumor size, cardia GC and adenocarcinoma. Furthermore, AA genotype was a protective factor for GC patients with smaller tumor size, non-cardia, and non-adenocarcinoma.
Limitations of this study existed. First, the relatively small sample size may yield false-positive results. Second, we did not investigate other SNPs of LEP gene. Third, gene-environment or gene–gene interaction was ignored. Fourth, the underlying mechanisms of the LEP G19A polymorphism in GC risk should be discovered. Fifth, we did collect the follow-up data of GC patients previously. Thus, we could not explore the correlation between GC prognosis and LEP G19A polymorphism at recent stage. Last, our findings should be validated in other populations in China and other races.
In sum, LEP G19A polymorphism is linked with a lower risk of GC in Chinese individuals. Overall, our findings may be generally helpful for early screening of individuals at high-risk of GC in Chinese Han population. Further studies are needed, which will help to comprehensively elucidate the potential role of LEP G19A polymorphism in the pathogenesis of GC.
Disclosure
The authors report no conflicts of interest in this work.
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