A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases. CRISPR-based base editors allow for single nucleotide genome editing in a range of organisms. Here the authors demonstrate the in vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors.

/pdf/efficient-generation-of-mouse-models-of-human-diseases-via-p9gafrxed2.pdf

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing

Most older individuals develop inflammageing, a condition characterized by elevated levels of blood inflammatory markers that carries high susceptibility to chronic morbidity, disability, frailty, and premature death. Potential mechanisms of inflammageing include genetic susceptibility, central obesity, increased gut permeability, changes to microbiota composition, cellular senescence, NLRP3 inflammasome activation, oxidative stress caused by dysfunctional mitochondria, immune cell dysregulation, and chronic infections. Inflammageing is a risk factor for cardiovascular diseases (CVDs), and clinical trials suggest that this association is causal. Inflammageing is also a risk factor for chronic kidney disease, diabetes mellitus, cancer, depression, dementia, and sarcopenia, but whether modulating inflammation beneficially affects the clinical course of non-CVD health problems is controversial. This uncertainty is an important issue to address because older patients with CVD are often affected by multimorbidity and frailty — which affect clinical manifestations, prognosis, and response to treatment — and are associated with inflammation by mechanisms similar to those in CVD. The hypothesis that inflammation affects CVD, multimorbidity, and frailty by inhibiting growth factors, increasing catabolism, and interfering with homeostatic signalling is supported by mechanistic studies but requires confirmation in humans. Whether early modulation of inflammageing prevents or delays the onset of cardiovascular frailty should be tested in clinical trials. Inflammageing is a chronic, pro-inflammatory state that develops with age and is a risk factor for cardiovascular disease, comorbidities, frailty, and death. In this Review, Ferrucci and Fabbri discuss whether therapies to modulate inflammageing can reduce the age-related decline in health.

Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty

Regulation and Function of the PD-L1 Checkpoint

The mitogen-activated protein kinases (MAPKs) in mammals include c-Jun NH2-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). These enzymes are serine-threonine protein kinases that regulate various cellular activities including proliferation, differentiation, apoptosis or survival, inflammation, and innate immunity. The compromised MAPK signaling pathways contribute to the pathology of diverse human diseases including cancer and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The JNK and p38 MAPK signaling pathways are activated by various types of cellular stress such as oxidative, genotoxic, and osmotic stress as well as by proinflammatory cytokines such as tumor necrosis factor-α and interleukin 1β. The Ras-Raf-MEK-ERK signaling pathway plays a key role in cancer development through the stimulation of cell proliferation and metastasis. The p38 MAPK pathway contributes to neuroinflammation mediated by glial cells including microglia and astrocytes, and it has also been associated with anticancer drug resistance in colon and liver cancer. We here summarize recent research on the roles of MAPK signaling pathways in human diseases, with a focus on cancer and neurodegenerative conditions.

Compromised MAPK signaling in human diseases: an update

Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders.

Amyloid plaque is the hallmark and primary cause of Alzheimer disease. Mutations of presenilin-1, the gamma-secretase catalytic subunit, can affect amyloid-beta (Abeta) production and Alzheimer disease pathogenesis. However, it is largely unknown whether and how gamma-secretase activity and amyloid plaque formation are regulated by environmental factors such as stress, which is mediated by receptors including beta(2)-adrenergic receptor (beta(2)-AR). Here we report that activation of beta(2)-AR enhanced gamma-secretase activity and thus Abeta production. This enhancement involved the association of beta(2)-AR with presenilin-1 and required agonist-induced endocytosis of beta(2)-AR and subsequent trafficking of gamma-secretase to late endosomes and lysosomes, where Abeta production was elevated. Similar effects were observed after activation of delta-opioid receptor. Furthermore, chronic treatment with beta(2)-AR agonists increased cerebral amyloid plaques in an Alzheimer disease mouse model. Thus, beta(2)-AR activation can stimulate gamma-secretase activity and amyloid plaque formation, which suggests that abnormal activation of beta(2)-AR might contribute to Abeta accumulation in Alzheimer disease pathogenesis.

Activation of β2-adrenergic receptor stimulates γ-secretase activity and accelerates amyloid plaque formation

Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) has been demonstrated to be an important player in various human malignancies; it is thought to promote tumor growth by cell cycle regulating. However, the roles of MALAT1 in esophageal squamous cell carcinoma(ESCC), and the mechanisms involved in cell cycle regulation remain poorly understood. Moreover, the factors contributing to its up-regulation in tumor tissues are still largely unclear. Expression of MALAT1 was determined from cell lines and clinical samples by qRT-PCR. The effects of MALAT1 knockdown on cell proliferation, cell cycle, apoptosis, migration, and invasion were evaluated by in vitro and in vivo assays. The potential protein expression changes were investigated by Western-blotting. The methylation status of the CpG island in the MALAT1 promoter was explored by bisulfite sequencing, while the copy numbers in tumor tissues and blood samples were detected by a well-established AccuCopyTM method. MALAT1 was over-expressed in 46.3% of ESCC tissues, mostly in the high-stage tumor samples. Enhanced MALAT1 expression levels were positively correlated with clinical stages, primary tumor size, and lymph node metastasis. Inhibition of MALAT1 suppressed tumor proliferation in vitro and in vivo, as well as the migratory and invasive capacity. MALAT1 depletion also induced G2/M phase arrest and increased the percentage of apoptotic cells. Western-blotting results implicated that the ATM-CHK2 pathway which is associated with G2/M arrest was phosphorylated by MALAT1 knockdown. No effects of CpG island methylation status on MALAT1 expression were found, whereas amplification of MALAT1 was found in 22.2% of tumor tissues, which correlated significantly with its over-expression. However, neither association between tissue copy number amplification and germline copy number variation, nor correlation between germline copy number variation and ESCC risk were identified in the case–control study. Our data suggest that MALAT1 serves as an oncogene in ESCC, and it regulates ESCC growth by modifying the ATM-CHK2 pathway. Moreover, amplification of MALAT1 in tumor tissues may play an important role for its up-regulation, and it seems that the gene amplification in tumor tissues emerges during ESCC progression, but is not derived from germline origins.

/pdf/up-regulation-of-long-noncoding-rna-malat1-contributes-to-4qrepibmov.pdf

Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma

Metastasis is the primary cause of cancer-related death all over the world. Metastasis is a process by which cancer spreads from the place at which it first arose to distant locations in the body. It is well known that several steps are necessary for this process, including cancer cell epithelial-mesenchymal transition (EMT), cell migration, resistance to anoikis, and angiogenesis. Therefore, investigating the molecular mechanism of regulating cancer metastasis progress may provide helpful insights in the development of efficient diagnosis and therapeutic strategy. Recent studies have indicated that long noncoding RNAs (lncRNAs) play important roles in cancer metastasis. lncRNAs are the nonprotein coding RNAs that have a size longer than 200 nucleotides. More and more studies have indicated that lncRNAs are involved in a broad range of biological processes and are associated with many diseases, such as cancer. The role of lncRNAs in cancer metastasis has been widely studied; however, lncRNAs are mainly involved in the EMT process on the current literature. This review focuses on the mechanisms underlying the role of lncRNAs in cancer metastasis.

Regulation of lncRNA and Its Role in Cancer Metastasis.

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. Recently, lncRNA CASC9 was shown to be dysregulated in many cancer types, but the mechanisms whereby this occurs remain largely unknown. In this study, we found that CASC9 was significantly upregulated in ESCC tissues, with further analysis revealing that elevated CASC9 expression was associated with ESCC prognosis and metastasis. Furthermore, we found that CASC9 knockdown significantly repressed ESCC migration and invasion in vitro and metastasis in nude mice in vivo. A microarray analysis and mechanical experiments indicated that CASC9 preferentially affected gene expression linked to ECM–integrin interactions, including LAMC2, an upstream inducer of the integrin pathway. We demonstrated that LAMC2 was consistently upregulated in ESCC and promoted ESCC metastasis. LAMC2 overexpression partially compromised the decrease of cell migration and invasion capacity in CASC9 knockdowns. In addition, we found that both CASC9 and LAMC2 depletion reduced the phosphorylation of FAK, PI3K, and Akt, which are downstream effectors of the integrin pathway. Moreover, the reduction in phosphorylation caused by CASC9 depletion was rescued by LAMC2 overexpression, further confirming that CASC9 exerts a pro-metastatic role through LAMC2. Mechanistically, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assay indicated that CASC9 could bind with the transcriptional coactivator CREB-binding protein (CBP) in the nucleus. Chromatin immunoprecipitation (ChIP) assay additionally illustrated that CASC9 increased the enrichment of CBP and H3K27 acetylation in the LAMC2 promoter, thereby upregulating LAMC2 expression. In conclusion, we demonstrate that CASC9 upregulates LAMC2 expression by binding with CBP and modifying histone acetylation. Our research reveals the prognostic and pro-metastatic roles for CASC9 in ESCC, suggesting that CASC9 could serve as a biomarker for prognosis and a target for metastasis treatment.

/pdf/lncrna-casc9-promotes-esophageal-squamous-cell-carcinoma-40ns97vhzp.pdf

LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein.

Abnormal expression of numerous long non-coding RNAs (lncRNAs) has been reported in esophageal squamous cell carcinoma (ESCC) recently, but the great majority of their roles and mechanisms remain largely unclear. We aim to identify the critical ESCC-associated lncRNAs and elucidate the functions and mechanisms in detail. Microarrays were used to analyze the differentially expressed lncRNAs in ESCC tissues. qRT-PCR was used to verify the result of microarrays. The effects of the most up-regulated lncRNA, cancer susceptibility candidate 9(CASC9), on cell growth, proliferation and cell cycle were investigated by in vivo and in vitro assays. Microarrays and recovery tests were used to discover the regulatory targets of CASC9. RNA FISH and subcellular fractionation assays were used to detect the subcellular location of CASC9. Finally, the mechanism of CASC9 regulating PDCD4 was explored by RIP, RNA-protein pull down and ChIP assays. ESCC tissue microarrays showed that CASC9 was the most up-regulated lncRNA. qRT-PCR analysis indicated that CASC9 expression was positively associated with tumor size and TNM stage, and predicted poor overall survival of ESCC patients. Knockdown of CASC9 inhibited ESCC cell growth in vitro and tumorigenesis in nude mice. Furthermore interfering CASC9 decreased cell proliferation and blocked cell cycle G1/S transition. CASC9-associated microarrays indicated that PDCD4 might be the target of CASC9. Consistent with this, PDCD4 expression was negatively associated with CASC9 expression in ESCC tissues and predicted good prognosis. Manipulating CASC9 expression in ESCC cells altered both PDCD4 mRNA and protein levels and cell cycle arrest caused by CASC9 knockdown could be rescued by suppressing PDCD4 expression. CASC9 located both in the nucleus and cytoplasm. Mechanistically, enhancer of zeste homolog2 (EZH2) could bind to both CASC9 and PDCD4 promoter region. Interfering CASC9 reduced the enrichment of EZH2 and H3K27me3 in the PDCD4 promoter region. Our study firstly demonstrates that lncRNA CASC9 functions as an oncogene by negatively regulating PDCD4 expression through recruiting EZH2 and subsequently altering H3K27me3 level. Our study implicates lncRNA CASC9 as a valuable biomarker for ESCC diagnosis and prognosis.

https://link.springer.com/content/pdf/10.1186/s12943-017-0715-7.pdf

Yun Bai

Papers

Activation of β2-adrenergic receptor stimulates γ-secretase activity and accelerates amyloid plaque formation

Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma

Regulation of lncRNA and Its Role in Cancer Metastasis.

LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein.

Up-regulation of lncRNA CASC9 promotes esophageal squamous cell carcinoma growth by negatively regulating PDCD4 expression through EZH2.