| BackgroundBreast cancer (BC) is the most common cancer among women all over the world, and threatens the health of women strongly. In the "World Cancer Report 2014", World Health Orgnization decleared that:the first 3 new diagnosed cancers of 2012 worldwide are lung cancer, breast cancer, colorectal cancer; there are 1677,000 new diagnosed breast cancer, the number of this case in China is 187,000. The number of China Cancer Annual Report is that the incidence of breast cancer in 2012 of China is 42.55/100,000. The standard prevalence rate of breast cancer in China is 207.7/100,000 in 2008, the cases of breast cancer in nearly 3 years among 10 years cases is 67.55%. The breast cancer incidence of China is increasing fast.Through the epidemiological investigation and basic study of breast cancer, we found that the proportion of obesity women in breast cancer patients is much more. Based on our own study,we found that obesity was related with breast cancer risk, OR=1.528. So we hypothesize that obesity and it’s related factors play important roles in breast cancer development.Obesity is a kind of systematic inflammational diseases. Fatty tissue can reserve fatty and has important endocrine function, can secret some kinds of peptide hormone, such as letpin, adiponectin, etc (all can be called adipocytokines). Obesity is related with many kinds of cancer, including colorectal cancer, breast cancer, esophageal cancer, etc. Epidemiological study and basic research show that, adipocytokines are the molecular link between obesity and cancer. Adiponectin is a special kind of adipocytokines, is one of the important factors between obesity and some kinds of cancer (breast cancer, esophageal cancer). Because breast tissue contains many fatty tissue, there are many the studies about breast cancer and adiponectin. Studies show that adiponectin is related with breast cancer risk, can influence the progress of breast cancer, is a valuable epidemiological marker of breast cancer susceptibility.1. Basic knowledge of adiponectin1.1 The structure and different forms of adiponectinHuman adiponectin gene (ADIPOQ) locates on 3q27, including 3 extrons and 2 introns. Adiponectin, also can be called as Acrp30, is a kind of proteins that is differentiated from mice’s fatty cell first. Adiponectin single peptide has 4 parts: N-secreting single sequence, reserved non-relix area, collagen-like area and C-globular protein area, including 247 amino acid (28kDa). Adiponectin can bind with receptors (ADIPOR1, ADIPOR2, T-caderin) to influence many biological functions.Adiponectin belongs to complement 1q family, is a kind of homopolymer protein. The adiponectin existing in human circulating system is not the same. There are high-molecular-weight adiponecin (HMW), middle-molecular-weight adipoenctin (MMW), low-molecular-weight adiponectin (LMW) in serum. The biological activity and affinity of receptors of different forms of adiponectin is not the same. In white fatty cell, adiponectin single peptide can form tri-polymer (LMW) by binding with collagen area; 2 LMW molecules can form a MMW by Cys-36 disulfide bond; MMW is the basic structure of HMW, by covalent bond, MMW can form HMW.The level of total adipoenctin in human serum is 0.5-30μg/ml, is about 0.01% of all protein in serum. Most adiponectin is formed and secreted by white fatty cell. When fatty cell is differentiated, the level of adiponectin is upregulated over 100 times. Insulin can also increase the expression of adiponectin. Adiponectin is the richest adipocytokine in blood.The serum concentration of adiponectin of male and female is different. Nishizawa’s study showed that, the serum adiponectin level of male was lower to 65% of female’s; but there is no difference between men and postmenopausal women. The adiponectin level of girl is 7.30μg/ml, while the number of boy is 6.72μg/ml.The expression level of adipoenctin is negatively related with BMI, age, height and IGF-1 level. Following the growth of boy, adiponectin level is decreasing, but there is no similar phenomenon in girl. Adiponectin level is negatively related with boy’s testosterone; however which is not related with estradiol in girl. The above studies show that androgen can influence the expression of adipoenctin directly.Serum adiponectin level is related with obesity, in negatively related with BMI. The adiponectin level of obesity women is lower than normal BMI women. The average adiponectin of normal BMI women is 7.6μg/ml, which of overweight and obesity women is 4.6μg/ml, and 4.7μg/ml.Adiponectin level is related with ADIPOQ SNP. rs 1501299 TG, GG polymorphism is related with decreased level of adiponectin, and also with the increasing risk of breast cancer(59%, OR=1.59,95%CI=1.03-2.48; 80%, OR=1.80, 95%CI=1.14-2.85). rs2241766 polymorphism can increase the level of adiponectin, also decreases the risk of breast cancer of about 39%(OR=0.61,95%CI=0.46-0.80). Virgina’s study showed that, rsl501299 was related with Caucasian race’s breast cancer risk, negatively related with serum adiponectin level. GG genotype’s breast cancer risk was 1.23 fold higher than those with TG.Adiponectin level is related with dietary habit. An article published in 2006 had showed that, Mediterranean style dietary pattern was related with higher serum adiponectin level, and this relationship was not dependent on age, obesity, energy intake, physical exercise, etc. Mediterranean style dietary pattern plus physical exercise can greatly upregulate the level of adiponectin of postmenopausal obesity women. Green tea can upregulate the serum level of adiponectin. Compared with the people who do not drink green tea, the serum adipoenctin level of the one who drink green tea 1-3 times a week is higher.Sex hormone-binding globulin (SHBG) is positively related with adiponectin level. Aline’s study showed that osteoblast’s Fra-2 protein could regulate the level of adiponectin, Fra-2 transgenic mice’s adipoenctin level is obviously low. 1.3 The main biological function of adiponectinADIPOQ’s location area is full of suspensiblity site of metabolic syndrome, type-II diabetes mellitus and coronary heart disease. The known functions of adiponectin are as follows:insulin regulation, anti-atherosclerosis, regulation of glucolipid metabolism, anti-proliferation, induction of apoptosis, anti-angiogenesis, etc. Adipoenctin is an important protective factor of human body. Serum adiponectin level is associated with type-II diabetes mellitus, metabolic syndrome, obesity and malignant diseases.1.3.1 Metabolic related diseaseAdipoenctin can influence some metabolic disease by the following related pathways:① Adiponectin is related with insulin sensitivity, insulin resistance:the higher of insulin resistance level, the lower of serum adiponectin level. By acetyl auxiliary A hydroxylase phosphorylation, adiponectin can inactive and decrease the level of malonyl CoA, stimulate the fatty acid oxidation, decrease free fatty acid level, improve insulin resistance. TNF-a can stimulate NF-κ B pathway to increase the adhesion related molecules, stimulate insulin resistance. Adiponectin can decrease the level of TNF-a of fatty cell.② Adiponectin has the function related to inflammation and Anti-atherosclerosis. Adiponectin can decrease the level of NF-κB pathway and expression of adhesion molecules to inhibit the adhesion of monocytes to endothelial cells. Adiponectin can decrease the scavenger receptor of macrophage of subendothelial cells and fatty accumulation to inhibit the change of macrophage to foam cells.1.3.2 Cancer related function① anti-angiogenesis:By casepase-2, casepase-8 and casepase-9, adiponectin can stimulate apoptosis of cell, and anti-angiogenesis.② Anti-proliferation:By AMPK or β-catenin-Wnt pathway, adiponectin can inhibit proliferation of cancer cells, induce the apoptosis of cell. This phenomenon can only be found in ER negative breast cancer cell lines.③ Ohers:Adiponectin can stimulate AMPK, PPAR-a activity, increase the intake of glucose and oxygen of fatty acid. By regulating the insulin-related pathway, adiponectin can improve insulin sensitivity, inhibit the occurence of cancer. Adipoenctin can also inhibit cancer by mTR、PI3K/Ak、MAPK、STAT3、NF-κ B pathway.Some studies show that adiponectin is the link bewteen obesity and breast cancer, especially postmenopausal breast cancer. Miyoshi Y showed the relationship between breast cancer and adiponectin at first time, and the study showed that lower adiponectin level could increase breast cancer risk. But similar studies do not show the same results. Touvier M’s study showed that the higher adiponectin in circulating system could increase the breast cancer risk; Shahar S’s study showed that higher adiponectin could decrease the breast cancer risk; Hancke K’s study showed there was no relationship between adiponectin and breast cancer risk. All in all, most studies’s results are declined to show that adiponectin is a breast cancer related factor independent of obesity and some other risk factors.Now there are three meta-analysis articles to research on this phenomenon, including our own meta-analysis. Two of these meta-analysis show that circulating adiponectin level is not related with breast cancer risk, but subgroup analysis shows that adiponectin is negatively related with breast cancer risk of postmenopausal women. Another meta-analysis published in 2013 showed that adiponectin was not related with breast cancer risk. Unclear conclusion limits the prevention related study based on adiponectin and breast cancer. We need to make clear that whether adiponectin is the breast cancer related (protective or risk) factor in Chinese women, or which kinds of adiponectin forms are the real influence factors for breast cancer.By browsing many articles, we think that the reasons of this phenomenon are as follows: ①The adiponectin in circulating system is not the same. Different forms of adiponectin has different biological activities, receptor affinity, etc. The examined marker of most study now is total adiponectin.②The sample of most studies based on Cell lines and animals is the purified adiponectin (for example, gAcrp、LMW、NHMW). This is not the same as human body. ③The adiponectin level of serum is different in different races. The study results of other races may be different from Chinese group. ④The number of study subject vary which may lead to some bias.As the most adiponectin form of serum, HMW adiponectin has some special function compared with other forms of adiponectin:①HMW adiponectin is related with postprandial blood glucose concentration more closely;②After oral administration of thiazolidinedione, the change of HMW/total adiponectin ratio is related with improved insulin sensitivity. But similar phenomenon has not found in total adiponectin;③LMW adiponectin is a factor related to anti-inflammation and HMW is related to pro-inflammation;④HMW adiponectin is related with heart disease more closely, compared with LMW adiponectin;⑤The increasing of non-HMW adiponectin level of serum is one of the reasons for patients with chronic hepatitis C to develop into hepatocellular cancer; ⑥Lower HMW adiponectin is an independent risk factor for postmenopausal breast cancer.Based on the above findings, we hypothesized that HMW adiponectin or HMW/total adiponectin ratio may be the true marker linked adiponectin with breast cancer. This study was designed to investigate the relationship between total adiponectin, HMW adiponectin, HMW/total adiponectin ratio and breast cancer by a hospital-based case-control study, to explore the cut-off for each marker. By this study, we could richen the adipoenctin related study of Chinese women; providing molecular epidemiological marker for our further study on breast cancer high risk model of Chinese women; providing basic theoretical foundation for the breast cancer prevention based on adiponectin.ObjectiveBreast cancer is the most common cancer among women all over the world, and threatens the health of women strongly. There are many factors which are related with breast cancer risk, including genetic factor, unhealthy diet, lack of physical exercise, etc. Obesity, central obesity are risk factors for breast cancer. For the past few years, the obesity incidence of Chinese women is increasing quickly. The relationship between obesity and breast cancer risk needs further research. By the Key Clinical Item of the Hospitals Affiliated to the Ministry of Public Health, China, the programme "The Establishment and Perfection of Breast Cancer High-risk Population Screening and Chemoprevention Study" (No.07090122), we conducted an cross-sectional epidemiological investigation in 3 provinces and 1 city. By analyzing the related factors with breast cancer risk, we could get the epidemiological characteristics of Chinese breast cancer, declearing the risk factors and protective factors.There are some obesity related factors in the questionnaire. By 1:3 matched case-control study, we want to analyze the relationship between these factors and breast cancer risk. By our study, we can provide theoretical basis for the prevention of breast cancer in China.l.The collecting and processing of study subjectsThe study subjects are coming from Shandong Province, Hebei Province, Jiangsu Province and Tianjin. By using cluster random sampling method, we select the sampling site in these three provinces and one city. The epidemiological investigation began at July,2008, ended at September,2008.The questionnaire contents include:①Basic questionnaire demographic characteristics and social and economic status, female physiology related factors, diseases history and family history, lifestyle and habits, breast cancer related knowledge. ②Clinical breast examination table including the apparent diagnosis of breast and palpation, primary diagnosis relevant suggestion which filled out by breast surgeons. ③Other physical examination table:including height, weight, breast ultrasound and X-ray mammography report, conventional pathology results. Each questionnaire had the only No. Epidata 3.1 was used to establish the database.2. The selection of 1:3 matched case-control studyIn the all study objects above, there are 320 breast cancer cases diagnosed within 2 years. Each case matched with 3 controls. The recruitment standards are:age± 2years; reside in the same place; neighbor or colleague.3. Statistical analysisSPSS22.0 software was used to analyze all data.Statistical methods, including t-test, x 2 test, univariate and multivariate conditional Logistic regression analyses, were used to identify the risk factors for breast cancer. The odds ratios (OR) with 95% confidence intervals were also calculated.Results1. The results of epidemiological investigation1.1 The charictristics of epidemiological investigationThe mean age of all study subjects are 44.2 (±11.6) years old. The mean age of all cases are 47.8 (±9.3) years old. Menopause, benign disease history of breast, first grade of breast cancer family history, secondary grade of breast cancer history, central obesity, overweight and obesity are related with breast cancer (P<0.05). Age of menarche, third grade of breast cancer history, height, waist-hip ratio and age at first full-term pregnancy are not related with breast cancer (P>0.05).1.2 The logistic regression results of epidemiological investigationThe results of the univariate analysis indicated that the breast-cancer-related factors included obesity, central obesity, menopause, benign disease history of breast, first grade of breast cancer family history, secondary grade of breast cancer history (P <0.05); age of menarche. third grade of breast cancer history are not related with breast cancer risk (P>0.05) (Table 2).Multivariate Logistic regression analysis identified five variables related to breast cancer:obesity, menopause, benign disease history of breast, first grade of breast cancer family history, secondary grade of breast cancer history. The OR of obesity is 2.183,95% CI:1.631-2.921 (Table 3)We took central obesity instead of obesity in multivariate Logistic regression analysis and identified five variables related to breast cancer:central obesity, menopause, benign disease history of breast, first grade of breast cancer family history, secondary grade of breast cancer history. The OR of obesity is 2.045,95%CI: 1.614-2.592 (Table 4)2. The results of 1:3 matched case-control study2. IThe characteristics of 1:3 matched case-control studyThe mean age of all study subjects (n=492) are 49.94 (±9.44) years old. The mean age of all cases are 49.95 (±9.48) years old. The mean age of all controls are 49.92 (±9.37) years old. Central obesity, obesity, benign disease history of breast, first grade of breast cancer family history are related with breast cancer (P<0.05). Age of menarche, menopause, secondary grade of breast cancer history, height and diabetes mellitus are not related with breast cancer (P>0.05) (Table 5).2.2 The logistic regression results of 1:3 matched case-control studyThe results of the univariate analysis indicated that the breast-cancer-related factors included obesity, central obesity, benign disease history of breast, first grade of breast cancer family history (P<0.05); diabetes mellitus, age of menarche, secondary grade of breast cancer history were not related with breast cancer risk (P>0.05) (Table 6).Multivariate Logistic regression analysis identified three variables related to breast cancer:obesity, benign disease history of breast, first grade of breast cancer family history. The OR of obesity was 2.476,95%CI:1.410-4.347 (Table 7)We took central obesity instead of obesity in multivariate Logistic regression analysis and identified four variables related to breast cancer:central obesity, menopause, benign disease history of breast, first grade of breast cancer family history, secondary grade of breast cancer history. The OR of obesity was 1.631,95%CI: 1.053-2.524 (Table 8)ConclusionObesity, central obesity are related with breast cancer risk in China. The factors related to obesity or central obesity might affect thr risk of breast cancer.ObjectiveBased on our hospital-based case-control study, to identify the expression status of plasma total adiponectin, HMW adiponectin, HMW/total adiponectin ratio of Chinese women; to make clear the relationship between total adiponectin, HMW adiponectin, HMW/total adiponectin ratio and breast cancer risks.Research Methods1.The collecting and processing of study subjectsThe project has passed the second hospital of Shandong university ethics committee review and approval before carrying out. This study chooses breast cancer cases and controls during April 2012 to April 2013 at the Second Hospital of Shandong University and other 20 hospitals as the research subjects. The case group and control group are formulated strict inclusion criteria and exclusion criteria. Basic questionnaire data was acquired by a face-to-face questionnaire survey. Pathology and related auxiliary examination data was collected from the corresponding medical documents (mainly including medical history, physical examination report) after obtaining the consent from the object of study. The questionnaire contents include: ①Basic questionnaire demographic characteristics and social and economic status, female physiology related factors, diseases history and family history, lifestyle and habits, breast cancer related knowledge. ②Clinical breast examination table including the apparent diagnosis of breast and palpation, primary diagnosis relevant suggestion which filled out by breast surgeons.③Other physical examination table:including height, weight, breast ultrasound and X-ray mammography report, conventional pathology results(Included detailed immunohistochemical results:ER, PR, Her-2, Ki67, etc) and the current treatment situation.④Breast cancer patients quality of life scale FACT-B(V4.0). Before any form of local and systemic treatment, took the venous blood 4 ml from study objects in the routine blood test tubes on the basis of informed consent. Marked venous blood and separated blood cells and plasma, and then saved in-80℃ for later use.2. The ELISA test of total adiponectin and HMW aidponectinThe test kit for total adiponectin is Human Total Adiponectin/Acrp30 Immunoassay (R&D system); the test kit for HMW adiponectin is Human HMW Adiponectin/Acrp30 Immunoassay (R&D system). The experimental steps are all in strict accordance with the kit instructions. Input the data into SPSS22.0 database, and calculate the HMW/total adiponectin ratio.3. Statistical analysisSPSS22.0 software was used to analyze all data.1167 sets of case-control study data were disposed to hierarchical data according to the subgroup stratification. Data hierarchy index were premenopausal, post-menopausal, having family history of breast cancer, without a family history of breast cancer, obesity, overweight, normal body weight, obese and overweight. Each subgroup was no longer using the original pair. And changed into not-matched case-control study data. Detailed analysis methods are similar as above. For continuous numerical variables, we used the t test to analyze. Classification variables were analysed by Chi-square test. Logistic regression analysis was used for correlation analysis between the factors and breast cancer. Then to determine the cut-off point value of adiponectin related indicators according to the results of ROC curve and Youden index. Analyze the binary data by univariate Logistic regression analysis. The multivariate Logistic regression analysis was conducted if necessary.1.Basic Total and HMW adiponectin results of 1167 setsThe results of this part are all analyzed by paired-t test. In 1167 sets, the mean concentration of plasma total adiponectin of case group is 6.34 (±3.54)μg/ml; the mean concentration of plasma total adiponectin of control group is 6.56 (±3.72) u g/ml, P=0.150. In 1167 sets, the mean concentration of plasma HMW adiponectin of case group is 2.52 (±1.88) u g/ml; the mean concentration of plasma HMW adiponectin of control group is 2.58(±1.88) u g/ml, P=0.150. In 1167 sets, the mean concentration of plasma HMW/total adiponectin ratio of case group is 0.38 (±0.15); the mean concentration of plasma HMW/total adiponectin ratio of control group is 0.38 (±0.15), P=0.590. As continuous numerical variables, total adiponecin, HMW adiponectin, HMW/total adiponectin ratio have no statistical differences between case and control groups.2. Logistic regression analysis results of 1167setsBy ROC curve and maximal Youden index method, we set 4.46μg/ml as cut-off for total adiponectin; 1.42μg/ml as cut-off for HMW adiponectin; 0.39 as cut-off for HMW/total adiponectin.By univariate analysis, we analyze the related factors (postmenopausal status or not, central obesity, BMI, DM or not, with family history of breast cancer or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. The factors whose P is less than 0.05 are included in multivariate analysis. Higher HMW adiponectin is an protective factor for breast cancer,.P=0.048, OR=0.828, 95%CI:0.687-0.998.3. Subgroup analysis results3.1 Pre-menopausal subgroupBy univariate analysis, we analyze the related factor (central obesity, BMI, DM or not, with family history of breast cancer or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Central obesity, higher BMI and positive breast cancer family history are the risk factors for this subgroup; others are not. Because there are no positive results of adiponectin-related factors in univariate analysis, we do not conduct multivariate Logistic regression analysis in this subgroup.3.2 Postmenopausal subgroupBy univariate analysis, we analyze the related factors (central obesity, BMI, DM or not, with family history of breast cancer or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Higher HMW adiponectin and higher HMW/total adiponectin ratio are protective factor for this subgroup; others are not. Because there are only positive results of adiponectin-related factors in univariate analysis, we do not conduct multivariate Logistic regression analysis for this subgroup.3.3 The subgroup of subjects with breast cancer family historyBy univariate analysis, we analyze the related factors (central obesity, postmenopausal status or not, BMI, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Higher HMW adiponectin and higher HMW/total adiponectin ratio are the risk factors for this subgroup; others are not. Because there are no positive results of adiponectin-related factor in univariate analysis, we do not conduct multivariate Logistic regression analysis for this subgroup.3.4 The subgroup of subjects without breast cancer family historyBy univariate analysis, we analyze the related factors (central obesity, postmenopausal status or not, BMI, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Central obesity, postmenopausal status and BMI>=24.0 are risk factor of this subgroup; higher HMW adiponectin is the protective factor for this subgroup; whereas others are not. The factors whose P is less than 0.05 are included in multivariate analysis. Higher HMW adiponectin is a protective factor for breast cancer, P=0.013, OR=0.785,95%CI 0.649-0.951.3.5 BMI normal subgroupBy univariate analysis, we analyze the related factors (central obesity, postmenopausal status or not, with family history of breast cancer or not, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Postmenopausal status and with family history of breast cancer are the risk factor for this subgroup; higher total adiponectin is the protective factor for this subgroup; others are not. The factors whose P is less than 0.05 are included in multivariate analysis. Higher total adiponectin is not an protective factor for breast cancer, P=0.096.3.6 Overweight and obesity subgroupBy univariate analysis, we analyze the related factors (central obesity, postmenopausal status or not, with family history of breast cancer or not, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Higher HMW adiponectin level is the protective factor for this subgroup; others are not. Central obesity is risk factor of this subgroup. The factors whose P is less than 0.05 are included in multivariate analysis. Higher HMW adiponectin is not an protective factor for breast cancer, P=0.060.3.7 Central obesity subgroupBy univariate analysis, we analyze the related factors (BMI, postmenopausal status or not, with family history of breast cancer or not, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. None of the above factors are related with breast cancer risk in this subgroup.3.8 Non-central obesity subgroupBy univariate analysis, we analyze the related factors (BMI, postmenopausal status or not, with family history of breast cancer or not, DM or not, menarche early or not; higher total adiponectin or not, higher HMW adiponectin or not, higher HMW/total adiponectin ratio or not, etc) with breast cancer. Postmenopausal status and positive family history of breast cancer are risk factors for this subgroup.ConclusionDifferent forms of adiponectin may play different roles in breast cancer risk.1.Higher HMW adiponectin level (>1.42μg/ml) is the protective factor of breast cancer.2.For positive breast cancer family history subgroup, the role of higher HMW adiponectin is special, it is the risk factor of breast cancer. |
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