Lung cancer is the leading cause of cancer death in China and across the globe. Statistics show that there are about 18.1 million malignant tumors in 2018 and 9.6 million deaths of malignant tumors worldwide. Lung cancer is the most commonly diagnosed cancer (2.1 million new cases, 11.6% of the total cancer cases) and the leading cause of cancer death (1.8 million deaths, 18.4% of the total cancer deaths). The incidence and death rates will continue to rise, mainly as a result of an increase in global tobacco use, particularly in Asia. Non-small cell lung cancer (NSCLC) accounts for approximately 80% to 85% of lung cancer cases. NSCLC is not diagnosed until advanced-stage disease is present, resulting in delays that may adversely affect survival. Despite advances in the treatment of NSCLC in recent years, the prognosis for NSCLC patients is still poor compared with the other types of cancer (eg, breast cancer and colorectal cancer), especially in China, with the 5-year survival rate < 20%. Therefore, it is of great significance to identify the biomarkers of NSCLC in order to identify individuals at high risk.
Retinol-binding protein 4 (RBP4) is a secretory molecule in the RBP family synthesized in the liver. It is secreted mainly by adipocytes and hepatocytes and has an important effect in assisting retinol to play its normal physiological function. It has been shown that circulating RBP4 has been implicated as a mediator in the development of insulin resistance and the metabolic disease. Animal study in mice suggested that elevated RBP4 levels may play an important role in the development of insulin resistance. RBP4 involves in the occurrence of insulin resistance and obesity, which can lead to disorders of glycolipid metabolism, and plays an important role in fatty liver, diabetes and cardiovascular diseases.[4–6]
In addition as a transport protein, RBP4 might also serve as a signaling molecule binding to the membrane receptor stimulated by retinoic acid 6 and triggering downstream activation of pro-oncogenic pathways, and thus RBP4 may be associated with cancer risk. Epidemiological studies showed that RBP4 was related to the occurrence and development of various malignant tumors, and it was highly expressed in liver cancer, pancreatic cancer, acute leukemia and other tumors.[7–10] For example, comparing with those in the lowest quartile, participants in the highest quartile of serum RBP4 levels was associated with 2.07 (95% confidence interval [CI]: 1.07–4.00) times increased risk of breast cancer. However, there is no report on the relationship between RBP4 and NSCLC.
Thus, this study aimed to assess association between the level of serum RBP4 and the risk factors of NSCLC through a case-control study among a Chinese population. In addition, we also investigated whether the association between serum RBP4 levels and NSCLC risk was influence by body mass index (BMI) and smoking status.
2.1 Study population
This was a case-control study conducted between June 2018 and June 2019. The NSCLC cases recruited from Central Hospital of Xiaogan. Inclusion criteria for NSCLC cases:
- (1) new cases (no more than 3 months prior to the diagnosis) of NSCLC without treatment, the first diagnosis time was between August 2017 and January 2019;
- (2) confirmed by histological examination from the physician and medical records of hospitals at or above the county level; and
- (3) signed informed consent, voluntary participation in research and good compliance. Participants were excluded if they simultaneously suffered a history of any other types of cancer.
Frequency matching method was used to select healthy residents who were similar to the case group in sex (5-year interval) and age, and who lived in the same area for more than 5 years as the control group. Except for a diagnosis, the same selection criteria for the cases were applied for controls. In total, 256 pair cases and controls were successfully recruited and had blood samples.
A written informed consent form was signed by all study participants. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and was approved by the Ethical Committee of Xiaogan Central Hospital.
2.2 Data collection
Investigators used a unified questionnaire to investigate the general situation (e.g., gender, age, height, nationality, education level, and occupation), family history of NSCLC and behavioral factors (eg, smoking status). Individuals were grouped as current smokers if a person who has smoked daily or occasionally in the last 30 days for at least 6 months, or as former smokers if a person who has a history of smoking for at least 6 months and currently stopped. Height and weight were measured and BMI was calculated by dividing weight (kg) by height (m2).
The subjects were all in the morning state of fasting, 5 mL of peripheral venous blood was collected in a vacuum tube containing ethylenediaminetetraacetic acid and centrifuged at room temperature for 3 000 r/min for 10 minute. The upper serum was taken for measurement within 4 hours. Serum lipids, such as total cholesterol (TC), high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, and total triglycerides (TG), as well as fasting glucose (FBG) were measured with a Hitachi 7600–010 automatic analyzer.
The serum RBP4 level was determined by enzyme-linked immune absorbent assay. The kit was purchased from Kanto Chemical Company of Japan. Inter- and intra-assay coefficients of variation (Epercentages) were 5.9% and 3.0%.
2.3 Statistical analysis
The survey data was carried out using SPSS 21.0 software. Means and standard deviation was used for continuous variables, and independent t-test was used for comparison between the 2 groups. Categorical variables were expressed by presented as number and percentage and the difference was assessed using Pearson Chi square test. Serum RBP4 was categorized into tertiles (T1–T3) based on the distribution among controls for men and women separately.
We used unconditional logistic regression models to assess the odds ratios (ORs) and 95% CIs for the associations between serum RBP4 and NSCLC risk, using the lowest tertile as the reference group. Potential confounders were selected into multivariable adjusted models. In model 1, we only adjusted age and gender. In model 2, we further adjusted BMI, smoking status, education, household income, family history of cancer, TC, TG, HDL, LDL, and FBG. Tests for trend were calculated by including the categorical variables as continuous variables.
Stratified analysis by BMI (<25 kg/m2 vs ≥25 kg/m2) and smoking status (current, ever or never) were also conducted. The interaction was assessed using the likelihood ratio test with a multiplicative interaction term.
Statistical analyses were conducted using the SPSS 13.0 software (Chicago, IL). Significance was set at 0.05 (2-sided).
The characteristics for all patients are displayed in Table 1. NSCLC cases were more likely to report higher proportions of current smoking and family history of NSCLC than those in control group (all P < .05). The serum levels of TC, TG, LDL and RBP4 in the case group were higher than those in the control group (all P < .05), and HDL was lower than those in the control group (P < .05). There were no significant differences in sex, age, height, nationality, BMI, BMI grade, education level, occupation and FBG between the 2 groups (all P > .05). The mean serum RBP4 level was (36.05 ± 8.28) ug/mL for cases and (29.54 ± 7.71) ug/mL for controls, and the difference was significant (P < .001).
After adjusting age and sex (Table 2), participants with higher serum RBP4 level had increased risk of NSCLC (P trend < .001). The associations were attenuated but remained significant after further adjustments (P trend = .001) (Table 2). Comparing participants in the highest tertile, the odds ratio (95% CI) for those in lowest tertile were 1.85 (95% CIs 1.07–3.2) (P = .029) for the second tertile and 2.18 (95% CIs 1.37–3.45) (P = .001) for the highest tertile, respectively.
Multivariate linear regression analysis was conducted with RBP4 as strain and metabolic indices and basic clinical characteristics as independent variables (Table 3). The level of RBP4 in serum of NSCLC patients and controls were both positively correlated with BMI (P = .003 and .048) and the level of RBP4 in control group was also positively correlated with TG (P = .041).
Stratified analysis by BMI showed that serum RBP4 level was positively associated with NSCLC risk among those with BMI < 25 kg/m2 and ≥25 kg/m2, whereas no significant association was found (P trends: .002 and .012; P interaction = .584) (Table 4). Similarly, after stratification by smoking status, higher serum RBP4 level was associated with increased NSCLC risk irrespective of smoking status, and the P trends were .015, .024, and .020 (P interaction = .357).
In this case–control study, a significant positive association was found between serum RBP4 level and the risk of NSCLC. Stratified analysis by BMI and smoking status showed that the positive association of RBP4 level with NSCLC risk was found for all strata.
NSCLC is a serious threat to the health of every individual around the globe. The etiology of NSCLC is not completely clear up to now. A lot of data shows that there is a very close relationship between long-term smoking and the occurrence of NSCLC. Proven by previous studies, the probability of NSCLC in long-term heavy smokers is 10–20 times higher than that in non-smokers, and the younger the age at which smokers begin to smoke, the higher the risk of NSCLC.[11,12] Furthermore, smoking not only directly affects personal health, but also has a negative impact on the health of the surrounding population, leading to a significant increase in the incidence of NSCLC among passive smokers.[13,14] The incidence of NSCLC in urban residents is higher than that in rural areas, which may be related to urban air pollution and the carcinogens contained in smoke and dust. Therefore, non-smoking should be advocated and urban environmental sanitation should be strengthened. Likewise, ionizing radiation, gene and other lung diseases such as pulmonary tuberculosis and bronchiectasis increase the risk of NSCLC.[16–18] This study shows that family history and smoking history of NSCLC can increase the incidence of NSCLC, which is consistent with many studies.And the serum lipids TC, TG, LDL, RBP4 in NSCLC patients are higher than those in normal people, and HDL is lower than that of normal people. It shows that hyperlipidemia is related to the incidence of NSCLC.
Early diagnosis of NSCLC is of great significance because only by early diagnosis and treatment of NSCLC can we obtain better curative effect. There is no typical symptom in the early stage of NSCLC, chest X-ray screening should be carried out regularly for people over 40 years old, and patients with primary or metastatic symptoms of NSCLC should be examined by chest X-ray or chest CT in time. When lung mass shadow is found, the diagnosis of NSCLC should be considered first, and further examination should be made to make a definite diagnosis through histopathological examination. At present, there is no good serum markers for diagnosis and differential diagnosis of NSCLC, so it is very important to find serum markers for diagnosis of NSCLC.
RBP4 is a kind of protein responsible for binding and transporting active metabolites of vitamin A retinol in vivo, which plays an important role in assisting vitamin A to play its physiological role. RBP4 can specifically bind all-trans retinol to vitamins and it is mainly synthesized by hepatocytes, followed by adipocytes. After RBP4 was released into blood, it binds to retinol and thyroxine carrier proteins and forms a retinol-RBP4-TTR ternary complex in the form of 1:1:1, which transports retinol to target tissues for specific physiological functions. Early studies found that RBP4 was associated with fatty liver, glycolipid metabolic diseases such as diabetes mellitus and cardiovascular diseases.[4,24] It was deemed that RBP4 was a fat-derived factor in the blood and was considered as an important target for the treatment of type 2 diabetes mellitus (T2DM), insulin resistance, non-alcoholic fatty liver and visceral obesity. Yamaaki et al indicated that the level of RBP4 in serum was independently and positively correlated with remnant-like particles triglyceride in subjects with T2DM, which suggested that RBP4 was involved in regulating the pathway of glucose metabolism in patients with T2DM. It has been reported that a region near the RBP4 gene on human chromosome 10q is closely related to the high risk of T2DM. Saucedo et al used data from100 pregnant women and 100 normal women, the results showed that the 2 variant genotypes of RBP4 (rs3758539 and rs34571439) were closely related to insulin level and insulin resistance in gestational diabetic women. Healthy non-diabetic high-risk population, Bose et al showed that high levels of serum RBP4 was positively correlated with serum insulin and blood sugar, which could be used as a predictor of diabetes. In recent years, studies have shown that RBP4 is related to the occurrence and development of tumors. RBP4 is highly expressed in many malignant tumors, such as breast cancer, pancreatic cancer, colon cancer and is related to the invasion and metastasis of tumors. Studies have shown that the higher the expression level of RBP in ovarian cancer, the higher its metastasis and invasion ability.
The results showed that the level of RBP4 was correlated with the risk of NSCLC, and the risk of NSCLC rose with the increase of RBP4 level. It is suggested that RBP4 level is an independent risk factor, and early detection of RBP4 may have a good predictive value for the detection of high-risk population of NSCLC. Smoking and BMI are important confounding factors in the analysis of the association between RBP4 and NSCLC. In order to reduce the residual confounding interference of smoking and BMI, this study made a detailed stratified analysis of the relationship between RBP4 and NSCLC adjusted the possible confounding factors. Our results show that the serum RBP4 level of NSCLC patients is higher than that of the control group regardless of whether they smoke or quit smoking. The increase of serum RBP4 level in patients with NSCLC also increases the risk of NSCLC, indicating that serum RBP4 level is positively correlated with the occurrence of NSCLC and is a risk factor for NSCLC. It is also worth noting, we found that after adjusting for age, sex, smoking and family history, serum RBP4 levels in NSCLC patients were positively correlated with BMI, suggesting that serum RBP4 levels in NSCLC patients were also related to metabolic factors such as obesity, which was consistent with relevant reports.
As a case-control study, this study inevitably has some limitations. First, selection bias, such as admission bias, is difficult to rule out in a case-control study. The NSCLC cases were recruited from only 1 hospital. Second, we collected the serum samples after the cases diagnosed, thus the associations do not necessarily indicate causality. To minimize this bias, we included only newly diagnosed cases and interviewed them as we collected samples before treatment. Third, although some known confounders that might be strongly associated with the risk of NSCLC (eg, gender and smoking status) were adjusted, there was still the possibility of residual confounding from additional factors. Fourth, random measurement error for serum RBP4 is also of concern in the estimation of usual level, since this misclassification bias is likely to lead the OR to the null, and thus our results may tend to be conservative.
To sum up, our study indicated that serum RBP4 level was positively related to the risk of NSCLC. However, the use of serum RBP4 as an indicator for NSCLC needs to be confirmed by further large-scale and prospective studies.
Conceived and designed the research: Shaomin Wang.
Conducted the research: Xiaoping Hu, Wenjun Huang, Feng Wang, Yifei Dai, Xiaocong Hu
Data analysis and interpretation: Xiaoping Hu.
Drafting of the manuscript: Xiaoping Hu.
. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin 2018;68:394–424.
. Duma N, Santana-Davila R, Molina JR. Non-small cell lung cancer
: epidemiology, screening, diagnosis, and treatment. Mayo Clin Proc 2019;94:1623–40.
. Norden A, Burling KA, Zeni L, et al. A new estimate of the glomerular sieving coefficient for retinol-binding protein 4 suggests it is not freely filtered. Kidney Int Rep 2019;4:1017–8.
. Karamfilova V, Gateva A, Alexiev A, et al. The association between retinol-binding protein 4 and prediabetes in obese patients with nonalcoholic fatty liver disease. Arch Physiol Biochem 2019;1–6.
. Sun HX, Ji HH, Chen XL, et al. Serum retinol-binding protein 4 is associated with the presence and severity of coronary artery disease in patients with subclinical hypothyroidism[J]. Aging (Albany NY) 2019;11:4510–20.
. Fan J, Yin S, Lin D, et al. Association of serum retinol-binding protein 4 levels and the risk of incident type 2 diabetes in subjects with prediabetes. Diabetes Care 2019;42:1574–81.
. Wlodarczyk B, Gasiorowska A, Borkowska A, et al. Evaluation of insulin-like growth factor (IGF-1) and retinol binding protein (RBP-4) levels in patients with newly diagnosed pancreatic adenocarcinoma (PDAC)[J]. Pancreatology 2017;17:623–8.
. Wang DD, Zhao YM, Wang L, et al. Preoperative serum retinol-binding protein 4 is associated with the prognosis of patients with hepatocellular carcinoma after curative resection[J]. J Cancer Res Clin Oncol 2011;137:651–8.
. Tang W, Li X, Ma ZZ, et al. Significance of retinol-binding protein expression in patients with acute myeloid leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2018;26:417–21.
. Jiao C, Cui L, Ma A, et al. Elevated serum levels of retinol-binding protein 4 are associated with breast cancer risk: a case-control study
. PLoS One 2016;11:e167498.
. Trofor AC, Papadakis S, Lotrean LM, et al. Knowledge of the health risks of smoking and impact of cigarette warning labels among tobacco users in six European countries: Findings from the EUREST-PLUS ITC Europe Surveys. Tob Induc Dis 2018;16:A10.
. Pintarelli G, Noci S, Maspero D, et al. Cigarette smoke alters the transcriptome of non-involved lung tissue in lung adenocarcinoma patients. Sci Rep 2019;9:13039.
. Su CP, Syamlal G, Tamers S, et al. Workplace secondhand tobacco smoke exposure among U.S. Nonsmoking workers, 2015. MMWR Morb Mortal Wkly Rep 2019;68:604–7.
. Kristina SA, Permitasari N, Krisnadewi KI, et al. Incidence and mortality of cancers related to secondhand smoking in Southeast Asia countries. Asian Pac J Cancer Prev 2019;20:971–6.
. Wang N, Mengersen K, Tong S, et al. Short-term association between ambient air pollution and lung cancer mortality. Environ Res 2019;179(Pt A):108748.
. Shahhoseini E, Feltis BN, Nakayama M, et al. Combined effects of gold nanoparticles and ionizing radiation on human prostate and lung cancer cell migration. Int J Mol Sci 2019;20:4488.
. Stirling ER, Cook KL, Roberts DD, et al. Metabolomic analysis reveals unique biochemical signatures associated with protection from radiation induced lung injury by lack of cd47 receptor gene expression. Metabolites 2019;9:218.
. Kim YW, Lee CH, Jin KN, et al. The regional association between bronchiectasis and lung cancer in chest CT. BMC Pulm Med 2016;16:151.
. Hidaka A, Sawada N, Svensson T, et al. Family history of cancer and subsequent risk of cancer: a large-scale population-based prospective study in Japan. Int J Cancer 2020;147:331–7.
. Nishi S, Zhou J, Okereke I, et al. Use of imaging and diagnostic procedures after low dose computed tomography screening for lung cancer[J]. Chest 2020;157:427–34.
. Sobotka R, Capoun O, Kalousova M, et al. Prognostic importance of Vitamins A, E and retinol-binding protein 4 in renal cell carcinoma patients. Anticancer Res 2017;37:3801–6.
. Elenewski JE, Hackett JC. Free energy landscape of the retinol/serum retinol binding protein complex: a biological host-guest system. J Phys Chem B 2010;114:11315–22.
. Cioffi CL, Racz B, Varadi A, et al. Design, synthesis, and preclinical efficacy of novel nonretinoid antagonists of retinol-binding protein 4 in the mouse model of hepatic steatosis. J Med Chem 2019;62:5470–500.
. Kwanbunjan K, Panprathip P, Phosat C, et al. Association of retinol binding protein 4
and transthyretin with triglyceride levels and insulin resistance in rural thais with high type 2 diabetes risk. BMC Endocr Disord 2018;18:26.
. Yamaaki N, Yagi K, Kobayashi J, et al. Impact of serum retinol-binding protein 4 levels on regulation of remnant-like particles triglyceride in type 2 diabetes mellitus. J Diabetes Res 2013;2013:143515.
. Saucedo R, Zarate A, Basurto L, et al. RBP4 gene variants are associated with insulin resistance in women with previous gestational diabetes. Dis Markers 2014;2014:269208.
. Bose KS, Gupta SK, Singh S. Is serum retinol binding protein-4: a predictor for diabetes in genetically high risk population? J Res Med Sci 2012;17:1015–9.
. Abola MV, Thompson CL, Chen Z, et al. Serum levels of retinol-binding protein 4 and risk of colon adenoma. Endocr Relat Cancer 2015;22:L1–4.
. Wang Y, Wang Y, Zhang Z. Adipokine RBP4 drives ovarian cancer cell migration. J Ovarian Res 2018;11:29.
. Kanoriya D, Pradeep AR, Mallika A, et al. Correlation of crevicular fluid and serum levels of retinol-binding protein 4 and leptin in chronic periodontitis and obesity
. Clin Oral Investig 2017;21:2319–25.