RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes.

Increasing evidence suggests that diverse RNA binding proteins (RBPs) interact with regulatory RNAs to regulate transcription. RBFox2 is a well-characterized pre-mRNA splicing regulator, but we now encounter an unexpected paradigm where depletion of this RBP induces widespread increase in nascent RNA production in diverse cell types. Chromatin immunoprecipitation sequencing (ChIP-seq) reveals extensive interaction of RBFox2 with chromatin in a nascent RNA-dependent manner. Bayesian network analysis connects RBFox2 to Polycomb complex 2 (PRC2) and H3K27me3, and biochemical experiments demonstrate the ability of RBFox2 to directly interact with PRC2. Strikingly, RBFox2 inactivation eradicates PRC2 targeting on the majority of bivalent gene promoters and leads to transcriptional de-repression. Together, these findings uncover a mechanism underlying the enigmatic association of PRC2 with numerous active genes, highlight the importance of gene body sequences to gauge transcriptional output, and suggest nascent RNAs as critical signals for transcriptional feedback control to maintain homeostatic gene expression in mammalian genomes.

ß-Catenin Sustains and Is Required for YES-associated Protein Oncogenic Activity in Cholangiocarcinoma.

BACKGROUND & AIMS: YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/ß-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and ß-Catenin cascades in iCCA. METHODS: Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, ß-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine ß-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or ß-Catenin. Immunostaining of total and nonphosphorylated/activated ß-Catenin staining was performed in mouse and human iCCAs. RESULTS: We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, ß-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by ß-Catenin. Importantly, activated/nonphosphorylated ß-Catenin was detected in more than 80% of human iCCAs. CONCLUSION: YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with ß-Catenin. ß-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and ß-Catenin pathways in cholangiocarcinogenesis.

Liver Fibrosis Resolution: From Molecular Mechanisms to Therapeutic Opportunities.

The liver is a critical system for metabolism in human beings, which plays an essential role in an abundance of physiological processes and is vulnerable to endogenous or exogenous injuries. After the damage to the liver, a type of aberrant wound healing response known as liver fibrosis may happen, which can result in an excessive accumulation of extracellular matrix (ECM) and then cause cirrhosis or hepatocellular carcinoma (HCC), seriously endangering human health and causing a great economic burden. However, few effective anti-fibrotic medications are clinically available to treat liver fibrosis. The most efficient approach to liver fibrosis prevention and treatment currently is to eliminate its causes, but this approach's efficiency is too slow, or some causes cannot be fully eliminated, which causes liver fibrosis to worsen. In cases of advanced fibrosis, the only available treatment is liver transplantation. Therefore, new treatments or therapeutic agents need to be explored to stop the further development of early liver fibrosis or to reverse the fibrosis process to achieve liver fibrosis resolution. Understanding the mechanisms that lead to the development of liver fibrosis is necessary to find new therapeutic targets and drugs. The complex process of liver fibrosis is regulated by a variety of cells and cytokines, among which hepatic stellate cells (HSCs) are the essential cells, and their continued activation will lead to further progression of liver fibrosis. It has been found that inhibiting HSC activation, or inducing apoptosis, and inactivating activated hepatic stellate cells (aHSCs) can reverse fibrosis and thus achieve liver fibrosis regression. Hence, this review will concentrate on how HSCs become activated during liver fibrosis, including intercellular interactions and related signaling pathways, as well as targeting HSCs or liver fibrosis signaling pathways to achieve the resolution of liver fibrosis. Finally, new therapeutic compounds targeting liver fibrosis are summarized to provide more options for the therapy of liver fibrosis.

Loss of Apc Cooperates with Activated Oncogenes to Induce Liver Tumor Formation in Mice.

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/ß-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development in vivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of ß-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional ß-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations might benefit from therapeutic strategies targeting the Wnt/ß-catenin pathway.

Pharmacokinetics and food impact assessment of ademetionine enteric-coated tablet as an endogenous substance drug in healthy Chinese volunteers.

WHAT IS KNOWN AND OBJECTIVES: Ademetionine 1,4-Butanedisulfonate (SAMe) enteric-coated tablets are widely used for treatment of pre-cirrhotic and cirrhotic intrahepatic cholestasis, as well as intrahepatic cholestasis of pregnancy (ICP), but incomplete clinical data and interference from endogenous substances pose numerous challenges for clinical trial of ademetionine. The objective of this study was to evaluate the pharmacokinetic profile of SAMe enteric-coated tablets and to assess its food impact and safety in healthy Chinese subjects. METHODS: A randomized, open-label, single-dose study was carried out to determine the pharmacokinetics of SAMe enteric-coated tablets administered in both fasted and postprandial conditions. Baseline collection and data adjustment were required to reduce the effect of endogenous substances. Relevant pharmacokinetic data from subjects administered the reference formulation will be disclosed and utilized in this thesis. RESULTS: Twenty-four subjects with a body mass index (BMI) of 19-24 kg/m2 were enrolled in the study and all completed the trial. The impact of food on the drug was noticeable, with faster absorption in the fasting group (Tmax , 4.50 ± 1.07 and 7.50 ± 1.58 for the fasting and postprandial groups, respectively) but higher exposure in the postprandial group (AUC0-inf , 4021.02 ± 3377.13 and 5087.28 ± 3539.26 for the fasting and postprandial groups, respectively). No serious adverse effects were observed in the fasted and postprandial conditions. WHAT IS NEW AND CONCLUSIONS: The pharmacokinetic profile of SAMe enteric-coated tablets in healthy Chinese subjects was partially complemented in this study. SAMe enteric-coated tablets showed promising safety in fasted and postprandial conditions. However, the impact of food on the drug was significant and might access to the absorption site and affect biochemical reactions.

Non-Coding RNA Related to MAPK Signaling Pathway in Liver Cancer.

The advancement in high-throughput sequencing analysis and the evaluation of chromatin state maps have revealed that eukaryotic cells produce many non-coding transcripts/RNAs. Further, a strong association was observed between some non-coding RNAs and cancer development. The mitogen-activated protein kinases (MAPK) belong to the serine-threonine kinase family and are the primary signaling pathways involved in cell proliferation from the cell surface to the nucleus. They play an important role in various human diseases. A few non-coding RNAs associated with the MAPK signaling pathway play a significant role in the development of several malignancies, including liver cancer. In this review, we summarize the molecular mechanisms and interactions of microRNA, lncRNA, and other non-coding RNAs in the development of liver cancer that are associated with the MAPK signaling pathway. Further, we briefly discuss the therapeutic strategies for liver cancer related to ncRNA and the MAPK signaling pathway.

Application of the Non-dominated Sorting Genetic Algorithm II in Multi-objective Optimization of Orally Disintegrating Tablet Formulation.

In the context of increasing application of modelling methods in the field of pharmaceutics, this study aims to reduce the weight of sildenafil orally disintegrating tablets (ODTs) and optimize their formulation through modelling methods. To achieve the goal, the back-propagation neural network (BPNN)-based non-dominated sorting genetic algorithm II (NSGA-II) was introduced to establish the models and to optimize the percentage of magnesium stearate (MgSt), crospovidone (PVPP), and croscarmellose sodium (CCNa) to obtain satisfactory candidate ODTs. Ultimately, the bioequivalence trial was conducted to verify the effectiveness of the formulation. With the support of the neural network, the model showed satisfactory results in the prediction of hardness and disintegration time of ODTs, and the pareto front obtained by the NSGA-II suggested that there was a strong "competition" between disintegration time and hardness. Since disintegration time should be given the priority, the optimal formulation was determined as 1% MgSt, 6% CCNa, and 2.6% PVPP. The bioequivalence trial results indicated a bioequivalence between the test and the reference formulations of sildenafil, and better medication experience for the test formulation. A bioequivalent formulation with better medication experience is successfully prepared using the NSGA-II. It proves that the NSGA-II is applicable to multi-objective optimization of the drug formulation.

A light-weight periodic plate with embedded acoustic black holes and bandgaps for broadband sound radiation reduction.

Conceiving lightweight structures with low vibration and sound radiation properties is an important topic. The concept of Acoustic Black Hole (ABH) offers new impetus to tackle this problem. Most existing ABH structures are based on simple ABH cells. Apart from the reduced structural strength, systematic ABH effects occur typically above the cut-on frequency of the ABH element, which is perceived as a bottlenecking problem. To tackle the problem, this paper examines the sound radiation properties of a plate comprising periodically tangled ABH cells. Through combining ABH effects with sub-wavelength bandgaps (BGs), numerical and experimental studies show that the plate exhibits reduced sound radiation properties in an ultra-broad frequency range, far below the cut-on frequency of an ABH element. This is owing to the tangled nature of the ABH elements, which extends the actual dimension of the ABH, lowers its onset frequency and reduces the sound radiation efficiency through creating slow waves. Inside the BGs, the reduced sound radiation is mainly due to the redistribution of the vibration energy, basically confined to the excitation area. Capitalizing on the combined ABH and BG features alongside improved mechanical properties, the proposed structure shows promise as a light-weight solution for broadband noise reduction.

Nerve Growth Factor: A Potential Therapeutic Target for Lung Diseases.

The lungs play a very important role in the human respiratory system. However, many factors can destroy the structure of the lung, causing several lung diseases and, often, serious damage to people's health. Nerve growth factor (NGF) is a polypeptide which is widely expressed in lung tissues. Under different microenvironments, NGF participates in the occurrence and development of lung diseases by changing protein expression levels and mediating cell function. In this review, we summarize the functions of NGF as well as some potential underlying mechanisms in pulmonary fibrosis (PF), coronavirus disease 2019 (COVID-19), pulmonary hypertension (PH), asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Furthermore, we highlight that anti-NGF may be used in future therapeutic strategies.

Substrate Stiffness Drives Epithelial to Mesenchymal Transition and Proliferation through the NEAT1-Wnt/ß-Catenin Pathway in Liver Cancer.

BACKGROUND: Extracellular matrix (ECM)-derived mechanical stimuli regulate many cellular processes and phenotypes through mechanotransduction signaling pathways. Substrate stiffness changes cell phenotypes and promotes angiogenesis, epithelial to mesenchymal transition (EMT), and metastasis in tumors. Enhanced liver tissue matrix stiffness plays a crucial role in the tumorigenesis and malignant development of liver cancer and is associated with unfavorable survival outcomes. However, how liver cancer cells sense changes in ECM stiffness and the underlying molecular mechanisms are largely unknown. METHODS: Seeding HepG2 cells on the micropillar gels, HepG2 cells were assessed for responsiveness to mechanotransduction using Western blot and immunofluorescence. CONCLUSIONS: We found that higher substrate stiffness dramatically enhanced malignant cell phenotypes and promoted G1/S transition in HepG2 cells. Furthermore, nuclear paraspeckle assembly transcript 1 (NEAT1) was identified as a matrix stiffness-responsive long non-coding RNA (lncRNA) regulating proliferation and EMT in response to increasing matrix stiffness during the progression of HepG2 cells towards liver cancer phenotypes. Higher matrix stiffness contributed to enhancing NEAT1 expression, which activated the WNT/ß-catenin pathway. ß-catenin translocates and enters the nucleus and the EMT transcription factor zinc finger E-box binding homeobox 1 (ZEB1) was upregulated to trigger EMT. Additionally, the proteins required for matrix stiffness-induced proliferation and resistance were strikingly upregulated in HepG2 cells. Therefore, our findings provide evidence that ECM-derived mechanical signals regulate cell proliferation and drive EMT through a NEAT1/WNT/ß-catenin mechanotransduction pathway in the tumor microenvironment of liver cancer.

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway.

hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.

The role of cold-inducible RNA binding protein in cell stress response.

Cold-inducible RNA binding protein (CIRP) was discovered after the cells were exposed to a moderate cold shock because its production was induced. Other cellular stresses such as ultraviolet light radiation and hypoxia also could increase its expression. Under stress conditions, CIRP could up regulate its own expression by self-transcriptional activation of alternative promoters. After relocating into cytoplasm from nucleus, CIRP assists cells in adapting to novel environmental conditions via stabilizing specific mRNAs and facilitating their translation. It not only participates in anti-apoptosis processes under mild hypothermia condition, but also protects cells from ultraviolet radiation and hypoxia induced senescence process. This article focuses on the possible mechanisms of its inducible expression, cytoprotective functions and carcinogenesis. In addition, extracellular CIRP has been shown to be a novel danger-associated molecular patter (DAMP) member and is able to induce inflammatory response. Finally, based on the distinct roles of CIRP in intracellular and extracellular conditions, a possible model of CIRP-mediated cell fate has been proposed.

The mechanism of CIRP in inhibition of keratinocytes growth arrest and apoptosis following low dose UVB radiation.

UV induces CIRP expression and subsequent Stat3 activation, but the biological function and mechanism of CIRP and Stat3 in mediating UVB-induced skin carcinogenesis have not been fully elucidated. In this study, we demonstrate that CIRP is elevated in all tested melanoma and non-melanoma skin cancer cell lines; and the expression of CIRP is upregulated in keratinocytes after being irradiated with relatively low dose (<5 mJ/cm2 ), but not high dose (50 mJ/cm2 ), UVB acutely and chronically. The increased expression of CIRP, either induced by UVB or through overexpression, leads to resistance of keratinocytes to UVB-induced growth arrest and death; and reduced expression of CIRP by RNA knockdown sensitizes keratinocyte cells to the low dose UVB radiation. We also demonstrated that CIRP expression is required for the low dose UVB-induced Tyr705-phosphorylation, but not total amount, of Stat3. The p-Stat3 level is correlated with the expression levels of cyclin D1 and VEGF, two known downstream cell growth regulators of Stat3, as well as Bag-1/S, an apoptosis regulator. Inhibition of Stat3 DNA-binding activity by S3I-201 leads to a reduction of the p-Stat3 and Bag-1/S along with growth and survival of keratinocytes post-UVB; and the effect of S3I-201 on the UVB-irradiated cells can be partially inhibited by overexpression of CIRP or Bag-1/S. Furthermore, the overexpression of Bag-1/S can totally inhibit UVB-induced PARP cleavage and caspase 3 activation. The results presented above led us to propose that CIRP-p(705)Stat3 cascade promotes cell proliferation and survival post-UVB via upregulating the expression of cyclin D1 and Bag-1/S, respectively. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

A novel inducible lentiviral system for multi-gene expression with human HSP70 promoter and tetracycline-induced promoter.

Despite lentiviral system's predominance, its ultimate potential for gene therapy has not been fully exploited. Currently, most lentivirus vectors are non-inducible expression system or single-gene-induced system, which limits the extensive application in gene therapy. In this study, we designed a novel lentiviral vector containing HSP70 promoter and TRE promoter. Compared to traditional lentiviral vectors and inducible vectors, our controllable system has many advantages. Firstly, it contains multiple gene or shRNA targets. Secondly, genes expression is on/off in response to heat shock and DOX induction in time of need respectively with high effectivity and sensitivity. Thirdly, TRE promoter and HSP70 promoter can work with no interference from each other in the same inducible lentiviral vector. In addition, our study also shows that our novel vector has a higher downstream gene expression efficiency than co-transfection method and can co-position multi-genes in single cell effectively. Finally, we propose four derived models based on our vector at the end, which may be useful in biological research and clinical research in the future. Therefore, we believe that this novel lentiviral system could be promising in gene therapy for tumor.

Ultrawide band gaps in beams with double-leaf acoustic black hole indentations.

Band gaps in conventional phononic crystals (PCs) are attractive for applications such as vibration control, wave manipulation, and sound absorption. Their practical implementations, however, are hampered by several factors, among which the large number of cells required and their impractically large size to ensure the stopbands at reasonably low frequencies are on the top of the list. This paper reports a type of beam carved inside with two double-leaf acoustic black hole indentations. By incorporating the local resonance effect and the Bragg scattering effect generated by a strengthening stud connecting the two branches of the indentations, ultrawide band gaps are achieved. Increasing the length of the stud or reducing the residual thickness of the indentation allows the tuning of the band gaps to significantly enlarge the band gaps, which can exceed 90% of the entire frequency range of interest. Experimental results show that with only three cells, the proposed beam allows considerable vibration energy attenuation within an ultra-broad frequency range including the low frequency range, which conventional PCs can hardly reach. Meanwhile, the proposed configuration also enhances the structural integrity, thus pointing at promising applications in vibration control and a high performance wave filter design.

Inducible RNAi system and its application in novel therapeutics.

RNA interference (RNAi) was discovered as a cellular defense mechanism more than decade ago. It has been exploited as a powerful tool for genetic manipulation. Characterized with specifically silencing target gene expression, it has great potential application for disease treatment. Currently, there are human clinical trials in progress or planned. Despite the excitement regarding this prominent technology, there are many obstacles and concerns that prevent RNAi from being widely used in the therapeutic field. Among them, the non-spatial and non-temporal control is the most difficult challenge, as well as off-target effects and triggering type I immune responses. Inducible RNAi technology can effectively regulate target genes by inducer-mediated small hairpin RNA expression. Combination with inducible regulation systems this makes RNAi technology more sophisticated and may provide a wider application field. This review discusses approaches of inducible RNAi systems, the potential problem areas and solutions and their therapeutic applications. Given the limitations discussed herein being resolved, we believe that inducible RNAi will be a major therapeutic modality within the next several years.

Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future.

A novel conditional gene silencing method using a tumor-specific and heat-inducible siRNA system.

RNAi technology is an invaluable tool for investigating gene function. However, the non-temporal and non-spatial control is the primary limitation, which leads to siRNA leakiness and off-target effects. In this study, we inserted three kinds of HSE into tumor specific promoter hTERT, which aims to construct a temperature-inducible and tumor-specific RNAi plasmid vector. In our system, the expression of mature siRNA is tightly controlled by the heat shock-inducible and tumor-specific promoters. From the expression level of RNA and protein, we determined the efficiency of the inducible siRNA system by targeting SNCG gene in HepG2 and MCF-7 cells. Results showed that the controllable siRNA system could be induced to initiate siRNA expression by heat-induce. The silencing effect of SNCG is on a relative low level (10 %) at 37 °C, while it is significantly increased to 50 or 60 % after heat inducing at 43 °C. This new conditional siRNA system provides a novel approach to drive the siRNA expression by heat-inducible and tumor-specific promoter.

CRIM1, a newfound cancer-related player, regulates the adhesion and migration of lung cancer cells.

CRIM1 is a member of the bone morphogenetic protein (BMP) antagonists; however, the role of CRIM1 in controlling cancer cell behavior remains unknown. This study investigated its function in the A549 cell line in vitro. The results show that treating cells with CRIM1 peptide could increase the migration and adhesion of A549. Consistently, silencing the CRIM1 expression decreased the migration and adhesion of A549. Furthermore, the CRIM1 protein expression was increased in A549 which were treated with transforming growth factor beta 1 to induced EMT. However, CRIM1 peptide treatment could increase the expression of N-CAD and E-CAD expression. Finally, overexpression of the oncogene YAP1 resulted in an up-regulation of the CRIM1 expression in A549, suggesting that CRIM1 was a target of the Hippo pathway. These observations provide evidence for the first time that CRIM1 plays a role in cancer cells by enhancing the migration and adhesion and increasing the expression of N-CAD and E-CAD.