Showing papers in "Molecular and Cellular Biology in 2021"

Exosome-Mediated Transfer of circ_0000338 Enhances 5-Fluorouracil Resistance in Colorectal Cancer through Regulating MicroRNA 217 (miR-217) and miR-485-3p.

Abstract: Exosomes are microvesicles secreted by body cells for intercellular communication. The circular RNA circ_0000338 was found to be present in extracellular vesicles and improve the chemoresistance of colorectal cancer (CRC) cells. However, the role of exosomal circ_0000338 in 5-fluorouracil (5-FU) resistance in CRC is largely unknown. The levels of circ_0000338, microRNA 217 (miR-217), and miR-485-3p were detected using quantitative real-time PCR (qRT-PCR). The 50% inhibitory concentration (IC50) values of cells for 5-FU, cell proliferation, and apoptosis were evaluated using cell counting kit 8 (CCK-8), colony formation, flow cytometry, and Western blot assays. The interaction between miR-217 or miR-485-3p and circ_0000338 was confirmed by RNA immunoprecipitation (RIP), dual-luciferase reporter, and pulldown assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), Nanosight tracking analysis (NTA), and Western blotting. Xenograft models were performed to analyze whether circ_0000338-loaded exosomes could increase resistance of CRC cells to 5-FU in vivo The circ_0000338 level was elevated in 5-FU-resistant CRC tissues and cells, and circ_0000338 knockdown sensitized 5-FU-resistant CRC cells to 5-FU through enhancing apoptosis and decreasing proliferation in vitro Mechanistically, circ_0000338 directly bound to miR-217 and miR-485-3p, and the inhibition of miR-217 or miR-485-3p reversed the effects of circ_0000338 knockdown on cell 5-FU resistance in CRC. Additionally, extracellular circ_0000338 could be incorporated into secreted exosomes and transmitted to 5-FU-sensitive cells. Treatment-sensitive cells with exosomes containing circ_0000338 reduced the 5-FU response in CRC both in vitro and in vivo Besides that, the exosomal circ_0000338 concentration was higher in patients exhibiting resistance to 5-FU and showed good diagnostic efficiency in 5-FU-resistant CRC. The delivery of circ_0000338 via exosomes enhanced 5-FU resistance in CRC through negative regulation of miR-217 and miR-485-3p, indicating a promising diagnostic and therapeutic marker for 5-FU-based chemotherapy in CRC patients.

Translational Control during Cellular Senescence.

Abstract: Senescence is a state of long-term cell cycle arrest that arises in cells that have incurred sublethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence-associated secretory phenotype (SASP). On the other hand, these progrowth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.

Circular RNA 100146 Promotes Colorectal Cancer Progression by the MicroRNA 149/HMGA2 Axis.

Abstract: Colorectal cancer (CRC) has developed into the third leading cause of cancer-associated death worldwide. Studies have confirmed that circular RNAs (circRNAs) absorb microRNAs (miRNAs) to regulate the function of downstream genes. This study aimed to explore the underlying mechanism of circRNA 100146 in CRC. The expression of circRNA 100146, miRNA 149 (miR-149), and high mobility group AT-Hook 2 (HMGA2) was detected by quantitative real-time PCR (RT-qPCR). A series of biofunctional effects (cell viability, apoptosis, migration/invasion) were evaluated by the use of methyl thiazolyl tetrazolium (MTT), flow cytometry, and transwell assays. Protein levels were measured by Western blot assay. A xenograft model was established for in vivo experiments. The interactions among circRNA 100146, miR-149, and HMGA2 were evaluated by dual-luciferase reporter assay, RNA immunoprecipitation assays, or RNA pulldown assay. circRNA 100146 was upregulated in CRC tissues and cells. circRNA 100146 knockdown inhibited cell proliferation, promoted apoptosis, and suppressed migration and invasion in vitro and impeded tumor growth in vivo. Also, miR-149 was negatively regulated by circRNA 100146 and was targeted to HMGA2 and mediated its expression. Moreover, miR-149 interference abrogated the activities of silenced circRNA 100146 in proliferation, apoptosis, migration, and invasion. Furthermore, HMGA2 overexpression abated the effects described above caused by circRNA 100146 silencing, while the mutations on miR-149 binding sites in the 3′ untranslated region (3′-UTR) of HMGA2 led to its loss of this ability. circRNA 100146 knockdown repressed proliferation, enhanced apoptosis, and hindered migration and invasion in SW620 and SW480 cells through targeting the miR-149/HMGA2 axis.

The Cell Biology of LRRK2 in Parkinson's Disease.

Abstract: Point mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD) and are implicated in a significant proportion of apparently sporadic PD cases Clinically, LRRK2-driven PD is indistinguishable from sporadic PD, making it an attractive genetic model for the much more common sporadic PD In this review, we highlight recent advances in understanding LRRK2's subcellular functions using LRRK2-driven PD models, while also considering some of the limitations of these model systems Recent developments of particular importance include new evidence of key LRRK2 functions in the endolysosomal system and LRRK2's regulation of and by Rab GTPases Additionally, LRRK2's interaction with the cytoskeleton allowed elucidation of the LRRK2 structure and appears relevant to LRRK2 protein degradation and LRRK2 inhibitor therapies We further discuss how LRRK2's interactions with other PD-driving genes, such as the VPS35, GBA1, and SNCA genes, may highlight cellular pathways more broadly disrupted in PD

NRF2-dependent bioactivation of mitomycin C as a novel strategy to target KEAP1-NRF2 pathway activation in human cancer

Abstract: Activating mutations in the KEAP1-NRF2 pathway are found in approximately 25% of lung tumours, where the hijacking of NRF2's cytoprotective functions results in aggressive tumour growth, chemoresistance, and a poor prognosis for patients. There are currently no approved drugs which target aberrant NRF2 activation, which means that there is an urgent clinical need to target this orphan oncogenic pathway in human tumours. In this study, we used an isogenic pair of wild-type and Keap1 knockout cells to screen a range of chemotherapeutic and pathway targeted anti-cancer drugs in order to identify compounds which display enhanced toxicity towards cells with high levels of Nrf2 activity. Through this approach, complemented by validation across a panel of eight human cancer cells lines from a range of different tissues, we identified the DNA damaging agent mitomycin C to be significantly more toxic in cells with aberrant Nrf2 activation. Mechanistically, we found that the NRF2 target genes cytochrome P450 reductase, NQO1, and enzymes in the pentose phosphate pathway, are all responsible for the NRF2-dependent enhanced bioactivation of mitomycin C. As mitomycin C is already approved for clinical use, it represents as excellent drug repositioning candidate to target the currently untreatable NRF2 activation in human tumours.

Silencing of hsa_circ_0009035 Suppresses Cervical Cancer Progression and Enhances Radiosensitivity through MicroRNA 889-3p-Dependent Regulation of HOXB7.

Abstract: Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, have been identified as critical regulators in human carcinogenesis. Here, we investigated the precise actions of hsa_circ_0009035 in the progression and radioresistance of cervical cancer (CC). The levels of hsa_circ_0009035, microRNA (miR)-889-3p and homeobox B7 (HOXB7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease R (RNase R) and Actinomycin D assays were used to assess the stability of hsa_circ_0009035. Cell proliferation, cell cycle progression, apoptosis, migration and invasion were gauged by the Cell Counting Kit-8 (CCK-8), flow cytometry and transwell assays, respectively. Cell colony formation and survival were determined by the colony formation assay. Targeted correlations among hsa_circ_0009035, miR-889-3p and HOXB7 were examined by the dual-luciferase reporter, RNA immunoprecipitation (RIP) or RNA pull-down assay. Animal studies were performed to evaluate the impact of hsa_circ_0009035 on tumor growth. We found that hsa_circ_0009035 was highly expressed in CC tissues and cells, and it was associated with the radioresistance of CC patients. Moreover, the silencing of hsa_circ_0009035 inhibited CC cell proliferation, migration, invasion, and enhanced apoptosis and radiosensitivity in vitro and weakened tumor growth in vivo Mechanistically, hsa_circ_0009035 directly targeted miR-889-3p by binding to miR-889-3p, and hsa_circ_0009035 modulated HOXB7 expression through miR-889-3p. HOXB7 was a functional target of miR-889-3p in regulating CC progression and radioresistance in vitro, and hsa_circ_0009035 modulated CC progression and radioresistance in vitro by miR-889-3p. Our current study first identified hsa_circ_0009035 as an important regulation of CC progression and radioresistance at least in part through targeting the miR-889-3p/HOXB7 axis, highlighting its significance as a potential therapeutic target for CC treatment.

Canonical and non-canonical autophagy pathways in microglia.

Abstract: Besides the ubiquitin-proteasome-system, autophagy is a major degradation pathway within cells. It delivers invading pathogens, damaged organelles, aggregated proteins and other macromolecules from the cytosol to the lysosome for bulk degradation. This so-called canonical autophagy activity contributes to the maintenance of organelle, protein and metabolite homeostasis as well as innate immunity. Over the past years, numerous studies rapidly deepened our knowledge on the autophagy machinery and its regulation; driven by the fact that impairment of autophagy is associated with several human pathologies including cancer, immune diseases and neurodegenerative disorders. Unexpectedly, components of the autophagic machinery were also found to participate in various processes that did not involve lysosomal delivery of cytosolic constituents. These functions are hereafter defined as non-canonical autophagy. Regarding neurodegenerative diseases, most research was performed in neurons, while for a long-time microglia received considerably less attention. Concomitant with the notion that microglia greatly contribute to brain health, the understanding of the role of autophagy in microglia expanded. To facilitate an overview of the current knowledge, we present herein the fundamentals as well as the recent advances of canonical and non-canonical autophagy functions in microglia.

STAT5 Regulation of Sex-Dependent Hepatic CpG Methylation at Distal Regulatory Elements Mapping to Sex-Biased Genes.

Abstract: Growth hormone-activated STAT5b is an essential regulator of sex-differential gene expression in mouse liver; however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5b (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were downregulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were derepressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG methylation were seen in STAT5-deficient liver, where sex differences were abolished at 88% of ∼1,500 sex-differentially methylated regions, largely due to increased DNA methylation upon STAT5 loss. STAT5-dependent CpG hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (histone H3 lysine 4 monomethylation [H3K4me1] and histone H3 lysine 27 acetylation [H3K27ac]), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. Thus, the sex-dependent binding of STAT5 to liver chromatin is closely linked to the sex-dependent demethylation of distal regulatory elements linked to STAT5-dependent genes important for liver sex bias.

Understanding individual SARS-CoV-2 proteins for targeted drug development against COVID-19.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic, responsible for millions of deaths globally. Even with effective vaccines, SARS-CoV-2 will likely maintain a hold in the human population through gaps in efficacy, percent vaccinated, and arising new strains. Therefore, understanding how SARS-CoV-2 causes widespread tissue damage and the development of targeted pharmacological treatments will be critical in fighting this virus and preparing for future outbreaks. Herein, we summarize the progress made thus far by using in vitro or in vivo models to investigate individual SARS-CoV-2 proteins and their pathogenic mechanisms. We have grouped the SARS-CoV-2 proteins into three categories: host entry, self-acting, and host interacting. This review focuses on the self-acting and host-interacting SARS-CoV-2 proteins and summarizes current knowledge on how these proteins promote virus replication and disrupt host systems, as well as drugs that target the virus and virus interacting host proteins. Encouragingly, many of these drugs are currently in clinical trials for the treatment of COVID-19. Future coronavirus outbreaks will most likely be caused by new virus strains that evade vaccine protection through mutations in entry proteins. Therefore, study of individual self-acting and host-interacting SARS-CoV-2 proteins for targeted therapeutic interventions is not only essential for fighting COVID-19 but also valuable against future coronavirus outbreaks.

A Leucine-Rich Repeat Protein Provides a SHOC2 the RAS Circuit: a Structure-Function Perspective.

Abstract: SHOC2 is a prototypical leucine-rich repeat protein that promotes downstream receptor tyrosine kinase (RTK)/RAS signaling and plays important roles in several cellular and developmental processes Gain-of-function germ line mutations of SHOC2 drive the RASopathy Noonan-like syndrome, and SHOC2 mediates adaptive resistance to mitogen-activated protein kinase (MAPK) inhibitors Similar to many scaffolding proteins, SHOC2 facilitates signal transduction by enabling proximal protein interactions and regulating the subcellular localization of its binding partners Here, we review the structural features of SHOC2 that mediate its known functions, discuss these elements in the context of various binding partners and signaling pathways, and highlight areas of SHOC2 biology where a consensus view has not yet emerged

Long Noncoding RNA LINC01006 Facilitates Cell Proliferation, Migration, and Epithelial-Mesenchymal Transition in Lung Adenocarcinoma via Targeting the MicroRNA 129-2-3p/CTNNB1 Axis and Activating Wnt/β-Catenin Signaling Pathway.

Abstract: Lung adenocarcinoma (LUAD) is a common type of malignancy of lung cancers. Long intergenic noncoding RNAs (lincRNAs) have emerged as crucial regulators of various cancers, including LUAD. LINC01006 is a newly discovered long noncoding RNA (lncRNA) whose function in LUAD remains to be explored. This study is to explore the role of LINC01006 in LUAD. Quantitative real-time PCR (RT-qPCR) analysis and Western blotting were used to determine the expression levels and protein levels, respectively. Functional assays and animal experiments investigated the role of LINC01006 both in vivo and in vitro. Moreover, TOP/FOP assay was performed to detect the activation of the Wnt/β-catenin signaling pathway. The interaction between LINC01006 and microRNA 29-2-3-p (miR-29-2-3-p)/catenin beta 1 (CTNNB1) was explored by RNA binding protein immunoprecipitation (RIP), RNA pulldown, luciferase reporter assays, and rescue experiments. According to the results, LINC01006 was highly expressed in LUAD tissues and cell lines. LINC01006 knockdown significantly suppressed cell proliferative, migratory, and epithelial-mesenchymal transition (EMT) capacities and tumor development. Moreover, LINC01006 enhanced CTNNB1 via sequestering miR-129-2-3p and activated the Wnt/β-catenin pathway in LUAD. Overall, LINC01006 promotes LUAD development via activating the Wnt/β-catenin pathway, implying that LINC01006 might be a promising biomarker for LUAD treatment.

Nuclear Lamin A/C Expression Is a Key Determinant of Paclitaxel Sensitivity.

Abstract: Paclitaxel is a key member of the Taxane (paclitaxel [originally named taxol], docetaxel/Taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anticancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from lamin A/C-null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wild-type cells. Forced overexpression of lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved by either the withdrawal of serum or the addition of mitotic inhibitors. These results provide a new understanding for the mitotis-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear lamin A/C protein levels correlate with nuclear shape deformation and are a key determinant of paclitaxel sensitivity of cancer cells.

CEBPA-AS1 Knockdown Alleviates Oxygen-Glucose Deprivation/Reperfusion-Induced Neuron Cell Damage by the MicroRNA 24-3p/BOK Axis.

Abstract: Cerebral ischemia/reperfusion (I/R) can lead to serious brain function impairments. Long noncoding RNA (lncRNA) CCAAT enhancer binding protein α antisense RNA 1 (CEBPA-AS1) was shown to be upregulated in human ischemic stroke. This study investigated the function and mechanism of CEBPA-AS1 in I/R. An oxygen-glucose deprivation/reperfusion (OGD/R) model was used to induce I/R injury in SH-SY5Y cells in vitro. RT-qPCR examined the expression of CEBPA-AS1, microRNA 24-3p (miR-24-3p), and Bcl-2-related ovarian killer (Bok). The cell viability, apoptosis, oxidative stress in OGD/R-treated cells were detected using CCK-8, flow cytometry, Western blotting, and enzyme-linked immunosorbent assays. The relationship among genes was tested by RNA pulldown and luciferase reporter assays. We found that OGD/R upregulated CEBPA-AS1 expression in SH-SY5Y cells. Functionally, CEBPA-AS1 depletion ameliorated OGD/R-induced apoptosis and oxidative stress in SH-SY5Y cells by reducing reactive oxygen species production and superoxide dismutase and glutathione. Mechanistic investigations indicated that CEBPA-AS1 acts as a sponge for miR-24-3p, and miR-24-3p binds to BOK. Moreover, miR-24-3p upregulation or BOK downregulation antagonized the protective role of CEBPA-AS1 depletion in SH-SY5Y cells exposed to OGD/R. Overall, downregulation of CEBPA-AS1 exerts protective functions against OGD/R-induced injury by targeting the miR-24-3p/BOK axis.

HNRNPU-AS1 Regulates Cell Proliferation and Apoptosis via the MicroRNA 205-5p/AXIN2 Axis and Wnt/β-Catenin Signaling Pathway in Cervical Cancer.

Abstract: Long noncoding RNAs (lncRNAs) have key functions in modulating cervical cancer (CC) genesis and progression. This work focused on exploring lncRNA HNRNPU-AS1's function in CC and the underlying mechanism. HNRNPU-AS1, AXIN2, and microRNA 205-5p (miR-205-5p) levels in CC cases were measured through reverse transcription-quantitative PCR. The relationship between miR-205-5p and AXIN2 or HNRNPU-AS1 was validated through a dual-luciferase assay. Cell proliferation was examined by CCK-8 and cell apoptosis by colony formation and flow cytometry analysis. HNRNPU-AS1 expression loss could be observed in CC patients and cell lines, which predicted the dismal prognosis of CC cases. Moreover, it was identified that the miR-205-5p level was upregulated, which acted as an inhibitory target of HNRNPU-AS1 and AXIN2. HNRNPU-AS1 inhibited cell proliferation and promoted apoptosis. As revealed by Kaplan-Meier curve, CC cases showing low HNRNPU-AS1, high miR-205-5p, and low AXIN2 levels had the poorest prognosis. AXIN2 reversed the CC cell proliferation-promoting, apoptosis-inhibiting, and Wnt/β-catenin signaling-activating behavior mediated by miR-205-5p or HNRNPU-AS1 knockout. In conclusion, the overexpression of lncRNA HNRNPU-AS1 suppressed CC progression by inhibiting the Wnt/β-catenin pathway through the miR-205-5p/AXIN2 axis.

Saikosaponin-d Alleviates Renal Inflammation and Cell Apoptosis in a Mouse Model of Sepsis via TCF7/FOSL1/Matrix Metalloproteinase 9 Inhibition.

Abstract: Evidence exists reporting that saikosaponin-d (Sa) can prevent experimental sepsis, and this study aims to illustrate the molecular events underlying its renoprotective effects on lipopolysaccharide (LPS)-induced renal inflammation simulating sepsis. Through network pharmacology analysis and bioinformatics analysis, we identified that Sa may influence sepsis development by mediating TCF7. Dual luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were used to explore the interactions between TCF7, FOSL1, and matrix metalloproteinase 9 (MMP9). The experimental data suggest that Sa attenuated LPS-induced renal injury, as evidenced by the reduced production of proinflammatory cytokines as well as cell apoptosis in the renal tissues of LPS-induced mice. Mechanically, Sa inhibited FOSL1 by inhibiting TCF7, which reduced the expression of inflammatory factors in renal cells. TCF7 activated the FOSL1 expression and consequently promoted the expression of MMP9. Also, Sa reduced cell apoptosis and the expression of inflammatory factors by inhibiting the TCF7/FOSL1/MMP9 axis in vivo. In conclusion, Sa suppresses FOSL1 transcription by downregulating TCF7, thereby inhibiting MMP9 expression and ultimately reducing the renal inflammation and cell apoptosis induced by sepsis.

The Non-structural Protein NSs of SFTSV Causes Cytokine Storm Through the Hyper-activation of NF-κB.

Abstract: Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging highly pathogenic phlebovirus. The syndrome is characterized by the substantial production of inflammatory cytokines and chemokines, described as cytokine storm, which correlates with multi-organ failure and high mortality. SFSTV nonstructural (NSs) protein was suggested to mediate the pathogenesis by inhibiting antiviral interferon signaling in the host. However, whether SFTSV NSs protein mediates the induction of fatal cytokine storm remains unaddressed. We demonstrated that SFTSV NSs promotes the hyper-induction of cytokine/chemokine genes in vitro, reminiscent of cytokine storm. Using gene deletion and pharmacological intervention, we found that the induced cytokine storm is driven by the transcription factor NF-κB. Our investigation revealed that TANK-binding kinase 1 (TBK1) suppresses NF-κB signaling and cytokine/chemokine induction in its kinase activity-dependent manner, and that NSs sequesters TBK1 to prevent it from suppressing NF-κB, thereby promoting the activation of NF-κB and its target cytokine/chemokine genes. Of note, NF-κB inhibition suppressed the induction of pro-inflammatory cytokines in SFTSV-infected type I interferon (IFN-I) receptor 1-deficient (Ifnar1-/-) mice. These findings establish the essential role of NSs in SFTS pathogenesis and suggest NF-κB as a possible therapeutic target.

The Herpes Simplex Virus 1 Protein ICP4 Acts as both an Activator and a Repressor of Host Genome Transcription during Infection.

Abstract: Infection by herpes simplex virus 1 (HSV-1) impacts nearly all steps of host cell gene expression. The regulatory mechanisms by which this occurs, and the interplay between host and viral factors, have yet to be fully elucidated. We investigated how the occupancy of RNA polymerase II (Pol II) on the host genome changes during HSV-1 infection and is impacted by the viral immediate early protein ICP4. Pol II ChIP-seq experiments revealed ICP4-dependent decreases and increases in Pol II levels across the bodies of hundreds of genes. Our data suggest ICP4 represses host transcription by inhibiting recruitment of Pol II and activates host genes by promoting release of Pol II from promoter proximal pausing into productive elongation. Consistent with this, ICP4 was required for the decrease in levels of the pausing factor NELF-A on several HSV-1-activated genes after infection. In the absence of infection, exogenous expression of ICP4 activated, but did not repress, transcription of some genes in a chromatin-dependent context. Our data support the model that ICP4 decreases promoter proximal pausing on host genes activated by infection and that ICP4 is necessary, but not sufficient, to repress transcription of host genes during viral infection.

Cellular Nrf2 Levels Determine Cell Fate during Chemical Carcinogenesis in Esophageal Epithelium.

Abstract: Nrf2 is essential for cytoprotection against carcinogens, and through systemic Nrf2 knockout mice, Nrf2-deficient cells were shown to be susceptible to chemical carcinogens and prone to developing cancers. However, the oncogenic potential of Nrf2-deficient epithelial cells surrounded by normal cells in the esophagus could not be assessed by previous models, and the fate of Nrf2-deficient cells in such situations remains elusive. In this study, therefore, we generated mice that harbor almost equal levels of cells with Nrf2 deleted and those with Nrf2 intact in the basal layer of the esophageal epithelium, utilizing inducible Cre-mediated recombination of Nrf2 alleles in adults through moderate use of tamoxifen. In this mouse model, epithelial cells with Nrf2 deleted were maintained with no obvious decrease or phenotypic changes for 12 weeks under unstressed conditions. Upon exposure to the carcinogen 4-nitroquinoline-1-oxide (4NQO), the cells with Nrf2 deleted accumulated DNA damage and selectively disappeared from the epithelium, so almost all 4NQO-induced tumors originated from cells with Nrf2 intact and not from those with Nrf2 deleted. We propose that cells with Nrf2 deleted do not undergo carcinogenesis due to selective elimination upon exposure to 4NQO, indicating that cellular Nrf2 abundance and the epithelial environment determine the cell fate or oncogenic potential of esophageal epithelial cells in 4NQO-induced carcinogenesis.

Reduce, Retain, Recycle: Mechanisms for Promoting Histone Protein Degradation versus Stability and Retention.

Abstract: The eukaryotic genome is packaged into chromatin. The nucleosome, the basic unit of chromatin, is composed of DNA coiled around a histone octamer. Histones are among the longest-lived protein species in mammalian cells due to their thermodynamic stability and their associations with DNA and histone chaperones. Histone metabolism plays an integral role in homeostasis. While histones are largely stable, the degradation of histone proteins is necessary under specific conditions. Here, we review the physiological and cellular contexts that promote histone degradation. We describe specific known mechanisms that drive histone proteolysis. Finally, we discuss the importance of histone degradation and regulation of histone supply for organismal and cellular fitness.

Scaffolding Protein IQGAP1 is Dispensable But Its Overexpression Promotes Hepatocellular Carcinoma via YAP1 Signaling

Abstract: IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffolding protein that is overexpressed in a number of cancers, including liver cancer, and is associated with protumorigenic processes, such as cell proliferation, motility, and adhesion IQGAP1 can integrate multiple signaling pathways and could be an effective antitumor target Therefore, we examined the role of IQGAP1 in tumor initiation and promotion during liver carcinogenesis We found that ectopic overexpression of IQGAP1 in the liver is not sufficient to initiate tumorigenesis Moreover, we report that the tumor burden and cell proliferation in the diethylnitrosamine-induced liver carcinogenesis model in Iqgap1-/- mice may be driven by MET signaling In contrast, IQGAP1 overexpression enhanced YAP activation and subsequent NUAK2 expression to accelerate and promote hepatocellular carcinoma (HCC) in a clinically relevant model expressing activated (S45Y) β-catenin and MET Here, increasing IQGAP1 expression in vivo does not alter β-catenin or MET activation; instead, it promotes YAP activity Overall, we demonstrate that although IQGAP1 expression is not required for HCC development, the gain of IQGAP1 function promotes the rapid onset and increased liver carcinogenesis Our results show that an adequate amount of IQGAP1 scaffold is necessary to maintain the quiescent status of the liver

Sirtuin 5 is Regulated by the SCF-Cyclin F Ubiquitin Ligase and is Involved in Cell Cycle Control

Abstract: The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

MicroRNA 617 Targeting SERPINE1 Inhibited the Progression of Oral Squamous Cell Carcinoma.

Abstract: Serpin family E member 1 (SERPINE1) is a serine proteinase inhibitor (serpin) upregulated in diverse types of cancer, including oral squamous cell carcinoma (OSCC), and it functions in an oncogenic role. Hence, exploring pathological mechanism underlying high expression of SERPINE1 is crucial to the targeted therapy of OSCC. Bioinformatics analysis was performed to identify the microRNA (miRNA) and the candidate gene contributing to OSCC progression. The viability, proliferation, and apoptosis of the OSCC cell were evaluated using Cell Counting Kit-8 (CCK-8) assay, BrdU assay, and cell apoptosis assay, respectively. The RNA pulldown assay and luciferase reporter assay were conducted to verify the relationship between SERPINE1 and miRNA 617 (miR-617). SERPINE1 was aberrantly upregulated in OSCC tissues and cell lines. Genetically inhibiting SERPINE1 led to reduction of OSCC cell viability and proliferation and elevation of OSCC cell apoptosis. According to bioinformatics analysis, miR-617 contained a response element for SERPINE1 overexpression, which is validated by the RNA pulldown and luciferase reporter assays. Furthermore, miR-617 was detected to be downregulated in OSCC tissues and cell lines, and it displayed a negative correlation with advanced stages. Besides, miR-617 mimic or inhibitor transfection could suppress or boost the SERPINE1 expression. More importantly, miR-617 mimic could block the effect of SERPINE1 overexpression on OSCC cell proliferation, viability, and apoptosis. SERPINE1 acted as a proproliferative oncogenic factor that is partly regulated by miR-167 downregulation in OSCC cells. Therefore, the miR-617/SERPINE1 axis is a potential therapeutic target against OSCC.

Distinct Regulations of HO-1 Gene Expression for Stress Response and Substrate Induction

Abstract: Heme oxygenase 1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas HO-1 gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which HO-1 gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of HO-1 utilize similar or distinct regulatory pathways, we developed an HO-1-DsRed-knock-in reporter mouse in which the HO-1 gene is floxed by loxP sites and the DsRed gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1-DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1-DsRed expression in the whole body in vivo and ex vivo. HO-1 inducers upregulated HO-1-DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1-DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated HO-1 induction and NRF2-independent substrate-mediated HO-1 induction. Thus, the HO-1 gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.

Circular RNA circ-ERBB2 Elevates the Warburg Effect and Facilitates Triple-Negative Breast Cancer Growth by the MicroRNA 136-5p/Pyruvate Dehydrogenase Kinase 4 Axis.

Abstract: Triple-negative breast cancer (TNBC) is an aggressive histological subtype of breast cancer. It has been reported that the circular RNA (circRNA) circ-ERBB2 (circBase identifier hsa_circ_0007766) is mainly distributed in the cytoplasm of TNBC cells and promotes the proliferation and invasion of TNBC cells. This study aimed to explore the molecular mechanism of circ-ERBB2 regulating the progression of TNBC. The expression of circ-ERBB2 was detected by quantitative real-time PCR (qRT-PCR). Loss-of-function experiments were performed to investigate the function of circ-ERBB2 in TNBC cells in vitro and in vivo. The regulatory mechanism of circ-ERBB2 was surveyed by bioinformatics analysis and dual-luciferase reporter and RNA immunoprecipitation (RIP) or RNA pulldown assays. We observed that circ-ERBB2 was overexpressed in TNBC, and TNBC patients with high circ-ERBB2 expression levels had a poor prognosis. Functionally, circ-ERBB2 knockdown constrained TNBC growth in vivo, reduced the Warburg effect, accelerated apoptosis, and repressed the proliferation, migration, and invasion of TNBC cells in vitro. Mechanically, circ-ERBB2 sponged microRNA 136-5p (miR-136-5p) to elevate pyruvate dehydrogenase kinase 4 (PDK4) expression. In conclusion, circ-ERBB2 facilitated the Warburg effect and malignancy of TNBC cells by the miR-136-5p/PDK4 pathway, at least in part. This study supports circ-ERBB2 as a prognostic indicator for TNBC.

Overexpression of MicroRNA 142-5p Suppresses the Progression of Cervical Cancer through Targeting Phosphoinositol-3-Kinase Adaptor Protein 1 Expression.

Abstract: The aim of current study was to explore the mechanism of microRNA 142-5p (miR-142-5p) in cervical cancer through mediating the phosphoinositol-3-kinase adaptor protein 1 (PIK3AP1)/PI3K/AKT axis. To this end, reverse transcription-quantitative PCR (RT-qPCR) and Western blot analysis results revealed that miR-142-5p was poorly expressed, whereas PIK3AP1 was highly expressed, in cervical cancer tissues and cells. Furthermore, miR-142-5p was hypermethylated in cervical cancer, as reflected by methylation-specific PCR (MS-PCR) and chromatin immunoprecipitation (ChIP) assessment of enrichment of DNMT1/DNMT3a/DNMT3b in the promoter region of miR-142-5p. A target binding relationship between miR-142-5p and PIK3AP1 was established, showing that miR-142-5p targeted and inhibited the expression of PIK3AP1. Loss- and gain-of-function assays were conducted to determine the roles of miR-142-5p and PIK3AP1 in cervical cancer cells. CCK-8, flow cytometry, and Transwell assay results revealed that overexpression of miR-142-5p in cervical cancer cells downregulated PIK3AP1 and inhibited the PI3K/AKT signaling pathway, leading to reduced proliferation, migration, and invasion capacity of cervical cancer cells but enhanced apoptosis. Collectively, epigenetic regulation of miR-142-5p targeted PIK3AP1 to inactivate the PI3K/AKT signaling pathway, thus suppressing development of cervical cancer, which presents new targets for the treatment of cervical cancer.

GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells.

Abstract: MicroRNAs (miRNAs) are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is retarded by Ago2-interacting protein GW182B. The GW182 group of proteins are localized to GW182 bodies or RNA processing bodies in mammalian cells, and GW182B-dependent retardation of miRNA export depends on GW body integrity and is independent of the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.

Genomic landscapes of non-coding RNAs regulating VEGFA and VEGFC expression in endothelial cells

Abstract: Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular related diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of non-coding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of non-coding RNAs, long ncRNA (lncRNA) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC By integrating genome-wide global run-on (GRO-seq) and Hi-C data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. RNA-seq data from lncRNA deletions showed downstream factors implicated in VEGFA and VEGFC linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.

FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair.

Abstract: Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR in vivo: cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

Inhibition of Long Noncoding RNA SNHG20 Improves Angiotensin II-Induced Cardiac Fibrosis and Hypertrophy by Regulating the MicroRNA 335/Galectin-3 Axis.

Abstract: Cardiac fibrosis is a hallmark of various heart diseases and ultimately leads to heart failure. Although long noncoding RNA (lncRNA) SNHG20 has been reported to play important roles in various cancers, its function in cardiac fibrosis remains unclear. The expression of SNHG20 and microRNA 335 (miR-335) in heart tissues of angiotensin II-induced mice and angiotensin II-stimulated mouse cardiomyocyte cell line HL-1 were detected by quantitative real-time PCR (qRT-PCR). Cell viability was evaluated by cell counting kit-8 assay. The expression of galectin-3, fibrosis-related proteins (fibronectin, collagen IaI, and α-SMA), and apoptosis-related proteins [cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP)] was detected by Western blotting. Bioinformatics prediction, luciferase reporter assay, and RNA pulldown assay were performed to determine the relationship between SNHG20 and miR-335 as well as miR-335 and Galectin-3. Gain- and loss-function assays were performed to determine the role of SNHG20/miR-335/Galectin-3 in cardiac fibrosis. SNHG20 was significantly upregulated and miR-335 was downregulated in heart tissues of angiotensin II-treated mice and angiotensin II-stimulated HL-1 cells. Downregulation of SNHG20 effectively enhanced cell viability and decreased cell size of HL-1 cells and the expression levels of fibrosis-related proteins (fibronectin, collagen IaI, and α-SMA) and apoptosis-related proteins (cleaved caspase-3 and cleaved PARP), which were induced by angiotensin II treatment. Furthermore, SNHG20 elevated the expression levels of Galectin-3 by directly regulating miR-335. Our study revealed that downregulation of SNHG20 improved angiotensin II-induced cardiac fibrosis by targeting the miR-335/Galectin-3 axis, suggesting that SNHG20 is a therapeutic target for cardiac fibrosis and hypertrophy.

Morphogenesis of the islets of Langerhans is guided by extra-endocrine Slit2/3 signals.

Abstract: The spatial architecture of the islets of Langerhans is vitally important for their correct function, and alterations in islet morphogenesis often result in diabetes mellitus. We have previously reported that Roundabout (Robo) receptors are required for proper islet morphogenesis. As part of the Slit-Robo signaling pathway, Robo receptors function in conjunction with Slit ligands to mediate axon guidance, cell migration, and cell positioning in development. However, the role of Slit ligands in islet morphogenesis has not yet been determined. Here we report that Slit ligands are expressed in overlapping and distinct patterns in both endocrine and non-endocrine tissues in late pancreas development. We show that function of either Slit2 or Slit3, which are predominantly expressed in the pancreatic mesenchyme, is required and sufficient for islet morphogenesis, while Slit1, which is predominantly expressed in the β-cells, is dispensable for islet morphogenesis. We further show that Slit functions as a repellent signal to β-cells. These data suggest that clustering of endocrine cells during islet morphogenesis is guided, at least in part, by repelling Slit2/3 signals from the pancreatic mesenchyme.