Stanniocalcin (STC), a glycoprotein hormone, is expressed in a wide variety of tissues to regulate Ca2+ and PO4- homeostasis. STC2, a member of STC family, has been reported to be associated with tumor development. In this study, we investigated whether the expression of STC2 is associated with migration and invasion of breast cancer cells. We found that breast cancer cell line 231 HM transfected with STC2 shRNA displayed high motility, fibroblast morphology, and enhanced cell migration and invasion. Introduction of STC2 in 231 cells reduced cell migration and invasion. In response to irradiation, silencing of STC2 in 231 HM cells reduced apoptosis, whereas overexpression of STC2 in 231 cells promoted apoptosis, compared with in control cells. Mechanistic study showed that STC2 negatively regulated PKC to control the expression of Claudin-1, which subsequently induced the expressions of EMT-related factors including ZEB1, ZO-1, Slug, Twist, and MMP9. Suppression of PKC activity by using a PKC inhibitor (Go 6983) restored the normal motility of STC2-silenced cells. Furthermore, in vivo animal assay showed that STC2 inhibited tumorigenesis and metastasis of breast cancer cells. Collectively, these results indicate that STC2 may inhibit EMT at least partially through the PKC/Claudin-1-mediated signaling in human breast cancer cells. Thus, STC2 may be exploited as a biomarker for metastasis and targeted therapy in human breast cancer.

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

The previous study in our team found that supplementation of probiotic Bacillus amyloliquefaciens (Ba) instead of antibiotics promote growth performance of piglets. Hence, the present study was carried out to further demonstrate the effect of Ba replacement of antibiotics on digestive and absorption enzyme activity and intestinal microbiota population of piglets. A total of 90 piglets were selected and divided into three groups: G1 group was fed with basal diet supplemented with 150 mg/Kg aureomycin, G2 group was fed with 1 × 108 cfu/Kg Ba and half dose of aureomycin, G3 group was used the diet with 2 × 108cfu/Kg Ba replaced aureomycin. Each treatment had three replications of 10 pigs per pen. Results indicated that Ba replacement significantly increased the activities of amylase, disaccharides and Na+/K+-ATPase. And chymotrypsin activity in different section of intestine was dramatically enhanced in half replacement of aureomycin with Ba. Moreover, Ba replacement maintained the intestinal integrity with the significantly decreased activity of DAO compared with aureomycin group. Besides, supplementation with Ba increased the β-diversity of intestinal microbiota. Taken together, the current study indicated that diet supplementation with Ba instead of aureomycin increased the growth performance of piglets by improving the digestive and absorb enzyme activities, enhancing the intestinal integrity and regulating the population of intestinal micrbiota.

PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 signaling pathway and to identify genes whose loss-of-function sensitizes cancer cells to PRMT5 inhibition, we performed a CRISPR/Cas9 genetic screen in the presence of a PRMT5 inhibitor. We identified known components of the PRMT5 writer/reader pathway including PRMT5 itself, MEP50/WDR77, PPP4C, SMNDC1 and SRSF3. Interestingly, loss of PRMT1, the major asymmetric arginine methyltransferase, also sensitizes cells to PRMT5 inhibition. We investigated the interplay between PRMT5 and PRMT1, and found that combinatorial inhibitor treatment of small cell lung cancer and pancreatic cancer cell models have a synergistic effect. Furthermore, MTAP-deleted cells, which harbor an attenuated PRMT5-MEP50 signaling pathway, are generally more sensitive to PRMT1 inhibition. Together, these findings demonstrate that there is a degree of redundancy between the PRMT5 and PRMT1 pathways, even though these two enzymes deposit different types of arginine methylation marks. Targeting this redundancy provides a vulnerability for tumors carrying a co-deletion of MTAP and the adjacent CDKN2A tumor suppressor gene.

Brain-derived neurotrophic factor is associated with the insulin signaling pathway and glucose tabolism. We hypothesized that expression of brain-derived neurotrophic factor and its receptor may be involved in glucose intolerance following ischemic stress. To verify this hypothesis, this study aimed to observe the changes in brain-derived neurotrophic factor and tyrosine kinase B receptor expression in glucose metabolism-associated regions following cerebral ischemic stress in mice. At day 1 after middle cerebral artery occlusion, the expression levels of brain-derived neurotrophic factor were significantly decreased in the ischemic cortex, hypothalamus, liver, skeletal muscle, and pancreas. The expression levels of tyrosine kinase B receptor were decreased in the hypothalamus and liver, and increased in the skeletal muscle and pancreas, but remained unchanged in the cortex. Intrahypothalamic administration of brain-derived neurotrophic factor (40 ng) suppressed the decrease in insulin receptor and tyrosine-phosphorylated insulin receptor expression in the liver and skeletal muscle, and inhibited the overexpression of gluconeogenesis-associated phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver of cerebral ischemic mice. However, serum insulin levels remained unchanged. Our experimental findings indicate that brain-derived neurotrophic factor can promote glucose metabolism, reduce gluconeogenesis, and decrease blood glucose levels after cerebral ischemic stress. The low expression of brain-derived neurotrophic factor following cerebral ischemia may be involved in the development of glucose intolerance.

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.

Histone modifications play important roles in regulating eukaryotic gene expression and have been used to model expression levels. Here, we present a regression model to systematically infer mRNA stability by comparing transcriptome profiles with ChIP-seq of H3K4me3, H3K27me3 and H3K36me3. The results from multiple human and mouse cell lines show that the inferred unstable mRNAs have significantly longer 3'Untranslated Regions (UTRs) and more microRNA binding sites within 3'UTR than the inferred stable mRNAs. Regression residuals derived from RNA-seq, but not from GRO-seq, are highly correlated with the half-lives measured by pulse-labeling experiments, supporting the rationale of our inference. Whereas, the functions enriched in the inferred stable and unstable mRNAs are consistent with those from pulse-labeling experiments, we found the unstable mRNAs have higher cell-type specificity under functional constraint. We conclude that the systematical use of histone modifications can differentiate non-expressed mRNAs from unstable mRNAs, and distinguish stable mRNAs from highly expressed ones. In summary, we represent the first computational model of mRNA stability inference that compares transcriptome and epigenome profiles, and provides an alternative strategy for directing experimental measurements.

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

Postmenopausal women with metabolic syndrome (MetS) have increased cardiovascular morbidity and left ventricular diastolic dysfunction (LVDD). The various protective effects of astragalus membranaceus (AM) have been described in previous studies. Therefore, this study aimed to evaluate the effects of different doses of AM on diastolic function in postmenopausal hypertensive women with MetS.

Kaempferol (kaem) is a dietary flavonoid found in a variety of fruits and vegetables. The inhibitory effects of kaem on primary tumour growth have been extensively investigated; however, its effects on tumour metastasis are largely unknown. In the present study, we found that kaem significantly suppresses both primary tumour growth and lung metastasis in mouse breast tumour model. Furthermore, decreased expression of citrullinated histone H3 (H3-cit), a biomarker of neutrophil extracellular traps (NETs), had been founded in metastatic lung upon treated with kaem. The reduction of H3-cit is not, however, due to the cytotoxicity of kaem on neutrophils since the frequency of CD11b+ Ly6G+ neutrophils did not change in lung, tumour or blood in the presence of kaem. We then confirm the anti-NETs effects of kaem in vitro by co-culturing mouse neutrophils and kaem. Supplementing the neutrophils with GSK484, a potent NET inhibitor, totally abrogated the inhibitory effects of kaem on tumour metastasis while having little or no impact on primary tumour growth, indicating the specificity of kaem acting on NET formation and tumour metastasis. We also found that kaem suppressed ROS production in mouse bone-marrow derived neutrophils. Supplementing with the ROS scavenger DPI abrogated kaem's effects on NET formation, suggesting the involvement of kaempferol in NADPH/ROS-NETs signalling. Finally, we applied the kaem on NET-deficient PAD4-/- mice and found decreased primary tumour volume and weight but similar lung metastatic tumour with kaempferol treatment. Therefore, our findings reveal a novel mechanism of kaem in breast cancer development by targeting NETs induced tumour metastasis.

Prefrontal GABAergic interneurons (INs) are crucial for social behavior by maintaining excitation/inhibition balance. However, the underlying neuronal correlates and network computations are poorly understood. We identified distinct firing patterns of prefrontal parvalbumin (PV) INs and somatostatin (SST) INs upon social interaction. Moreover, social interaction closely correlated with elevated gamma rhythms particularly at low gamma band (20 to 50 Hz). Pharmacogenetic inhibition of PV INs, instead of SST INs, reduced low gamma power and impaired sociability. Optogenetic synchronization of either PV INs or SST INs at low gamma frequency improved sociability, whereas high gamma frequency or random frequency stimulation had no effect. These results reveal a functional differentiation among IN subtypes and suggest the importance of low gamma rhythms in social interaction behavior. Furthermore, our findings underscore previously unrecognized potential of SST INs as therapeutic targets for social impairments commonly observed in major neuropsychiatric disorders.

Dasatinib, as a second-generation broad-spectrum tyrosine kinase inhibitor, presents an antitumor effect by inhibiting tyrosine kinases. However, dasatinib causes serious side effects, such as gastrointestinal bleeding and liver toxicity, possibly through the activation of ROCK kinase and MLC phosphorylation. At present, there is no effective prevention and treatment method. Previous research studies have shown that YQFM (YiQiFuMai powder injection) protects the blood-brain barrier by inhibiting the ROCK/MLC signaling pathway; whether YQFM can alleviate the side effects of dasatinib is unknown. In this study, dasatinib was injected (i.p. 70 mg/kg) and YQFM (i.p. 0.336 g/kg, 0.672 g/kg, 1.342 g/kg) was given in advance for 3 days to mice, to explore the effect of YQFM on side effects induced by Dasatinib. The results confirmed that YQFM significantly decreased Evans blue leakage in the small intestine and increased intestinal blood flow, increased the expression of ZO-1, Occludin, and VE-cadherin, and reduced the contents of D-lactic acid, s-VE-cadherin, Alanine aminotransferase (ALT), and Aspartate aminotransferase (AST) in serum. Finally, YQFM inhibited the expression of ROCK-1 and phosphorylation of MLC induced by Dasatinib. These findings suggested that YQFM could improve the side effects caused by Dasatinib linked with the ROCK/MLC signaling pathway, as shown in the graphical abstract.

Advancements in immunotherapy have improved our understanding of the immune characteristics of breast cancer. Here, we analyzed gene expression profiles and clinical data obtained from The Cancer Genome Atlas database to identify genes that were differentially expressed between breast tumor tissues and normal breast tissues. Comparisons with the Immunology Database and Analysis Portal (ImmPort) indicated that many of the identified differentially expressed genes were immune-related. Risk scores calculated based on an eight-gene signature constructed from these immune-related genes predicted both overall survival and relapse-free survival outcomes in breast cancer patients. The predictive value of the eight-gene signature was validated in different breast cancer subtypes using external datasets. Associations between risk score and breast cancer immune characteristics were also identified; invitro experiments using breast cancer cell lines confirmed those associations. Thus, the novel eight-gene signature described here accurately predicts breast cancer survival outcomes as well as immune checkpoint expression and immune cell infiltration processes.

Hepatocellular carcinoma (HCC), a leading cause of cancer-related death with high invasive and metastatic potential, has a low survival rate. To improve the survival and quality of life in HCC patients, it is urgently needed to explore novel biomarkers for early diagnosis and prognosis of HCC, as well as therapeutic strategies. Circular RNAs (circRNAs) are a class of highly conserved, stable and abundant non-coding RNAs (ncRNAs) that can regulate gene expression at transcriptional or post-transcriptional levels. Recently, some circRNAs are identified to be potential biomarkers for HCC diagnosis and prognosis. Furthermore, some circRNAs are found to play oncogenic or suppressive roles in HCC progression by regulating various biological processes, including cell proliferation, migration, invasion and metastasis, epithelial-mesenchymal transition (EMT), as well as apoptosis. In this review, we summarize recent findings of deregulated circRNAs, their functions and molecular mechanisms in HCC, and discuss their potential roles as diagnostic biomarkers, prognostic biomarkers, as well as therapeutic targets for HCC.

The effects of TiO2 nanoparticles (nano-TiO2) together with antibiotics leaking into wastewater treatment plants (WWTPs), especially the partial nitrification (PN) process remain unclear. To evaluate the combined impact and mechanisms of nano-TiO2 and antibiotics on PN systems, batch experiments were carried out with six bench-scale sequencing batch reactors. Nano-TiO2 at a low level had minimal effects on the PN system. In combination with tetracycline and erythromycin, the acute impact of antibiotics was enhanced. Both steps of nitrification were retarded due to the decrease of bacterial activity and abundance, while nitrite-oxidizing bacteria were more sensitive to the inhibition than ammonia-oxidizing bacteria. Proteobacteria at the phylum level and Nitrosospira at the genus level remained predominant under single and combined impacts. The flow cytometry analysis showed that nano-TiO2 enhanced the toxicity of antibiotics through increasing cell permeability. Our results can help clarify the risks of nano-TiO2 combined with antibiotics to PN systems and explaining the behavior of nanoparticles in WWTPs.

Acute lung injury (ALI) is a severe inflammatory disease, underscoring the urgent need for novel treatments. Nauclea officinalis Pierre ex Pitard (Danmu in Chinese, DM) is effective in treating inflammatory respiratory diseases. However, there is still no evidence of its protective effect against ALI.

Mutations in CAD gene, encoding a multifunctional enzyme involved in de novo pyrimidine biosynthesis, has been reported to be associated with early-onset epileptic encephalopathy (EOEE). Herein, we generated an induced pluripotent stem cell (iPSC) line from the skin fibroblasts of a five-year-old boy with CAD deficiency, presented with developmental delay, refractory epilepsy, anemia with anisopoikilocytosis, and dramatic responsive to supplementation with oral uridine, carrying biallelic mutations, c.108delC (p.Tyr36Tyrfs*15) and c.3775G>A (p.Val1259Met) in CAD. These iPSCs exhibited stable amplification, expressed pluripotent markers, and differentiated spontaneously into three germ layers in vitro.

Cancer subtypes can improve our understanding of cancer, and suggest more precise treatment for patients. Multi-omics molecular data can characterize cancers at different levels. Up to now, many computational methods that integrate multi-omics data for cancer subtyping have been proposed. However, there are no consistent criteria to evaluate the integration methods due to the lack of gold standards (e.g., the number of subtypes in a specific cancer). Since comprehensive evaluation and comparison between different methods serves as a useful tool or guideline for users to select an optimal method for their own purpose, we develop a scalable platform, CEPICS, for comprehensively evaluating and comparing multi-omics data integration methods in cancer subtyping. Given a user-specified maximum number of subtypes, k-max, CEPICS provides (1) cancer subtyping results using up to five built-in state-of-the-art integration methods under the number of subtypes from two to k-max, (2) a report including the evaluation of each user-selected method and comparisons across them using clustering performance metrics and clinical survival analysis, and (3) an overall analysis of subtyping results by different methods representing a robust cancer subtype prediction for samples. Furthermore, users can upload subtyping results of their own methods to compare with the built-in methods. CEPICS is implemented as an R package and is freely available at https://github.com/GaoLabXDU/CEPICS.

Introduction: Long non-coding RNAs (lncRNAs) are key regulators in multiple cancers. lncRNA, SNHG1, was shown to be associated with tumorigenesis. However, little is known about the role SNHG1 plays in breast cancer. The aim of the study was to study the role and underlying mechanism of SNHG1 regulation in breast cancer. Methods: Quantitative real-time PCR was used to measure the levels of SNHG1, miR-382 and ZEB1 levels in breast cancer tissue or cells. The proliferation, colony formation, migration and invasion of breast cancer cells, under SNHG1 knockdown achieved by transfection of SNHG1-specific siRNAs, were assessed by Cell Counting Kit-8, colony forming, scratch wound and transwell assays. Bioinformatical analysis and luciferase assay were used to explore the interaction between SNHG1 and its potential miRNA target. Western blot was used to evaluate the expression of epithelial-to-mesenchymal transition (EMT) markers. MDA-MB-231 cells with or without SNHG1 knockdown were used to initiate tumor xenografts in vivo. Tumor growth and expression of SNHG1, miR-382-5p and EMT markers were evaluated. Results: SNHG1 upregulation was observed in breast cancer tissues and cells. Knockdown of SNHG1 attenuated breast cancer proliferation, colony formation, migration and invasion. A miRNA, miR-382-5p, was identified as the target of SNHG1. A reciprocal negative regulation was found between SNHG1 and miR-382-5p. SNHG1 knockdown attenuated EMT both in vitro and in vivo. miR-382-5p transfection reversed the tumor-promoting role by SNHG1. In vivo, SNHG1 knockdown decreased breast tumor growth. Conclusion: SNHG1 promotes breast cancer through the regulation of miR-382-5p and EMT markers. Our results report SNHG1 as a novel miRNA that govern the progression of breast cancer, providing a potential new therapeutic target in breast cancer.