[Gene-lifestyle interaction on coronary heart disease in adult twins of China].

Objective: To explore the gene-lifestyle interaction on coronary heart disease (CHD) in adult twins of China. Methods: Participants were selected from twin pairs registered in the Chinese National Twin Registry (CNTR). Univariate interaction model was used to estimate the interaction, via exploring the moderation effect of lifestyle on the genetic variance of CHD. Results: A total of 20 477 same-sex twin pairs aged ≥25 years were recruited, including 395 CHD cases, and 66 twin pairs both had CHD. After adjustment for age and sex, no moderation effects of lifestyles, including current smoking, current drinking, physical activity, intake of vegetable and fruit, on the genetic variance of CHD were found (P>0.05), suggesting no significant interactions. Conclusion: There was no evidence suggesting statistically significant gene-lifestyle interaction on CHD in adult twins of China.

[Regional and demographic differences on passive smoking among non-smokers aged 30-79 years in 10 regions of China].

Objective: To describe the regional and demographic differences on passive non-smokers from 10 regions involved in the China Kadoorie Biobank (CKB) study. Methods: Detailed information regarding passive smoking behaviors related to 317 486 non-smokers who were 30-79 years old from the 10 study regions were gathered and analyzed. Results: Following the standardization of the 2010 China national population, the prevalence rate of passive smoking was 56.7%, and the prevalence rate of living with smokers was 66.5% among the Chinese adults. Both of the aforementioned rates were higher in rural than in urban areas. Meanwhile, the regional distribution of weekly passive smoking frequency and cumulative duration of passive smoking per week and cumulative duration of passive smoking per day were significantly different. The cumulative passive smoking duration per week increased along with the weekly frequency in people living in urban areas. Among women, the weekly passive smoking frequency was the highest, and the cumulative durations per week and per day appeared the lowest in Hunan, opposite to the situation in Henan. The prevalence of passive smoking among participants living with smokers was 2.27 times (95%CI: 2.24-2.29) of those who were not and the association appeared stronger in women (OR=2.61, 95%CI: 2.58-2.64) but not in men (OR=1.01, 95%CI: 0.95-1.06). Almost all the indicators seemed higher in women than those in men, except for the cumulative duration per day. Furthermore, these indicators appeared higher among those who were at younger age or with less education. The prevalence rates of passive smoking and living with smokers were lower but the cumulative duration per day was higher among those with lower household income. And the two rates were higher in married women and lower in married men, as compared to their counterparts. Conclusion: Regional and demographic differences in passive smoking were noticed among study population of CKB in the 10 regions.

[Correlation between fasting plasma glucose, HbA1c and DNA methylation in adult twins].

OBJECTIVE: To explore the cytidine-phosphate-guanosine (CPG) sites associated with fas-ting plasma glucose (FPG) and glycated haemoglobin (HbA1c) in twins. METHODS: In the study, 169 pairs of monozygotic twins were recruited in Qingdao, Zhejiang, Jiangsu, Sichuan and Heilongjiang in June to December of 2013 and June 2017 to October 2018. The methylation was detected by Illumina Infinium HumanMethylation450 BeadChip and Illumina Infinium MethylationEPIC BeadChip. According to the Linear Mixed Effect model (LME model), fasting plasma glucose and HbA1c were taken as the main effects, the methylation level (ß value) was taken as the dependent variable, continuous variables, such as age, body mass index (BMI), blood pressure, components of blood cells, surrogate variables generated by SVA, and categorical variables, such as gender, smoking and drinking status, hypoglycemic drugs taking, were included in the fixed effect model as covariates, and the identity numbers (ID) of the twins was included in the random effect model. The intercept was set as a random. Regression analysis was carried out to find out the CpG sites related to fasting blood glucose or HbA1c, respectively. RESULTS: In this study, 338 monozygotic twins (169 pairs) were included, with 412 459 CpG loci. Among them, 114 pairs were male, and 55 pairs were female, with an average age of (48.2±11.9) years. After adjustment of age, gender, BMI, blood pressure, smoking, drinking, blood cell composition, and other covariates, and multiple comparison test, 7 CpG sites (cg19693031, cg01538969, cg08501915, cg04816311, ch.8.1820050F, cg06721411, cg26608667) were found related to fasting blood glucose, 3 of which (cg08501915, ch.8.1820050f, cg26608667) were the newly found sites in this study; whereas 10 CpG sites (cg19693031, cg04816311, cg01538969, cg01339781, cg01676795, cg24667115, cg09029192, cg20697417, ch.4.1528651F, cg16097041) were found related to HbA1c, and 4 of which(cg01339781, cg24667115, cg20697417, and ch.4.1528651f) were new. We found that cg19693031 in TXNIP gene was the lowest P-value site in the association analysis between DNA methylation and fas-ting plasma glucose and HbA1c (PFPG=2.42×10-19, FDRFPG<0.001; PHbA1c=1.72×10-19, FDRHbA1c<0.001). CONCLUSION: In this twin study, we found new CpG sites related to fasting blood glucose and HbA1c, and provided some clues that partly revealed the potential mechanism of blood glucose metabolism in terms of DNA methylation, but it needed further verification in external larger samples.

[Fujian Tulou Family Cohort Study: study design and characteristics of participants and pedigrees in baseline investigation].

Objective: To describe the study design, the characteristics of participants as well as the pedigrees included in the baseline survey of Fujian Tulou Family Cohort Study. Methods: Fujian Tulou Family Cohort Study was a prospective open cohort study with a biological sample bank. A baseline survey was conducted in Tulou areas of Nanjing county in Fujian province from 2015 to 2018, including questionnaire survey, physical and biochemical indicators examinations, and blood sample collection in adults aged ≥18 years. In addition, family relationship of the participants was also recorded. The pedigree information of the juveniles under 18 years old were also collected. Results: The baseline survey included 2 727 individuals in two clans, of whom 2 373 (87.0%) were adults, and 2 126 participants completed questionnaires, physical examinations and biochemical tests. The average age of the 2 126 participants was (57.9±13.3) years, with 39.4% being males. The current smoking rates in male and female participants were 41.2% and 2.1%, respectively. The corresponding rates of current alcohol consumption were 19.0% and 2.6%. For common chronic diseases, the prevalence rates were 51.3% for hypertension, 9.7% for diabetes and 26.7% for hyperlipemia according to the self-reported disease diagnoses, health examination results and biochemical examination results in class Ⅱ or Ⅲ hospitals. Based on the family relationship information and genealogical data, 710 pedigrees were finally identified, consisting of 5 087 family members. The numbers of five, four, three, and two generations pedigrees were 3, 88, 238 and 381, respectively. The pairs of the first to the fifth degree relatives were 12 039, 2 662, 1 511, 202 and 31, respectively. Conclusion: The establishment of Fujian Tulou Family Cohort provides valuable resources for exploring the genetic risk factors, environmental risk factors and gene-environment interactions contributing to the risk of common chronic diseases.

[Effect of family environment in childhood and adolescence on mental health in adulthood].

Objective: To explore the relationship of family environment in childhood and adolescence and mental health in adulthood. Methods: A total of 791 subjects aged ≥25 years were selected through the Chinese National Twin Registry (CNTR). The short-form of Family Environment Scale-Chinese Version (FES-CV) was used to evaluate the family environment during childhood and adolescence in three dimensions: relationship, system maintenance and personal growth. The mental health status in adulthood was assessed with the Chinese version of 6-item Kessler Psychological Distress Scale (K6). The generalized linear mixed model was used to examine their relationship. Results: About 4.6% of the subjects reported general or worse mental health status. Compared with the subjects with good mental status, statistical difference was observed only in parenting way among twins (living together or not). After adjusting the potential confounders, such as age, sex, zygosity, education and lifestyle (smoking, drinking and physical activity), good family relationship and system maintenance had a positive effect on mental health, with the OR (95%CI) of 0.66 (0.51-0.87) and 0.70 (0.50-0.98) respectively. Furthermore, parenting way did not modify the effect of family environment on mental health status in adulthood (interaction: P>0.05). In each scale, scores of cohesion and organization were positively correlated with mental health, while the score of conflict was negatively correlated with the mental health. Conclusion: Good family relationship and system maintenance in childhood and adolescence had a positive impact on mental health in adulthood.

MicroRNA-509-5p functions as an anti-oncogene in breast cancer via targeting SOD2.

OBJECTIVE: Breast cancer is one of the most common malignant tumors in women worldwide. Considering the poor therapeutic effect of breast cancer, we are supposed to dissect the functioning mode of miR-509-5p on breast cancer cell growth and metastasis, providing therapeutic targets for breast cancer. PATIENTS AND METHODS: Quantitative Real-time PCR (qRT-PCR) assay was employed to detect miR-509-5p expression level. CCK8 assay and colony formation assay were incorporated to assess cell viability and proliferation capacities. Cell migration and invasion assay were performed to investigate metastasis capacity of breast cancer cells. Flow cytometry was used to identify cell apoptosis and cell cycle distribution. Protein levels were assessed by Western blotting assay. The target gene was predicted and verified by bioinformatics analysis and luciferase assay. RESULTS: MiR-509-5p was obviously downregulated in breast cancer tissues when compared with pericarcinomatous tissues (n=76). Overexpressed miR-509-5p could attenuate breast cancer cell viability, proliferation, migration and invasion capacities, as well as promote cell apoptosis and induce cell cycle arrest at G0/G1 phase. Superoxide dismutase 2 (SOD2) was chosen as the target gene of miR-509-5p by bioinformatic analysis and Luciferase reporter assay. Moreover, restoration of SOD2 could rescue tumor suppression role of miR-509-5p on breast cancer tumorigenesis. CONCLUSIONS: MiR-509-5p exerted tumor-suppressive effects on breast cancer progression and metastasis via targeting SOD2 in vitro, which provided an innovative and candidate target for diagnose and treatment of breast cancer.

[Associations between airflow obstruction and total and cause-specific mortality in adults in China].

Objective: To examine the prospective associations between airflow obstruction and total and cause-specific mortality. Methods: The study was based on China Kadoorie Biobank, in which 199 099 men and 287 895 women aged 30-79 years at baseline survey were included after excluding those with heart disease, stroke and cancer. The Global Initiative on Obstructive Lung Disease (GOLD) guideline was used to classify airflow obstruction. Cox regression models were used to estimate adjusted HR and 95%CI. Results: During 3 494 079 person-years of follow-up between 2004 and 2013 (median 7.2 years), a total of 21 649 people died. Absolute mortality rates were 5.5, 9.9, 13.1, 32.4 and 63.3 deaths per 1 000 person-years for participants who had normal airflow, GOLD-1 to GOLD-4 airflow obstruction, respectively. After adjusting potential confounders, compared with participants with normal lung function, the HRs for death were 0.98 (95%CI: 0.88-1.09), 1.03 (95%CI: 0.97-1.09), 1.62 (95% CI: 1.53-1.73) and 2.83 (95% CI: 2.59-3.10) for those whose airflow obstruction were classified as GOLD-1 to GOLD-4, respectively. The airflow obstruction was also associated with increased risk for deaths due to ischemic heart disease, cerebrovascular disease and chronic obstructive pulmonary disease. Conclusion: Airflow obstruction is associated with total and certain cause-specific mortality, the higher the airflow obstruction degree is, the higher the death risk is.

[Influences of genetic and environmental factors on smoking related behaviors among male twin adults in China].

OBJECTIVE: To analyze the influences of genetic and environmental factors on smoking behavior, smoking cessation and onset age of smoking less than 20 years in male twin adults. METHODS: A face-to-face questionnaire was conducted to collect data from 6 458 pair male twins aged ≥25 years registered in 9 provinces(municipality)in China. The heritability of three smoking related behaviors were calculated by using structural equation models. RESULTS: The ACE models were the best models of the three dimensions of smoking, i.e. smoking behavior, smoking cessation and onset age of smoking less than 20 years for male twins, and the corresponding heritability of these behaviors were 0.26(0.19-0.34), 0.27(0.19-0.37)and 0.05(0.00-0.14), respectively. When adjusted for area and age, the heritability of these three behaviors were 0.26(0.19-0.34), 0.31(0.00-0.74)and 0.05(0.00-0.14), respectively. CONCLUSIONS: All the three smoking related behaviors were affected by genetic factors, but environment factors had more effect on them. For smoking cessation, the heritability was highest, but the influence of environmental factors was lowest. Meanwhile, for onset age of smoking, the influence of environmental factors was highest.

USP4 promotes invasion of breast cancer cells via Relaxin/TGF-ß1/Smad2/MMP-9 signal.

OBJECTIVE: Ubiquitin-specific protease 4 (USP4) is a deubiquitinating enzyme with key roles in the regulation of TGF-ß1 signaling, suggesting its importance in tumorigenesis. However, the underlying mechanisms causing this are not entirely clear. In the present study, we investigated the effect of USP4 on invasion and tumorigenesis of breast cancer cells, and explored its mechanism. MATERIALS AND METHODS: Effects of USP4 overexpression or USP4 silencing by small interfering RNA (USP4 siRNA) on invasion of breast cancer MDA-MB-231 and T47D cells in vitro was detected. Using siRNAs and inhibitors to examine the USP4 signaling pathway. RESULTS: The migration and invasion assays showed that USP4 promotes human breast cancer cell migration and invasion by USP4 overexpression, and knockdown of USP4 by siRNA inhibits human breast cancer cell migration and invasion. Treatment with RLX siRNAs, TGF-ß1 siRNAs, Smad2 siRNAs or BB94 (MMPs inhibitor) to USP4-overexpressing breast cancer cells revealed that USP4- induced RLX via TGF-ß1 pathway promotes the cell migration and invasion. Further studies demonstrated that USP4-mediated TGF-ß1 activation not only enhances the phosphorylation of Smad2 through TGF-ß, but also directly upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of breast cancer cells. CONCLUSIONS: Therapies targeting the USP4 inhibits invasion of breast cancer cells via Relaxin/TGF-ß1/Smad2/MMP-9 signal. These results indicate that USP4 is an attractive target for breast cancer therapy.

Aberrant DNA methylation of the P16, MGMT, and hMLH1 genes in combination with the methylenetetrahydrofolate reductase C677T genetic polymorphism and folate intake in gastric cancer.

Epidemiological studies have indicated that folate metabolism is correlated with increased risk of gastric cancer. Since methylenetetrahydrofolate reductase (MTHFR) is an important enzyme involved in folate metabolism, in this study, we examined whether polymorphisms and haplotypes of MTHFR are correlated with the risk of gastric cancer. The polymorphisms MTHFR C677T and MTHFR A1298C were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis in 285 patients and 570 healthy controls. Association analyses based on binary logistic regression were conducted to determine the odds ratio (OR) and its 95% confidence interval (95%CI) for each genotype. The MTHFR 677TT genotype was significantly related with a reduced risk of gastric cancer (OR = 0.60, 95%CI = 0.39-0.92) compared to the CC genotype. Similarly, the MTHFR 1298CC genotype was significantly associated with a decreased risk of cancer (OR = 0.52, 95%CI = 0.32- 0.81). Haplotype analysis showed that the TC haplotype was associated with a reduced risk of gastric cancer compared to the most common haplotype, CA (OR = 0.28, 95%CI = 0.12-0.60). Our results suggest that the MTHFR C677T and MTHFR A1298C polymorphisms are related to gastric cancer susceptibility in the Chinese population.

Overexpression of microRna-200c in CD44+CD133+ CSCS inhibits the cellular migratory and invasion as well as tumorigenicity in mice.

Cancer stem cells (CSCs) are believed to be responsible for drug resistance, metastasis of tumors. To investigate the biological characteristics of CD44+CD133+CSCs with over- expressing microRNA-200c (miR-200c), and to provide evidences for miR-200c as a tumor suppressor to treat melanoma. CD44+CD133+CSCs were isolated from the mouse melanoma B16F10 cell line by using immune magnetic activated cell sorting. The lentivirus miR-200c was transduced into the cells, and the effect of miR-200c overexpression on the biological characteristics of B16F10 CD44+ CD133+CSCs was analyzed by a series assays. The stable overexpression of miR-200c in B16F10 CD44+CD133+CSCs obviously resulted in downregulation of zinc-finger E-box binding homeobox 1 expression, reduction of the cell proliferation, colony forming, cell migratory and invasion ability in vitro as well as tumorigenicity in vivo compared with those of the B16F10 cells and B16F10 non-CD44+ CD133+CSCs. These findings suggest that the miR-200c overexpression as a novel strategy to target therapy of melanoma CSCs.

Relaxin enhances in-vitro invasiveness of breast cancer cell lines by upregulation of S100A4/MMPs signaling.

OBJECTIVES: Relaxin (RLX or RLN) levels are increased in cases of human breast cancer and has been shown to promote cancer cell migration in carcinoma cells of the breast; however, the cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. In human breast cancer cells, relaxin was shown to downregulate the metastasis-promoting protein S100A4, a highly significant prognostic factor for poor survival in breast cancer patients. RLX was also found to enhance in-vitro invasiveness of breast cancer cell lines by induction of matrix metalloproteinases (MMPs) expression. The aim of this study was to investigate the effects of relaxin on breast cancer cell invasion by S100A4 dependent MMPs pathway. MATERIALS AND METHODS: The human breast cancer MDA-MB-231 cells were treated with 100-500 µg/L porcine RLX, or/and transfected with S100A4 siRNA (20 ng), or/and treated with MMPs inhibitor FN439 (0.3 nM). RESULTS: We observed that incubation with porcine RLX increases in-vitro cell invasion and in vitro invasiveness. Enhanced invasiveness was accompanied by up-regulation of S100A4 and MMP-2 and MMP-9. The relaxin-induced increase in cell invasion was blocked almost when S100A4 expression was diminished using an S100A4 small interfering RNA knockdown approach or when MMPs was inhibited by MMPs inhibitor FN439. The relaxin-induced increase in MMP-2 and MMP-9 expression was blocked when S100A4 was inhibited by S100A4 siRNA transfection. CONCLUSIONS: Our data demonstrate that the RLX controls the in-vitro invasive potential of human breast cancer cells through S100A4 dependent MMPs regulation.

Poly(ADP-ribose) polymerase-1 plays a role in suppressing mammary tumourigenesis in mice.

The DNA strand break-binding molecule, poly(ADP-ribose) polymerase-1 (PARP-1), plays a role in DNA repair, chromosomal stability, transcription and cell death. Accumulating evidence suggests that dysfunction of PARP-1 contributes to tumorigenesis. Here, we report that PARP-1 deficiency causes mammary carcinoma formation in female mice, and that the introduction of Trp53 mutations accelerates the onset and shortens the latency of mammary tumorigenesis. We show that PARP-1 deficiency results in chromosomal aneuploidy and centrosome amplification, which are substantiated by the inactivation of Trp53 in primary mammary epithelial (PME) cells. In addition, PARP-1 deficiency compromises p53 activation and impairs BRCA1 recruitment to the sites of DNA damage in PME cells. PARP-1 complementation partly rescues the defective DNA damage response mediated by p53 and BRCA1. The present study thus identifies a role of PARP-1 in suppressing mammary tumorigenesis in vivo and suggests that dysfunction of PARP-1 may be a risk factor for breast cancer in humans.

Myeloid and erythroid hemopoiesis supported by human bone marrow fibroblasts in vitro.

The passages 0 and 3 purified human bone marrow fibroblast layers (FLs) were established by long-term liquid cultures. After 12-day coculture of stromal. cell-depleted marrow cell suspensions with passage 0 FLs, 68.33 +/- 4.04% of the hemopoietic colonies adhered to FLs was myeloid in nature and the other 31.67 +/- 4.04% was erythroid. There were still CFU-E (colony forming unit-erythroid), BFU-E (burst forming unit- erythroid), and CFU-GM (colony forming unit-granulocyte/macrophage) among the nonadherent cells. The media conditioned by passage 0 (F0-CM) and passage 3(F3-CM) FLs stimulated the growth of myeloid and erythroid (BFU-E) colonies. From these data, it is concluded that both FLs and the media conditioned by fibroblasts can stimulate myeloid and erythroid hemopoiesis.