Key mRNAs and lncRNAs of pituitary that affect the reproduction of FecB + + small tail han sheep.

BACKGROUND: The pituitary directly regulates the reproductive process through follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Transcriptomic research on the pituitaries of ewes with different FecB (fecundity Booroola) genotypes has shown that some key genes and lncRNAs play an important role in pituitary function and sheep fecundity. Our previous study found that ewes with FecB + + genotypes (without FecB mutation) still had individuals with more than one offspring per birth. It is hoped to analyze this phenomenon from the perspective of the pituitary transcriptome. RESULTS: The 12 Small Tail Han Sheep were equally divided into polytocous sheep in the follicular phase (PF), polytocous sheep in the luteal phase (PL), monotocous sheep in the follicular phase (MF), and monotocous sheep in the luteal phase (ML). Pituitary tissues were collected after estrus synchronous treatment for transcriptomic analysis. A total of 384 differentially expressed genes (DEGs) (182 in PF vs. MF and 202 in PL vs. ML) and 844 differentially expressed lncRNAs (DELs) (427 in PF vs. MF and 417 in PL vs. ML) were obtained from the polytocous-monotocous comparison groups in the two phases. Functional enrichment analysis showed that the DEGs in the two phases were enriched in signaling pathways known to play an important role in sheep fecundity, such as calcium ion binding and cAMP signaling pathways. A total of 1322 target relationship pairs (551 pairs in PF vs. MF and 771 pairs in PL vs. ML) were obtained for the target genes prediction of DELs, of which 29 DEL-DEG target relationship pairs (nine pairs in PF vs. MF and twenty pairs in PL vs. ML). In addition, the competing endogenous RNA (ceRNA) networks were constructed to explore the regulatory relationships of DEGs, and some important regulatory relationship pairs were obtained. CONCLUSION: According to the analysis results, we hypothesized that the pituitary first receives steroid hormone signals from the ovary and uterus and that VAV3 (Vav Guanine Nucleotide Exchange Factor 3), GABRG1 (Gamma-Aminobutyric Acid A Receptor, Gamma 1), and FNDC1 (Fibronectin Type III Domain Containing 1) played an important role in this process. Subsequently, the reproductive process was regulated by gonadotropins, and IGFBP1 (Insulin-like Growth Factor Binding Protein 1) was directly involved in this process, ultimately affecting litter size. In addition, TGIF1 (Transforming Growth Factor-Beta-Induced Factor 1) and TMEFF2 (Transmembrane Protein With EGF Like And Two Follistatin Like Domains 2) compensated for the effect of the FecB mutation and function by acting on TGF-ß/SMAD signaling pathway, an important pathway for sheep reproduction. These results provided a reference for understanding the mechanism of multiple births in Small Tail Han Sheep without FecB mutation.

A Retrospective Clinical Study on Cardiovascular Complications from Colorectal Cancer.

OBJECTIVE: To investigate the incidence and risk factors of cardiovascular complications amongst patients with colorectal cancer (CRC). METHODS: A retrospective cohort study was conducted on 2085 patients diagnosed with CRC in two tertiary hospitals in China between 2015 and 2020. The patients' medical records were reviewed to identify cardiovascular complications, including myocardial infarction, heart failure, stroke, hypertension, coronary heart disease, heart failure, and arrhythmia. The incidence rate of cardiovascular complications was calculated, and Cox proportional hazards regression analysis was used to identify risk factors. RESULTS: Of the 2085 CRC patients, 329 (15.8%) experienced cardiovascular complications during the follow-up period, with an incidence rate of 17.4 cases per 1000 person-years. The risk was significantly higher in patients who were older than 60 years (adjusted hazard ratio [HR] 2.04, 95% confidence interval [CI] 1.22-3.41), had a higher level of low-density lipoprotein cholesterol (LDL-C) (adjusted HR 2.32, 95% CI 1.31-4.10), had higher levels of serum C-reactive protein (CRP) (adjusted HR 1.57, 95% CI 1.21-2.04), or who underwent chemotherapy or radiotherapy. CRC patients with cardiovascular complications had significantly higher levels of oxidative stress markers, including malondialdehyde (MDA) (5.8 ± 1.2 µmol/L vs. 3.4 ± 0.9 µmol/L, p < 0.001), lower levels of superoxide dismutase (SOD) (85.2 ± 15.6 U/mg protein vs. 112.5 ± 21.3 U/mg protein, p < 0.001), and lower levels of glutathione peroxidase (GPx) (15.6 ± 3.2 U/mg protein vs. 20.4 ± 4.1 U/mg protein, p < 0.001) compared to those without complications. A progressive increase was observed in the proportion of CRC patients with cardiovascular complications over time, rising from 10% in the first year to 38% by the tenth year of follow-up. CONCLUSION: Cardiovascular complications pose a high risk in CRC patients, particularly amongst older patients and those with higher levels of LDL-C or CRP. Regular monitoring of cardiovascular function should be considered in the management of patients with CRC.

Construction of Prognostic Nomogram in Patients with N3-Stage Nasopharyngeal Carcinoma.

INTRODUCTION: The aim of the study was to retrospectively identify the metastatic influence factors and predict the prognosis and develop an individualized prognostic prediction model for patients with N3-stage nasopharyngeal carcinoma (NPC). METHODS: The study collected 446 NPC patients with N3 stage from the Surveillance, Epidemiology, and End Results database between 2010 and 2015. The patients were classified into subgroups based on the histological types and metastatic status. Multivariable logistic, Cox regression, and Kaplan-Meier method with the log-rank test were performed. The nomogram model was created using the prognostic factors identified from Cox regression analysis. The predictive accuracy was determined based on the concordance index (c-index) and calibration curves. RESULTS: The 5-year overall survival (OS) of the NPC patients with N3 stage was 43.9%, and the prognosis of patients without any distant metastases was largely longer than that with metastases. No difference was observed between different pathological types in the entire cohort. However, patients with non-keratinized squamous cell carcinoma had a better OS than that of the patients with keratinized squamous cell carcinoma in a nonmetastatic subgroup. Using the Cox regression analysis results, the nomogram successfully classified these patients into low- and high-risk subgroups and presented the survival difference. The c-index of the nomogram for predicting the prognosis was satisfactory. CONCLUSION: This study identified metastatic risk factors and developed a convenient clinical tool for the prognosis of NPC patients. This tool can be used for individualized risk classification and decision-making regarding treatment of NPC patients with N3 stage.

Lnc-SELPLG-2:1 enhanced osteosarcoma oncogenesis via hsa-miR-10a-5p and the BTRC cascade.

BACKGROUND: To investigate the potential role of Long Non-coding RNAs (lncRNAs) in the progression of osteosarcoma. METHODS: The candidate lncRNAs were screened with RNA-seq and confirmed with quantitative real-time PCR. Using MTS, transwell assay, and flow cytometric analysis, the effects of overexpressed lnc-SELPLG-2:1 on cell functions were determined. Immunohistochemical staining, fluorescence in situ hybridization, and luciferase reporter assay were used to evaluate the potential mechanism of lnc-SELPLG-2:1 in vivo and in vitro using a tumor model. Moreover, the effects of overexpression of hsa-miR-10a-5p on the functions of SaOS2 cells were determined using functional cell analysis. A response test was used to confirm the mechanism by which lnc-SELPLG-2:1 sponge hsa-miR-10a-5p promotes the expression of BTRC to regulate osteosarcoma. RESULTS: Lnc-SELPLG-2:1 was highly expressed in osteosarcoma compared to normal cells and bone and marrow samples. Inhibition of lnc-SELPLG-2:1 accelerated cell apoptosis and suppressed cell proliferation, migration, and invasion, whereas lnc-SELPLG-2:1 overexpression had the opposite effect. Moreover, inhibiting lnc-SELPLG-2:1 in an in vivo model decreased tumor size and suppressed the expression of cell migration-related proteins. The prediction, dual luciferase assay, and response test results indicated that hsa-miR-10-5p and BTRC were involved in the lnc-SELPLG-2:1 cascade. Unlike lnc-SELPLG-2:1, hsa-hsa-miR-10a-5p had opposite expression and function. Competitive binding of lnc-SELPLG-2:1 to hsa-hsa-miR-10a-5p prevented BTRC from miRNA-mediated degradation, thereby activating the expression of VIM, MMP9, and MMP2, promoting osteosarcoma cell proliferation, migration, and invasion, and inhibiting apoptosis. CONCLUSION: Lnc-SELPLG-2:1 is an oncogenesis activator in osteosarcoma, and its functions are performed via hsa-miR-10a-5p /BTRC cascade.

Clinical efficacy of irinotecan plus raltitrexed chemotherapy in refractory esophageal squamous cell cancer.

Our retrospective study assessed the efficacy and safety of irinotecan plus raltitrexed in esophageal squamous cell cancer (ESCC) patients who were previously treated with multiple systemic therapies. Between January 2016 and December 2018, records of 38 ESCC patients who underwent irinotecan plus raltitrexed chemotherapy after at least one line of chemotherapy were reviewed. Efficacy assessment was performed every two cycles according to the RECIST version 1.1. A total of 95 cycles of chemotherapy were administered, and the median course was 3 (range 2-6). There was no treatment-related death. Nine patients had partial response, 21 had stable disease and eight had progressive disease. The overall objective response rate was 23.68% (9/38) and the disease control rate was78.94% (30/38). After a median follow-up of 18.5 months, the median progression-free survival and overall survival were 105 and 221 days, respectively. There were five patients (13.15%) with grade 3/4 leukopenia, three patients (7.89%) with grade 3/4 neutropenia and one patient (2.63%) with grade 3/4 diarrhea. The combination of irinotecan plus raltitrexed was effective for pretreated ESCC patients. Further studies are needed to determine the optimal dose of the two drugs.

Extramedullary leukemia behaving as solid cancer: clinical, histologic, and genetic clues to chemoresistance in organ sites.

Recent studies of leukemic tumors in individual extramedullary sites showed they adopt the clinical and metastatic behavior of solid cancers originating in those sites. To elucidate features of leukemic tumors that render them resistant to agents effective against marrow leukemia, we analyzed a series of AML breast tumors by histology, immunohistochemistry, and RNA sequencing. Striking histologic similarities to solid cancers were found: a single-filing architectural pattern virtually identical to that of invasive lobular breast carcinoma and dense desmoplastic keloid-like fibrosis similar to colon, gallbladder, and pancreas carcinomas. Sequencing found 2157 genes significantly downregulated in AML breast tumors compared to normal breast. Comparison to triple-negative breast cancer found 859 genes similarly downregulated. At least 30 of these genes have been associated with poor prognosis in breast cancers. Five were reported in AML marrow studies to correlate with poor prognosis. The findings of this pilot study suggest the seed-and-soil interaction recognized in solid cancer growth may help explain how leukemic cells, in some patients, adopt solid tumor behavior in non-marrow sites. Transformed cells that metastasize from tumor to marrow can impart chemoresistance and be an unrecognized cause of treatment failure and death. Further studies comparing leukemic tumor to simultaneous marrow could potentially identify biomarkers that predict extramedullary resistance and lead to new therapeutic targets. Recognizing the potential for leukemia to adopt solid tumor phenotype, and implementation of body scanning and ablative tumor treatment, could decrease the persistently high rates of marrow resistance and treatment failure.

Regulator of G protein signaling 6 is a critical mediator of both reward-related behavioral and pathological responses to alcohol.

Alcohol is the most commonly abused drug worldwide, and chronic alcohol consumption is a major etiological factor in the development of multiple pathological sequelae, including alcoholic cardiomyopathy and hepatic cirrhosis. Here, we identify regulator of G protein signaling 6 (RGS6) as a critical regulator of both alcohol-seeking behaviors and the associated cardiac and hepatic morbidities through two mechanistically divergent signaling actions. RGS6(-/-) mice consume less alcohol when given free access and are less susceptible to alcohol-induced reward and withdrawal. Antagonism of GABA(B) receptors or dopamine D2 receptors partially reversed the reduction in alcohol consumption in RGS6(-/-) animals. Strikingly, dopamine transporter inhibition completely restored alcohol seeking in mice lacking RGS6. RGS6 deficiency was associated with alterations in the expression of genes controlling dopamine (DA) homeostasis and a reduction in DA levels in the striatum. Taken together, these data implicate RGS6 as an essential regulator of DA bioavailability. RGS6 deficiency also provided dramatic protection against cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia when mice were forced to consume alcohol. Although RGS proteins canonically function as G-protein regulators, RGS6-dependent, alcohol-mediated toxicity in the heart, liver, and gastrointestinal tract involves the ability of RGS6 to promote reactive oxygen species-dependent apoptosis, an action independent of its G-protein regulatory capacity. We propose that inhibition of RGS6 might represent a viable means to reduce alcohol cravings and withdrawal in human patients, while simultaneously protecting the heart and liver from further damage upon relapse.

Snail Enhances Glycolysis in the Epithelial-Mesenchymal Transition Process by Targeting FBP1 in Gastric Cancer.

BACKGROUND: Snail is a key regulator of epithelial-mesenchymal transition (EMT) in cancer. However, the regulatory role and underlying mechanisms of Snail in gastric cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by Snail in gastric cancer. METHODS: The impact of Snail on glucose metabolism was studied in vitro. Combining maximum standardized uptake value (SUVmax), which was obtained preoperatively via a PET/CT scan, with immunohistochemistry staining, we further analyzed the correlation between SUVmax and Snail expression in gastric cancer tissues. RESULTS: Increased expression of Snail promoted lactate production, glucose utilization, and decreased FBP1 expression at both mRNA and protein level. The expression level of Snail was positively associated with SUVmax in gastric cancer patients (P=0.022). Snail and FBP1 expression were inversely correlated at both mRNA and protein level (P=0.002 and P=0.015 respectively) in gastric cancer tissues. Further studies demonstrated that Snail inhibited the FBP1 gene expression at the transcriptional level. Restoring FBP1 expression reversed the effects of glycolysis and EMT induced by Snail in gastric cancer cells. CONCLUSIONS: Our results thus reveal that Snail serves as a positive regulator of glucose metabolism through regulation of the FBP1 in gastric cancer. Disrupting the Snail-FBP1 signaling axis may be effective to prevent primary tumor EMT and glycolysis process.

Regulator of G Protein Signaling 6 Protects the Heart from Ischemic Injury.

Gαi-coupled receptors play important roles in protecting the heart from ischemic injury. Regulator of G protein signaling (RGS) proteins suppress Gαi signaling by accelerating the GTPase activity of Gαi subunits. However, the roles of individual RGS proteins in modulating ischemic injury are unknown. In this study, we investigated the effect of RGS6 deletion on myocardial sensitivity to ischemic injury. Hearts from RGS6 knockout (RGS6-/-) and RGS6 wild-type (RGS6+/+) mice were subjected to 30 minutes of ischemia and 2 hours of reperfusion on a Langendorff heart apparatus. Infarcts in RGS6-/- hearts were significantly larger than infarcts in RGS6+/+ hearts. RGS6-/- hearts also exhibited increased phosphorylation of ß2-adrenergic receptors and G protein-coupled receptor kinase 2 (GRK2). Mitochondrial GRK2 as well as caspase-3 cleavage were increased significantly in RGS6-/- hearts compared with RGS6+/+ hearts after ischemia. Chronic propranolol treatment of mice prevented the observed increases in ischemic injury and the GRK2 phosphorylation observed in RGS6-/- hearts. Our findings suggest that loss of RGS6 predisposes the ventricle to prodeath signaling through a ß2AR-GRK2-dependent signaling mechanism, and they provide evidence for a protective role of RGS6 in the ischemic heart. Individuals expressing genetic polymorphisms that suppress the activity of RGS6 may be at increased risk of cardiac ischemic injury. Furthermore, the development of agents that increase RGS6 expression or activity might provide a novel strategy for the treatment of ischemic heart disease.

Rgs6 is required for adult maintenance of dopaminergic neurons in the ventral substantia nigra.

Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

Regulator of G protein signaling 6 (RGS6) in dopamine neurons promotes EtOH seeking, behavioral reward, and susceptibility to relapse.

Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). The neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6fl/fl; DAT-iCreER mice to determine the role of RGS6 in DA neurons on EtOH consumption, reward, and relapse behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse ventral tegmental area (VTA), and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6-/- mice. Strikingly, RGS6 loss from DA neurons before or after EtOH behavioral reward is established significantly reduced (~ 50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies identify DA neurons as the locus of RGS6 action in promoting EtOH consumption, preference, reward, and relapse. RGS6 is unique among R7 RGS proteins in promoting rather than suppressing behavioral responses to drugs of abuse and to modulate EtOH behavioral reward. This is a result of RGS6's pre-synaptic actions that we hypothesize promote VTA DA transmission by suppressing GPCR-Gαi/o-DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.

Regulator of G protein signaling 6 (RGS6) protein ensures coordination of motor movement by modulating GABAB receptor signaling.

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gßγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gß(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.

Defective retinal depolarizing bipolar cells in regulators of G protein signaling (RGS) 7 and 11 double null mice.

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gß5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gß5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gß5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gß5S is undetectable, whereas levels of the photoreceptor-specific Gß5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gß5S expression in retina, the DBC-related defects in Gß5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gß5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.

Regulator of G protein signaling 6 (RGS6) in ventral tegmental area (VTA) dopamine neurons promotes EtOH seeking, behavioral reward and susceptibility to relapse.

Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). EtOH is the most abused substance worldwide with chronic consumption often leading to the development of dependence and abuse. Unfortunately, the neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6 fl/fl ; DAT-iCreER mice to determine the role of RGS6 in VTA DA neurons on EtOH consumption and reward behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse VTA, and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in VTA DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6 -/- mice. Strikingly, RGS6 loss from VTA DA neurons before or after EtOH behavioral reward is established significantly reduced (∼50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies illuminate a critical role of RGS6 in the mesolimbic circuit in promoting EtOH seeking, reward, and reinstatement. We propose that RGS6 functions to promote DA transmission through its function as a negative modulator of GPCR-Gα i/o -DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.

Comparative Transcriptomics Identify Key Pituitary Circular RNAs That Participate in Sheep (Ovis aries) Reproduction.

CircRNAs have been found to play key roles in many biological processes and have diverse biological functions. There have been studies on circRNAs in sheep pituitary, and some important circRNAs have been found. But there are still few studies on circRNAs in sheep pituitary with different fecundity. In this study, we obtained the circRNAs expression profiles in the pituitary of FecB ++ genotype Small Tail Han sheep with different fecundity and estrous phases. A total of 34,878 circRNAs were identified in 12 pituitary samples, 300 differentially expressed circRNAs (DE circRNAs) (down: 104; up: 196) were identified in polytocous sheep in the follicular phase (PF) and monotocous sheep in the follicular phase (MF) (PF vs. MF), and 347 DE circRNAs (down: 162; up: 185) were identified in polytocous sheep in the luteal phase (PL) and monotocous sheep in the luteal phase (ML) (PL vs. ML). Cortisol synthesis and secretion pathway (follicular phase) and estrogen signaling pathway (luteal phase) were obtained by functional enrichment analysis of circRNAs source genes. Competing endogenous RNA (ceRNA) network analysis of key DE circRNAs revealed that oar-circ-0022776 (source gene ITPR2, follicular phase) targeted oar-miR-432, oar-circ-0009003 (source gene ITPR1, luteal phase) and oar-circ-0003113 (source gene PLCB1, luteal phase) targeted oar-miR-370-3p. We also explored the coding ability of DE circRNAs. In conclusion, our study shows that changes in the pituitary circRNAs may be related to the response of the pituitary to steroid hormones and regulate the reproductive process of sheep by affecting the pituitary function. Results of this study provide some new information for understanding the functions of circRNAs and the fecundity of FecB ++ genotype sheep.

Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method.

Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice (Atm TgC3001L, Nes-Cre, and Syn1-Cre) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.

Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization.

Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys(588) and Lys(591) of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.

Regulator of G protein signaling 6 (RGS6) induces apoptosis via a mitochondrial-dependent pathway not involving its GTPase-activating protein activity.

Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.

RGS6, a modulator of parasympathetic activation in heart.

RATIONALE: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G(i/o) proteins to promote G protein-coupled inwardly rectifying K(+) (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart. OBJECTIVE: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. METHODS AND RESULTS: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I(K)(ACh)) activation and deactivation, as well as the extent of I(K)(ACh) desensitization. CONCLUSIONS: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.

Levels and Significance of Tumor Markers and Cytokines in Serum and Peritoneal Lavage Fluid of Patients with Peritoneal Metastasis of Gastric Cancer.

The paper is written to investigate the levels and significance of tumor markers [carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), and carbohydrate antigen 19-9 (CA19-9)] and cytokines [interleukin-6 (IL-6), IL-4, and IL-2] in serum and peritoneal lavage fluid of patients with peritoneal metastasis of gastric cancer. For this research, 145 patients with gastric cancer treated in our hospital were divided into peritoneal metastasis group (n = 25), other metastasis group (n = 32), and nonmetastasis group (n = 88) according to the occurrence of metastasis. At the same time, the levels of serum tumor markers and cytokines and tumor markers and cytokines in intraoperative peritoneal lavage fluid were compared among the three groups. The results showed that the proportion of TNM stage III in peritoneal metastasis group and other metastasis group was 68.00% and 62.50%, respectively, and the proportion of tumor >5 cm was 64.00% and 59.38%, respectively, which was significantly higher than that in the control group. The 1-year survival rate of peritoneal metastasis group and other metastasis group was 44.00% and 40.63%, respectively, which was significantly lower than that of nonmetastasis group (P < 0.05).The serum levels of CEA, CA125, CA19-9, IL-6, IL-4, and IL-2 in peritoneal metastasis group and other metastasis group were higher than those in nonmetastasis group. The intraoperative peritoneal lavage fluid CEA, CA125, and IL-6 were 13.41 ± 3.72 ng/ml, 8.97 ± 1.33 U/ml, and 1.85 ± 0.44 pg/ml, respectively, which were higher than those in other metastasis groups and nonmetastasis groups (P < 0.05). There was no significant difference in the levels of CA19-9, IL-4, and IL-2 in peritoneal lavage fluid among peritoneal metastasis group, other metastasis groups, and nonmetastasis groups (P > 0.05); the areas under the ROC curve of intraoperative peritoneal lavage fluid CEA, CA125, and IL-6 in predicting peritoneal metastasis were 0.850, 0.902, and 0.806, respectively, P < 0.05. Thus, the conclusion is that peritoneal lavage fluid CEA, CA125, and IL-6 have certain application value in predicting and diagnosing peritoneal metastasis of gastric cancer, while the other indexes have no application value.