CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

Decoding the potential role of regulatory T cells in sepsis-induced immunosuppression.

Sepsis, a multiorgan dysfunction with high incidence and mortality, is caused by an imbalanced host-to-infection immune response. Organ-support therapy improves the early survival rate of sepsis patients. In the long term, those who survive the "cytokine storm" and its secondary damage usually show higher susceptibility to secondary infections and sepsis-induced immunosuppression, in which regulatory T cells (Tregs) are evidenced to play an essential role. However, the potential role and mechanism of Tregs in sepsis-induced immunosuppression remains elusive. In this review, we elucidate the role of different functional subpopulations of Tregs during sepsis and then review the mechanism of sepsis-induced immunosuppression from the aspects of regulatory characteristics, epigenetic modification, and immunometabolism of Tregs. Thoroughly understanding how Tregs impact the immune system during sepsis may shed light on preclinical research and help improve the translational value of sepsis immunotherapy.

CO2-Responsive Rosin-Based Supramolecular Hydrogels: Diverse Chiral Nanostructures and Their Application in In Situ Synthesis of Chiral Gold Nanoparticles.

Natural small molecules have demonstrated tremendous potential for the construction of supramolecular chiral nanostructures owing to their unique molecular structures and chirality. In this study, novel CO2-responsive supramolecular hydrogels were constructed using a series of rosin-based surfactants (CnMPAN, n = 10, 12, and 14). The macroscopic properties, rheological properties, nanostructures, and intermolecular interactions of the hydrogels were investigated using differential scanning calorimetry, rotational rheometry, cryogenic transmission electron microscopy, and Fourier transform infrared spectroscopy. Interestingly, diverse nanostructures containing helical nanofibers, interwoven nanofibers, and twisted nanoribbons were formed in the hydrogels, which were rarely observed in reported supramolecular hydrogels, and the strength of the hydrogels was significantly enhanced by increasing the CnMPAN concentration and the alkyl chain length. The obtained hydrogels exhibited excellent CO2-responsiveness, with no obvious variation in the nanostructures and rheological properties after response to CO2/N2 for five cycles. Taking advantage of the chiral nanostructures of hydrogels, gold nanoparticles (GNPs) were further prepared. The average particle sizes of the resulting GNPs were as low as 2.5 nm, and the GNPs also had a chiral structure. It is worth noting that no additional reductants and UV-light irradiation were used during the reduction process of GNPs. This study emphasizes that the unique molecular structure and chirality of rosin are critical for the preparation of hydrogels with chiral nanostructures. In addition, this study enriches the applications of forest resources.

Type III CRISPR-based RNA editing for programmable control of SARS-CoV-2 and human coronaviruses.

Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.

CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s.

BACKGROUND: Functional alveolar regeneration is essential for the restoration of normal lung homeostasis after acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Lung is a relatively quiescent organ and a variety of stem cells are recruited to participate in lung repair and regeneration after lung tissue injury. However, there is still no effective method for promoting the proliferation of endogenous lung stem cells to promote repair and regeneration. METHODS: Using protein mass spectrometry analysis, we analyzed the microenvironment after acute lung injury. RNA sequencing and image cytometry were used in the alveolar epithelial type 2 cells (AEC2s) subgroup identification. Then we used Sftpc+AEC2 lineage tracking mice and purified AEC2s to further elucidate the molecular mechanism by which CTGF regulates AEC2s proliferation both in vitro and in vivo. Bronchoalveolar lavage fluid (BALF) from thirty ARDS patients who underwent bronchoalveolar lavage was collected for the analysis of the correlation between the expressing of Krt5 in BALF and patients' prognosis. RESULTS: Here, we elucidate that AEC2s are the main facultative stem cells of the distal lung after ALI and ARDS. The increase of connective tissue growth factor (CTGF) in the microenvironment after ALI promoted the proliferation of AEC2s subpopulations. Proliferated AEC2s rapidly expanded and differentiated into alveolar epithelial type 1 cells (AEC1s) in the regeneration after ALI. CTGF initiates the phosphorylation of LRP6 by promoting the interaction between Krt5 and LRP6 of AEC2s, thus activating the Wnt signaling pathway, which is the molecular mechanism of CTGF promoting the proliferation of AEC2s subpopulation. CONCLUSIONS: Our study verifies that CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of AEC2s subpopulation.

VSIG2 promotes malignant progression of pancreatic ductal adenocarcinoma by enhancing LAMTOR2-mediated mTOR activation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable malignancies to overcome clinically due to its insidious onset as well as rapid progression. It is urgent to seek new diagnostic markers and therapeutic targets in order to furthest ameliorate the prognosis of patients with PDAC. V-set and immunoglobulin domain containing 2 (VSIG2) belongs to immunoglobulin superfamily (IgSF), which function as coinhibitory molecule to mediate immune evasion of tumors. Nevertheless, the role of VSIG2 in PDAC and related mechanism still keep unclear. METHODS: Different expression of VSIG2 in PDAC tissues and cells were detected by bioinformatic analysis, immunohistochemistry, real-time quantitative PCR as well as western blotting. CCK-8, colony formation, Transwell assay, and scratch experiment were utilized to assess proliferation, invasion and migration properties of PDAC cells. The relationship of VSIG2 with late endosomal/lysosomal adaptor, MAPK and MTOR activator 2 (LAMTOR2) and mechanistic target of rapamycin (mTOR) was identified using mass spectrometry, co-immunoprecipitation and immunofluorescence. GO and KEGG enrichment analysis were performed for further pathway verification using western blotting. Additionally, subcutaneous xenograft tumor model and clinical samples analysis were implemented to further elucidate the oncogenic effect of VSIG2 on PDAC in vivo and clinically. RESULTS: VSIG2 was highly expressed in PDAC tissues and cells. Overexpression of VSIG2 facilitated the proliferation, invasion and migration abilities of PDAC cells, while VSIG2-inhibition exerted opposite effects. Mechanistically, VSIG2 could simultaneously bind to LAMTOR2 and mTOR, thereby enhancing interaction between two molecules, which resulted in elevated phosphorylation-modificatory activation of mTOR and downstream key molecules. Clinically, up-regulation of VSIG2 was positively associated with advanced stage, overall survival and disease-free survival of PDAC patients. CONCLUSIONS: Our study disclosed that VSIG2 was overexpressed in PDAC, which promoted the proliferation, invasion and metastasis. Mechanically, VSIG2 acted as a scaffold to recruit LAMTOR2 and mTOR simultaneously, stabilize the interaction between them, thus enhancing LAMTOR2-mediated mTOR phosphorylated activation. Collectively, VSIG2 could be exploited as a biomarker for diagnosis and prognosis monitor of PDAC in the future, meanwhile, targeting VSIG2 in PDAC management is expected to be a novel strategy. Video Abstract. Video Abstract.

Cytokine Biomarker Phenotype for Early Prediction and Triage of Sepsis in Blunt Trauma Patients.

BACKGROUND: Altered levels of inflammatory markers secondary to severe trauma present a major problem to physicians and are prone to interfering with the clinical identification of sepsis events. This study aimed to establish the profiles of cytokines in trauma patients to characterize the nature of immune responses to sepsis, which might enable early prediction and individualized treatments to be developed for targeted intervention. METHODS: A 15-plex human cytokine magnetic bead assay system was used to measure analytes in citrated plasma samples. Analysis of the kinetics of these cytokines was performed in 40 patients with severe blunt trauma admitted to our trauma center between March 2016 and February 2017, with an Injury Severity Score (ISS) greater than 20 with regard to sepsis (Sepsis-3) over a 14-d time course. RESULTS: In total, the levels of six cytokines were altered in trauma patients across the 1-, 3-, 5-, 7-, and 14-d time points. Additionally, IL-6, IL-10, IL-15, macrophage derived chemokine (MDC), GRO, sCD40 L, granulocyte colony-stimulating factor (G-CSF), and fibroblast growth factor (FGF)-2 levels could be used to provide a significant discrimination between sepsis and nonsepsis patients at day 3 and afterward, with an area under the curve (AUC) of up to 0.90 through a combined analysis of the eight biomarkers (P < 0.001). Event-related analysis demonstrated 1.5- to 4-fold serum level changes for these cytokines within 72 h before clinically apparent sepsis. CONCLUSIONS: Cytokine profiles demonstrate a high discriminatory ability enabling the timely identification of evolving sepsis in trauma patients. These abrupt changes enable sepsis to be detected up to 72 h before clinically overt deterioration. Defining cytokine release patterns that distinguish sepsis risk from trauma patients might enable physicians to initiate timely treatment and reduce mortality. Large prospective studies are needed to validate and operationalize the findings. TRIAL REGISTRATION: Clinicaltrials, NCT01713205. Registered October 22, 2012, https://register. CLINICALTRIALS: gov/NCT01713205.

Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) αvß3 or αvß5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvß3 and αvß5 Using a preclinical model of sepsis, we found that CIRP-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of αvß3/αvß5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells.

Immunosuppression in response to severe sepsis remains a serious human health concern. Evidence of sepsis-induced immunosuppression includes impaired T lymphocyte function, T lymphocyte depletion or exhaustion, increased susceptibility to opportunistic nosocomial infection, and imbalanced cytokine secretion. CD4 T cells play a critical role in cellular and humoral immune responses during sepsis. Here, using an RNA sequencing assay, we found that the expression of T cell-containing immunoglobulin and mucin domain-3 (Tim-3) on CD4 T cells in sepsis-induced immunosuppression patients was significantly elevated. Furthermore, the percentage of Tim-3+ CD4 T cells from sepsis patients was correlated with the mortality of sepsis-induced immunosuppression. Conditional deletion of Tim-3 in CD4 T cells and systemic Tim-3 deletion both reduced mortality in response to sepsis in mice by preserving organ function. Tim-3+ CD4 T cells exhibited reduced proliferative ability and elevated expression of inhibitory markers compared with Tim-3-CD4 T cells. Colocalization analyses indicated that HMGB1 was a ligand that binds to Tim-3 on CD4 T cells and that its binding inhibited the NF-κB signaling pathway in Tim-3+ CD4 T cells during sepsis-induced immunosuppression. Together, our findings reveal the mechanism of Tim-3 in regulating sepsis-induced immunosuppression and provide a novel therapeutic target for this condition.

Early globalized industrial chain revealed by residual submicron pigment particles in Chinese imperial blue-and-white porcelains.

The success of early Chinese blue-and-white porcelains relied heavily on imported cobalt pigment from the West. In contrast to art-historical concept, which contains both typological evidence and literature records, it is assumed that imported Sumali blue was completely superseded by domestic Chinese asbolane ore based on the analytical results of the Fe/Mn ratio in imperial productions from the Xuande reign (1426 to 1435 CE) onward. Using a focused ion beam transmission electron microscopy technique to reassess this hotly debated question, we have identified two classes of residual submicron pigment particles in the blue glaze with diagnostic differences in morphology, chemical composition, and distribution behavior. Compared with the microstructural features of the blue-and-white porcelains of the Yuan and Qing dynasties, we show that a mixture of imported and domestic cobalt pigments was used for aesthetic reasons, indicating that the overseas supply chain of imported pigment remained consistent and adequate even though the authorities had terminated official trade and tributary activities after the death of Admiral Zheng He. This discovery further suggests that the globalized trading network and cross-regional industrial chain had been extensively established in the 15th century. Moreover, we provide analytical evidence against the fundamental assumption of the current Fe/Mn provenancing criteria, implying that the failures of previous chemical analyses can be attributed to elemental differentiation between the silicate glaze and the arsenic pigment. We propose an innovative method for directly assessing original mineralogic information from submicron residual pigment particles that provides a more reliable way to trace cobalt circulation and holds great promise for provenance studies.

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

Electrotaxis of alveolar epithelial cells in direct-current electric fields.

PURPOSE: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. METHODS: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. RESULTS: The successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. CONCLUSION: EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

Small-Molecule Inhibitor of 8-Oxoguanine DNA Glycosylase 1 Regulates Inflammatory Responses during Pseudomonas aeruginosa Infection.

The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises 8-oxo-7,8-dihydroguanine lesions induced in DNA by reactive oxygen species, has been linked to the pathogenesis of lung diseases associated with bacterial infections. A recently developed small molecule, SU0268, has demonstrated selective inhibition of OGG1 activity; however, its role in attenuating inflammatory responses has not been tested. In this study, we report that SU0268 has a favorable effect on bacterial infection both in mouse alveolar macrophages (MH-S cells) and in C57BL/6 wild-type mice by suppressing inflammatory responses, particularly promoting type I IFN responses. SU0268 inhibited proinflammatory responses during Pseudomonas aeruginosa (PA14) infection, which is mediated by the KRAS-ERK1-NF-κB signaling pathway. Furthermore, SU0268 induces the release of type I IFN by the mitochondrial DNA-cGAS-STING-IRF3-IFN-ß axis, which decreases bacterial loads and halts disease progression. Collectively, our results demonstrate that the small-molecule inhibitor of OGG1 (SU0268) can attenuate excessive inflammation and improve mouse survival rates during PA14 infection. This strong anti-inflammatory feature may render the inhibitor as an alternative treatment for controlling severe inflammatory responses to bacterial infection.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting.

PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION: AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

YEATS2 is a target of HIF1α and promotes pancreatic cancer cell proliferation and migration.

Hypoxia is involved in the development of pancreatic cancer (PC). The responses of hypoxia-associated genes and their regulated mechanisms are largely unknown. In this study, through bioinformatic analysis and quantitative real-time polymerase chain reaction, the YEATS domain containing 2 (YEATS2) was determined to be a key hypoxia-associated gene. It was increased in PC cells under hypoxia, upregulated in PC tissues, and predicted poor outcome. YEATS2 inhibition decreased the proliferation and migration of PC cells under both normoxia and hypoxia in vitro as well as proliferation and metastasis in vivo. We found that hypoxia-inducible factor 1α (HIF1α) regulated the expression of YEATS2 via binding to the hypoxia response element (HRE) of YEATS2 and coexpressed with YEATS2 in PC tissues. Overexpression of YEATS2 blocked the inhibitory effects of HIF1α silence on PC cell proliferation and migration under hypoxia. Collectively, our study revealed that YEATS2 is a target gene of HIF1α and promotes PC development under hypoxia.

CircSEC24A upregulates TGFBR2 expression to accelerate pancreatic cancer proliferation and migration via sponging to miR-606.

BACKGROUND: Circular RNA (circRNA), producing by special selective splicing, was widely expressed in the cytoplasm of eukaryotic cells as a newly non-coding RNAs. It played different roles in a variety of diseases including cancer and performed different functions. Nonetheless, reports on the specific function of circRNA in pancreatic cancer (PC) were still rarely so far. In particular, the role of circSEC24A in PC remains unclear. METHODS: Real-time fluorescent quantitative PCR was used to evaluate the expression level of circSEC24A in pancreatic cancer tissues and cell lines. Furthermore, we used some functional experiments, such as EDU and Transwell assays, to explore the effects of circSEC24A on the proliferation and invasiveness of pancreatic cancer. Finally, the corresponding relationship among circSEC24A, miR-606 and TGFBR2 was explored by dual luciferase reporter and other mechanism studies. RESULTS: The expression of circSEC24A in both pancreatic cancer tissues and cell lines was evidently up-regulated. Furthermore, knockdown of circSEC24A significantly inhibited the proliferative, migration and invasive capacity of pancreatic cancer cells, whereas miR-606 inhibitor obviously counteracted these effects. Further study confirmed that circSEC24A alleviated suppression on target TGFBR2 expression by directly sponging miR-606 and then influenced the tumorigenesis of pancreatic cancer. CONCLUSIONS: These findings indicated that the progression of pancreatic cancer can be driven by circSEC24A influencing miR-606/TGFBR2 axis. Therefore, circSEC24A might be used as a critical biomarker influencing the early diagnosis and prognosis of pancreatic cancer.

[6]-Paradol suppresses proliferation and metastases of pancreatic cancer by decreasing EGFR and inactivating PI3K/AKT signaling.

BACKGROUND: The underlying mechanism behind the tumorigenesis and progression of pancreatic cancer is not clear, and treatment failure is generally caused by early metastasis, recurrence, drug resistance and vascular invasion. Exploring novel therapeutic regimens is necessary to overcome drug resistance and improve patients outcomes. METHODS: Functional assays were performed to investigate the role of [6]-Paradol (6-P) in proliferation and metastasis of pancreatic cancer in vitro and in vivo. The interaction between EGFR and 6-P was tested by KEGG enrichment analysis and molecular docking analysis. qRT-PCR was performed to detect the mRNA expression of EGFR in 6-P treated groups. Involvement of the PI3K/AKT pathway was measured by western blotting. RESULTS: 6-P significantly suppressed pancreatic cancer cell proliferation and metastasis. KEGG enrichment analysis and molecular docking analysis suggested that there existed certain interaction between EGFR and 6-P. In addition, 6-P obviously decreased EGFR protein expression level but did not change the mRNA expression level of EGFR. 6-P could induce degradation of EGFR through decreasing the protein stability of EGFR and enhancing the ubiquitin-mediated proteasome-dependent degradation, 6-P-mediated EGFR degradation led to inactivation of PI3K/AKT signaling pathway. However, ectopic expression of EGFR protein resulted in resistance to 6-P-mediated inactivity of PI3K/AKT signaling and inhibition of malignant phenotype of pancreatic cancer. Inversely, erlotinib could enhance the 6-P-mediated anticancer activity. CONCLUSION: Our data indicated that 6-P/EGFR/PI3K/AKT signaling axis might become one of the potential therapies for the treatment of pancreatic cancer.

Upregulation of METTL14 mediates the elevation of PERP mRNA N6 adenosine methylation promoting the growth and metastasis of pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal human cancers. N6-methyladenosine (m6A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive. METHODS: LC/MS was used to profile m6A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m6A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14. RESULTS: We found that the m6A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m6A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo, via direct targeting of the downstream PERP mRNA (p53 effector related to PMP-22) in an m6A-dependent manner. Methylation of the target adenosine lead to increased PERP mRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells. CONCLUSIONS: Our data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m6A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.

MiR-608 Exerts Anti-inflammatory Effects by Targeting ELANE in Monocytes.

miR-608 has been indicated to play an important role in the pathogenesis of various inflammation-related diseases, including sepsis and several types of cancers. However, there is little information about the underlying mechanism, especially in inflammatory cells. In this study, an hsa-miR-608-inhibition cell model was constructed in U937 cells using a lentivirus, and gene expression profiles were determined by a cDNA microarray. Altogether, 682 genes showed a difference greater than 1.2-fold, including 184 genes downregulated and 498 genes upregulated. Among these genes, one potential miR-608-target gene, ELANE, was further investigated. A positive relationship between the expression of miR-608 and that of ELANE was found both in vivo and in vitro. In addition, decreased expression of miR-608 resulted in overexpression of ELANE at both the mRNA and protein levels. Cotransfection of HEK293T cells with a miR-608 mimic inhibited reporter activity, and mutation of the miRNA seed sequences abolished the repression of reporter activity. These results suggest that miR-608 is an important posttranscriptional regulator of ELANE expression in human monocytes and may play an important role in the process of inflammation. miR-608 and neutrophil elastase may be novel targets for the diagnosis or treatment of sepsis.

Expression of integrins to control migration direction of electrotaxis.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMß2, ß1, α2, αIIbß3, and α5 migrated to the cathode, whereas cells expressing ß3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6ß4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.