SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?

BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].

Cancer Incidence After Diagnosis of Abdominal Aortic Aneurysm-Brief Report.

BACKGROUND: Epidemiological and mechanistic data support a potential causal link between cardiovascular disease (CVD) and cancer. Abdominal aortic aneurysms (AAAs) represent a common form of CVD with at least partially distinct genetic and biologic pathogenesis from other forms of CVD. The risk of cancer and how this risk differs compared with other forms of CVD, is unknown among AAA patients. We conducted a retrospective cohort study using the IBM MarketScan Research Database to test whether individuals with AAA have a higher cancer risk independent of traditional shared risk factors. METHODS: All individuals ≥18 years of age with ≥36 months of continuous coverage between 2008 and 2020 were enrolled. Those with potential Mendelian etiologies of AAA, aortic aneurysm with nonspecific anatomic location, or a cancer diagnosis before the start of follow-up were excluded. A subgroup analysis was performed of individuals having the Health Risk Assessment records including tobacco use and body mass index. The following groups of individuals were compared: (1) with AAA, (2) with non-AAA CVD, and (3) without any CVD. RESULTS: The propensity score-matched cohort included 58 993 individuals with AAA, 117 986 with non-AAA CVD, and 58 993 without CVD. The 5-year cumulative incidence of cancer was 13.1% (12.8%-13.5%) in participants with AAA, 10.1% (9.9%-10.3%) in participants with non-AAA CVD, and 9.6% (9.3%-9.9%) in participants without CVD. Multivariable-adjusted Cox proportional hazards regression models found that patients with AAA exhibited a higher cancer risk than either those with non-AAA CVD (hazard ratio, 1.28 [95% CI, 1.23-1.32]; P<0.001) or those without CVD (hazard ratio, 1.32 [95% CI, 1.26-1.38]; P<0.001). Results remained consistent after excluding common smoking-related cancers and when adjusting for tobacco use and body mass index. CONCLUSIONS: Patients with AAA may have a unique risk of cancer requiring further mechanistic study and investigation of the role of enhanced cancer screening.

Time-varying associations of patient and tumor characteristics with cancer survival: an analysis of SEER data across 14 cancer sites, 2004-2017.

PURPOSE: Surveillance, Epidemiology, and End Results (SEER) cancer registries provides information about survival duration and cause of death for cancer patients. Baseline demographic and tumor characteristics such as age, sex, race, year of diagnosis, and tumor stage can inform the expected survival time of patients, but their associations with survival may not be constant over the post-diagnosis period. METHODS: Using SEER data, we examined if there were time-varying associations of patient and tumor characteristics on survival, and we assessed how these relationships differed across 14 cancer sites. Standard Cox proportional hazards models were extended to allow for time-varying associations and incorporated into a competing-risks framework, separately modeling cancer-specific and other-cause deaths. For each cancer site and for each of the five factors, we estimated the relative hazard ratio and absolute hazard over time in the presence of competing risks. RESULTS: Our comprehensive consideration of patient and tumor characteristics when estimating time-varying hazards showed that the associations of age, tumor stage at diagnosis, and race/ethnicity with risk of death (cancer-specific and other-cause) change over time for many cancers; characteristics of sex and year of diagnosis exhibit some time-varying patterns as well. Stage at diagnosis had the largest associations with survival. CONCLUSION: These findings suggest that proportional hazards assumptions are often violated when examining patient characteristics on cancer survival post-diagnosis. We discuss several interesting results where the relative hazards are time-varying and suggest possible interpretations. Based on the time-varying associations of several important covariates on survival after cancer diagnosis using a pan-cancer approach, the likelihood of the proportional hazards assumption being met or corresponding interpretation should be considered in survival analyses, as flawed inference may have implications for cancer care and policy.

Triglyceride-glucose index is associated with severe obstructive coronary artery disease and atherosclerotic target lesion failure among young adults.

BACKGROUND: Early diagnosis and treatment effectiveness of early-onset coronary artery disease (EOCAD) are crucial, and non-invasive predictive biomarkers are needed for young adults. We aimed to evaluate the usefulness of the triglyceride-glucose (TyG) index, a novel marker of insulin resistance, in identifying young CAD patients and predicting their risk of developing target lesion failure (TLF). METHODS: We recruited EOCAD patients (luminal narrowing ≥ 70%) and controls free from CAD (luminal narrowing < 30%), both aged 45 years or younger, from 38 hospitals in China between 2017 and 2020. EOCAD patients who underwent successful percutaneous coronary intervention were followed for incident TLF. TyG index was defined as Ln [fasting triglyceride (mg/dL) × fasting blood glucose (mg/dL)/2]. We used logistic regression and Cox proportional hazards modeling to evaluate the association of TyG index with prevalent EOCAD and incident TLF, respectively. The discriminatory ability of TyG index was assessed by the area under the receiver-operating characteristic curve (AUC). RESULTS: Among the included 1513 EOCAD patients (39.6 ± 4.4 years, 95.4% male) and 1513 age-matched controls (39.0 ± 4.4 years, 46.4% male), TyG index was positively associated with the prevalence of EOCAD (adjusted odds ratio: 1.40, 95% confidence interval [CI] 1.23-1.60, per standard deviation [SD] increase in TyG index). The addition of TyG index to an empirical risk model provided an improvement in diagnostic ability for EOCAD, with a net reclassification improvement of 0.10 (95% CI 0.03-0.17, p = 0.005). During a medium of 33 month (IQR: 31-34 months) follow-up, 43 (3.3%) patients experienced TLF. Multivariate Cox regression model revealed that TyG index was an independent risk factor for TLF (adjusted hazard ratio [HR]: 2.410, 95% CI 1.07-5.42 comparing the top to bottom TyG index tertile groups; HR: 1.30, 95% CI 1.01-1.73, per SD increase in TyG index). Compared with a model of conventional risk factors alone, the addition of the TyG index modestly improved the AUC (0.722-0.734, p = 0.04) to predict TLF. CONCLUSIONS: TyG index is positively associated with prevalent EOCAD and incident TLF. TyG index appeared to be a valuable component of future efforts to improve CAD risk stratification and TLF outcome prediction among young adults.

Maternal genetic intergenerational and transgenerational effects on hormone synthesis in ovarian granulosa cells of offspring exposed to cadmium during pregnancy.

This study aimed to investigate the maternally inherited intergenerational and transgenerational effects of cadmium (Cd) exposure on steroid hormone synthesis in the ovarian granulosa cells (GCs) of offspring rats. F1 rats were obtained by mating adult female Sprague-Dawley rats with healthy adult male rats and were exposed to 0, 0.5, 2.0, and 8.0 mg/kg CdCl2 during pregnancy. The adult female rats (PND 56) were mated with healthy adult male rats to produce F2 and F3 rats. The serum progesterone (Pg) and estradiol (E2) levels of the F2 adult female rats were decreased, while those of F3 rats were significantly increased. Moreover, hormone synthesis-related genes had different expression patterns in the F2 and F3 generations. F2 and F3 rat ovarian GCs exhibited altered miRNA expression profiles and DNA methylation patterns. Validation of miRNAs that regulate hormone synthesis-related genes in the cAMP/PKA signaling pathway suggested that miR-124-3p was downregulated in F2 and F3 rats, while miR-133a-5p and miR-150-5p were upregulated in F2 rats and downregulated in F3 rats. In summary, 1) there are maternal genetic intergenerational (GCs hormone synthesis disorder) and transgenerational (GCs hormone synthesis function repair change) effects on hormone synthesis function changes in offspring GCs induced by Cd exposure during pregnancy. 2) Changes in miRNAs and DNA methylation modifications associated with the genetic effects of altered hormone synthesis function in offspring GCs induced by Cd exposure during pregnancy are important. 3) Under the current environmental level of Cd exposure, the possible risk of maternal genetic intergenerational and transgenerational effects of offspring ovarian toxicity should be strongly considered.

Multigenerational genetic effects of paternal cadmium exposure on ovarian granulosa cell apoptosis.

To explore whether paternal cadmium (Cd) exposure causes ovarian granulosa cell (GC) apoptosis in offspring and the multigenerational genetic effects. From postnatal day 28 (PND28) until adulthood (PND56), SPF male Sprague-Dawley (SD) rats were gavaged daily with varying concentrations of CdCl2. (0, 0.5, 2, and 8 mg/kg). After treatment, the F1 generation was produced by mating with untreated female rats, and the F1 generation male rats were mated with untreated female rats to produce the F2 generation. Apoptotic bodies (electron microscopy) and significantly higher apoptotic rates (flow cytometry) were observed in both F1 and F2 ovarian GCs following paternal Cd exposure. Moreover, the mRNA (qRTPCR) or protein (Western blotting) levels of bax, bcl2, bcl-xl, caspase 3, caspase 8, and caspase 9 were changed to varying degrees. Apoptosis-related miRNAs (qRTPCR) and methylation modifications of apoptosis-related genes (bisulfite-sequencing PCR) in ovarian GCs were further detected. Compared with those of controls, the expression patterns of miRNAs in F1 and F2 offspring were different after paternal Cd exposure, while the average methylation level of apoptosis-related genes did not change significantly (except for individual loci). In summary, there are paternal genetic intergenerational and transgenerational effects on ovarian GC apoptosis induced by paternal Cd exposure. These genetic effects were related to the upregulation of BAX, BCL-XL, Cle-CASPASE 3, and Cle-CASPASE 9 in F1 and the upregulation of Cle-CASPASE 3 in F2 progeny. Important changes in apoptosis-related miRNAs were also observed.

The Wnt/ß-catenin pathway is involved in 2,5-hexanedione-induced ovarian granulosa cell cycle arrest.

N-Hexane causes significant ovarian toxicity, and its main active metabolite 2,5-hexanedione (2,5-HD) can induce ovarian injury through mechanisms such as inducing apoptosis in ovarian granulosa cells (GCs); however, the specific mechanism has not been fully elucidated. In this study, we investigated the effects on the cell cycle of rat ovarian GCs exposed in vitro to different concentrations of 2,5-HD (0 mM, 20 mM, 40 mM, and 60 mM) and further explored the mechanism by mRNA and miRNA microarray analyses. The flow cytometry results sindicated that compared with control cells, in ovarian GCs, there was significant cell cycle arrest after 2,5-HD treatment. Cell cycle- and apoptosis- related gene (Cdk2, Ccnd1, Bax, Bcl-2, Caspase3, and Caspase9) expression was altered. The mRNA and miRNA microarray results suggested that 5678 mRNAs and 32 miRNAs were differentially expressed in the 2,5-HD-treated group. A total of 262 target mRNAs were obtained by miRNA and mRNA coexpression analysis, forming 368 miRNA-mRNA coexpression relationship pairs with 27 miRNAs. GO and KEGG analyses showed that differentially expressed genes were significantly enriched in the cell cycle and Wnt signaling pathways. Furthermore, significant changes in the expression of Wnt signaling pathway and cell cycle- related genes (Fzd1, Lrp6, Tcf3, Tcf4, Fzd6, Lrp5, ß-catenin, Lef1, GSK3ß, and Dvl3) after 2,5-HD treatment were confirmed by qRT-PCR and Western blotting. Ther results of dual-luciferase assays indicated decreased ß-catenin/TCF transcriptional activity after 2,5-HD treatment. In addition, Wnt pathway-related miRNAs (rno-miR-145-5p, rno-miR-143-3p, rno-miR-214-3p, rno-miR-138-5p, and rno-miR-199a-3p) were changed significantly after 2,5-HD treatment. In summary, 2,5-HD induced cell cycle arrest in ovarian GCs, and the Wnt/ß-catenin signaling pathway may play a very critical role in this process. Alterations in the expression of miRNAs such as rno-miR-145-5p may have significant implications.

Cadmium disrupts mouse embryonic stem cell differentiation into ovarian granulosa cells through epigenetic mechanisms.

Cadmium (Cd) can influence germ cell development, and epigenetic events may be involved. However, there is no study on whether Cd can influence germ cells differentiation into ovarian granulosa cells (GCs), and more insight into the molecular mechanism of the effect of Cd on germ cell development from mouse embryonic stem (ES) cells into ovarian granulosa cells and investigation of appropriate epigenetic factors are of great importance. In this study, mouse ES cell differentiation into GCs was established in an in vitro model. Subsequently, different Cd concentrations of 0, 0.1, 0.3, and 1 and then 3.0, and 10.0 µmol/L were cultured in this in vitro model. We demonstrated that Cd treatment can interrupt ES cell differentiation into GCs by morphology and ultrastructure observation. Four specific markers (octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (Nanog), and Anti-müllerian hormone type II receptor (Amhr2)) were significantly changed as measured by quantitative real-time-PCR or Western blot (p < 0.05). Cd also significantly changed the DNA methylation of GC sites on the CpG island of Nanog according to the sequential mass ARRAYR methylation method (p < 0.05). The MeRIP-qPCR method was used to detect the levels of N6-methyladenosine (m6A) methylation modification of long noncoding RNA (lncRNA) 1281 and indicated that they were decreased (p < 0.05). Microarray chip analysis, miRNA screening, and bioinformatics were used to further explore the roles of marker regulation-related miRNAs, and 27 miRNAs were putatively related to Cd-interrupted differentiation in ES cells. These data indicated that Cd can interrupt ES cell differentiation into GCs and affect germ cell development, and the underlying mechanism may involve epigenetic mechanisms.

Scalable proximal methods for cause-specific hazard modeling with time-varying coefficients.

Survival modeling with time-varying coefficients has proven useful in analyzing time-to-event data with one or more distinct failure types. When studying the cause-specific etiology of breast and prostate cancers using the large-scale data from the Surveillance, Epidemiology, and End Results (SEER) Program, we encountered two major challenges that existing methods for estimating time-varying coefficients cannot tackle. First, these methods, dependent on expanding the original data in a repeated measurement format, result in formidable time and memory consumption as the sample size escalates to over one million. In this case, even a well-configured workstation cannot accommodate their implementations. Second, when the large-scale data under analysis include binary predictors with near-zero variance (e.g., only 0.6% of patients in our SEER prostate cancer data had tumors regional to the lymph nodes), existing methods suffer from numerical instability due to ill-conditioned second-order information. The estimation accuracy deteriorates further with multiple competing risks. To address these issues, we propose a proximal Newton algorithm with a shared-memory parallelization scheme and tests of significance and nonproportionality for the time-varying effects. A simulation study shows that our scalable approach reduces the time and memory costs by orders of magnitude and enjoys improved estimation accuracy compared with alternative approaches. Applications to the SEER cancer data demonstrate the real-world performance of the proximal Newton algorithm.

MicroRNA-204-5p regulates apoptosis by targeting Bcl2 in rat ovarian granulosa cells exposed to cadmium†.

This study aimed to investigate whether cadmium (Cd) cytotoxicity in rat ovarian granulosa cells (OGCs) is mediated through apoptosis or autophagy and to determine the role of microRNAs (miRNAs) in Cd cytotoxicity. To test this hypothesis, rat OGCs were exposed to 0, 10, and 20 µM CdCl2 in vitro. As the Cd concentration increased, OGC apoptosis increased. In addition, Cd promoted apoptosis by decreasing the mRNA and protein expression levels of inhibition of B-cell lymphoma 2 (Bcl2). However, under our experimental conditions, no autophagic changes in rat OGCs were observed, and the mRNA and protein expression levels of the autophagic markers microtubule-associated protein 1 light chain 3 alpha (Map1lc3b) and Beclin1 (Becn1) were not changed. Microarray chip analysis, miRNA screening, and bioinformatics approaches were used to further explore the roles of apoptosis regulation-related miRNAs. In total, 19 miRNAs putatively related to Cd-induced apoptosis in rat OGCs were identified. Notably, miR-204-5p, which may target Bcl2, was identified. Then, rat OGCs were cultured in vitro and used to construct the miR-204-5p-knockdown cell line LV2-short hairpin RNA (shRNA). LV2-shRNA cells were exposed to 20 µM Cd for 12 h, and the mRNA and protein expression levels of Bcl2 were increased. Our findings suggest that Cd is cytotoxic to rat OGCs, and mitochondrial apoptosis rather than autophagy mediates Cd-induced damage to OGCs. Cd also affects apoptosis-related miRNAs, and the underlying apoptotic mechanism may involve the Bcl2 gene.

The Protective Role of Natriuretic Peptide Receptor 2 against High Salt Injury in the Renal Papilla.

Mutations in natriuretic peptide receptor 2 (Npr2) gene cause a rare form of short-limbed dwarfism, but its physiological effects have not been well studied. Human and mouse genetic data suggest that Npr2 in the kidney plays a role in salt homeostasis. Herein, we described anatomic changes within renal papilla of Npr2 knockout (Npr2-/-) mice. Dramatic reduction was found in diuresis, and albuminuria was evident after administration of 1% NaCl in drinking water in Npr2-/- and heterozygous (Npr2+/-) mice compared with their wild-type (Npr2+/+) littermates. There was indication of renal epithelial damage accompanied by high numbers of red blood cells and inflammatory cells (macrophage surface glycoproteins binding to galectin-3) and an increase of renal epithelial damage marker (T-cell Ig and mucin domain 1) in Npr2-/- mice. Addition of 1% NaCl tended to increase apoptotic cells (cleaved caspase 3) in the renal papilla of Npr2-/- mice. In vitro, genetic silencing of the Npr2 abolished protective effects of C-type natriuretic peptide, a ligand for Npr2, against death of M-1 kidney epithelial cells exposed to 360 mmol/L NaCl. Finally, significantly lower levels of expression of the NPR2 protein were detected in renal samples of hypertensive compared with normotensive human subjects. Taken together, these findings suggest that Npr2 is essential to protect renal epithelial cells from high concentrations of salt and prevent kidney injury.

Cadmium induces ovarian granulosa cell damage by activating PERK-eIF2α-ATF4 through endoplasmic reticulum stress.

This study aimed to investigate whether cadmium induces ovarian granulosa cell damage by activating protein kinase R-like endoplasmic reticulum kinase (PERK)-eIF2α-ATF4 through endoplasmic reticulum (ER) stress and to elucidate the underlying regulation mechanism. Two models of cadmium exposure were established. In one model, ovarian granulosa cells isolated from 21-day-old female Sprague Dawley rats were cultured in vitro for 36 h and exposed to CdCl2 (0, 5, 10, and 20 µM), and in another model, a human ovarian granulosa tumor cell line (COV434) was used to construct the binding immunoglobulin protein (BIP)-knockdown cell line sh-BIP and exposed to 0 and 20 µM CdCl2. After exposure to cadmium for 12 h, the expression mRNA and protein levels of BIP, p-PERK, and p-eIF2α were determined in the two models. miRNAs related to BIP were also detected in granulosa cells after cadmium exposure. We found that mRNA and protein levels of all factors were upregulated in each cadmium-dose group, except for BIP mRNA expression in the 5 µM Cd group. The BIP gene was knocked down in COV434 cells before exposure to cadmium. All factors were upregulated in COV434 cells exposed to Cd, and the expression of the p-eIF2α protein was downregulated in sh-BIP cells exposed to Cd. In addition, no differences in BIP-related miRNAs were detected in cadmium-exposed rat ovarian granulosa cells versus the control group. Cadmium induces ovarian granulosa cell damage by inducing ER stress.

Effect of cadmium on kitl pre-mRNA alternative splicing in murine ovarian granulosa cells and its associated regulation by miRNAs.

In this study, we established an in vitro exposure model of murine ovarian granulosa cells to observe the effect of Cd on alternative splicing of the kitl pre-mRNA and subsequently to explore the role of kitl gene expression regulation-related miRNAs through miRNA prediction, miRNA chip, bioinformatics and real-time quantitative polymerase chain reaction analyses. Our results showed that the kitl1/kitl2 mRNA ratio was significantly different (P < 0.05) at different dosages and times. The miRNA chip analysis showed that the miRNA expression profiles for the Cd treatment were significantly changed, and the expression of 29 miRNAs involved in alternative splicing of the kitl pre-mRNA was changed. The gene ontology analysis showed that the target gene functions of these 29 miRNAs were mainly enriched in the biological processes of cell metabolism regulation, post-transcriptional regulation of mRNA, interleukin-6-mediated signal transduction, cell cycle, cell proliferation, differentiation and migration. The pathway enrichment analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in the Ras signaling pathway, the Rap1 signaling pathway, the Foxo signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the carcinogenic pathway. Polymerase chain reaction verification results showed that compared to the control group, the variation trends in the expression of mmu-miR-27a-3p, mmu-miR-34b-5p, mmu-miR-297a-3p, mmu-miR-129-5p and mmu-miR-107-3p in the 4 hour 10 µm Cd treatment group were basically the same as that of the chip result. Our results indicate that Cd exposure can affect alternative splicing of the kitl pre-mRNA in ovarian granulosa cells, and miRNAs play regulatory roles in the alternative splicing of kitl.

Di(2-ethylhexyl) phthalate (DEHP) influences follicular development in mice between the weaning period and maturity by interfering with ovarian development factors and microRNAs.

Although studies have shown that di(2-ethylhexyl) phthalate (DEHP) can disrupt ovarian function, few reports have focused on follicular development in mice between the weaning period and maturity, especially with respect to microRNA (miRNA) expression. In this study, 21-day-old ICR mice were administered DEHP at doses of 0, 100, 400, and 1600 mg/(kg d) for 6 weeks by gavage. After DEHP administration, a significant decrease in the expression of follicle development-related factors (including c-kit, kitl, gdf9, and atm) was observed by quantitative real-time PCR (RT-PCR), but no significant difference in the proteins encoded by these genes was observed by Western blot. Bisulfite sequencing suggested that the total methylation percentages of promoter regions of these genes were not notably altered after DEHP exposure. However, RT-PCR revealed a significantly increased expression of ovarian miRNAs (let-7b, miR-17-5p miR-181a, and miR-151), which inhibit follicular granulosa cell proliferation. Overall, this study showed that DEHP administration from weaning to maturity could suppress the mRNA expression of follicular development factors, and this effect was not achieved through changes in the methylation of DNA in CpG islands of development factors. In addition, DEHP was shown to induce miRNAs to inhibit the proliferation of follicular granulosa cells and the anti-apoptosis function of KITL and GDF9 while increasing bax/bcl2 expression to further promote the apoptosis of granulosa cells.

Expression and DNA methylation status of the Rap2B gene in human bronchial epithelial cells treated by cigarette smoke condensate.

BACKGROUND: The relationship between lung cancer and smoking has been demonstrated. The Rap2B gene is usually overexpressed in lung cancers. This study was aimed to investigate the Rap2B gene expression and its promoter methylation in human bronchial epithelial cells (16HBE) treated by cigarette smoke condensate (CSC). METHODS: 16HBE cells were treated with CSC (1/8 IC50). Soft ager assay, tumorigenicity test, chromosome aberrations analysis were used to identify the transformed cells. The expression level of mRNA and protein of Rap2B was detected using real time PCR and Western blotting, respectively. The genome DNA methylation level was detected using combined bisulfite restriction analysis (COBRA) and the methylation status of the target fragment in Rap2B gene promoter was determined by bisulfite sequencing PCR (BSP). RESULTS: The 16HBE cells were successfully malignant transformed after the chronic exposure to CSC. The expression of Rap2B gradually increased in the process of malignant transformation. Meanwhile, global DNA was hypomethylated. However, no obvious change was observed in the methylation level of Rap2B gene promoter in transformed 16HBE cells. CONCLUSIONS: Rap2B gene may play an important role in the process of lung cancer and global DNA hypomethylation might be an early event in tumorigenesis.

Effects of cadmium exposure during pregnancy on genome-wide DNA methylation and the CREB/CREM pathway in the testes of male offspring rats.

This experimental study explored the multigenerational and transgenerational effects of cadmium (Cd) exposure during pregnancy on the testicular tissue and spermatogenesis of male offspring rats. CdCl2 at different doses (0, 0.5, 1, 2 mg/kg/day) were dispensed to pregnant SD rats, thus producing generation F1. Adult females in F1 (PND 56) were mated with untreated fertile males so as to produce generation F2. Likewise, adult females in F2 were mated to produce generation F3. Damages to testicular tissue were observed in all the three generations, with serum testosterone (T) increased in F2 and F3. Notably, the genome-wide DNA methylation level in the testicular tissue of F1 was altered, as was the expression of F1-F3 methyltransferases. In addition, the expression of Creb/Crem pathway, a pathway critical for the metamorphosis from postmeiotic round spermatocytes to spermatozoa, was also remarkably altered in the three generations. In concludion, prenatal Cd exposure might bring multigenerational and transgenerational toxic effects to testes via genome-wide DNA methylation and the regulation of CREB/CREM pathway.

Apoptosis of rat ovarian granulosa cells by 2,5-hexanedione in vitro and its relevant gene expression.

Although n-hexane is toxic to the ovary, the related mechanism remains unknown. Apoptosis of rat ovarian granulosa cells is thought to be one of the important reasons for ovarian toxicity. Rat ovarian granulosa cells were exposed in vitro to different concentrations of 2,5-hexanedione (HD, a major metabolite of n-hexane; 0, 20, 40 and 60 mmol l(-1) ) to observe the apoptosis, and its relevant gene expression was investigated. It was found by MTT assay that different exposure doses and different exposure time could inhibit the viability of ovarian granulosa cells. After a 12 h exposure, we found the apoptosis rate of the ovarian granulosa cells increased notably with the increase of HD dose. The HD concentration of each exposure dose could inhibit the message RNA (mRNA) expression of both bcl-2 and xiap, and increase the mRNA expression of fasl without any influence on that of bcl-xl, while only an HD concentration of 40 mol l(-1) could raise the mRNA expression of bax. Concentrations of HD of 40 and 60 mol l(-1) could inhibit the protein expression of NF-κB, but only an HD concentration of 60 mol l(-1) could improve the activation of NF-κB. In summary, our results indicate that HD can induce the apoptosis of ovarian granulosa cell, and the occurrence of apoptosis is related to inhibition of protein expression of NF-κB, activation of NF-κB, upregulation of mRNA levels of fasl and bax, and downregulation of mRNA levels of xiap and bcl-2.