NSCLC Extracellular Vesicles Containing miR-374a-5p Promote Leptomeningeal Metastasis by Influencing Blood‒Brain Barrier Permeability.

Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Its diagnosis and monitoring can be challenging. Recently, extracellular vesicle (EV) miRNAs have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EV miRNAs in NSCLC-LM. Next-generation sequencing analysis revealed that miRNAs with differential expression of EVs in sera of patients with NSCLC with LM and non-LM were detected to identify biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EV miRNA promoting LM in NSCLC. In the present study, we first demonstrated that the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EV miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO1 and occludin in endothelial cells by targeting γ-adducin, increasing vascular permeability and promoting LM. Implications: These results suggest that serum NSCLC-derived EV miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of the blood-brain barrier to promote NSCLC-LM and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.

The Segmentation of Multiple Types of Uterine Lesions in Magnetic Resonance Images Using a Sequential Deep Learning Method with Image-Level Annotations.

Fully supervised medical image segmentation methods use pixel-level labels to achieve good results, but obtaining such large-scale, high-quality labels is cumbersome and time consuming. This study aimed to develop a weakly supervised model that only used image-level labels to achieve automatic segmentation of four types of uterine lesions and three types of normal tissues on magnetic resonance images. The MRI data of the patients were retrospectively collected from the database of our institution, and the T2-weighted sequence images were selected and only image-level annotations were made. The proposed two-stage model can be divided into four sequential parts: the pixel correlation module, the class re-activation map module, the inter-pixel relation network module, and the Deeplab v3 + module. The dice similarity coefficient (DSC), the Hausdorff distance (HD), and the average symmetric surface distance (ASSD) were employed to evaluate the performance of the model. The original dataset consisted of 85,730 images from 316 patients with four different types of lesions (i.e., endometrial cancer, uterine leiomyoma, endometrial polyps, and atypical hyperplasia of endometrium). A total number of 196, 57, and 63 patients were randomly selected for model training, validation, and testing. After being trained from scratch, the proposed model showed a good segmentation performance with an average DSC of 83.5%, HD of 29.3 mm, and ASSD of 8.83 mm, respectively. As far as the weakly supervised methods using only image-level labels are concerned, the performance of the proposed model is equivalent to the state-of-the-art weakly supervised methods.

CircHERC1 promotes non-small cell lung cancer cell progression by sequestering FOXO1 in the cytoplasm and regulating the miR-142-3p-HMGB1 axis.

BACKGROUND: Noncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. Non-small cell lung cancer (NSCLC) is one of the most common malignant cancers. Information on the functions and mechanism of circRNAs in lung cancer is limited; thus, the topic needs more exploration. The purpose of this study was to identify aberrantly expressed circRNAs in lung cancer, unravel their roles in NSCLC progression, and provide new targets for lung cancer diagnosis and therapy. METHODS: High-throughput sequencing was used to analyze differential circRNA expression in patients with lung cancer. qRT‒PCR was used to determine the level of circHERC1 in lung cancer tissues and plasma samples. Gain- and loss-of-function experiments were implemented to observe the impacts of circHERC1 on the growth, invasion, and metastasis of lung cancer cells in vitro and in vivo. Mechanistically, dual luciferase reporter assays, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down experiments were performed to confirm the underlying mechanisms of circHERC1. Nucleocytoplasmic localization of FOXO1 was determined by nucleocytoplasmic isolation and immunofluorescence. The interaction of circHERC1 with FOXO1 was verified by RNA pull-down, RNA immunoprecipitation (RIP) and western blot assays. The proliferation and migration of circHERC1 in vivo were verified by subcutaneous and tail vein injection in nude mice. RESULTS: CircHERC1 was significantly upregulated in lung cancer tissues and cells, ectopic expression of circHERC1 strikingly facilitated the proliferation, invasion and metastasis, and inhibited the apoptosis of lung cancer cells in vitro and in vivo. However, knockdown of circHERC1 exerted the opposite effects. CircHERC1 was mainly distributed in the cytoplasm. Further mechanistic research indicated that circHERC1 acted as a competing endogenous RNA of miR-142-3p to relieve the repressive effect of miR-142-3p on its target HMGB1, activating the MAPK/ERK and NF-κB pathways and promoting cell migration and invasion. More importantly, we found that circHERC1 could bind FOXO1 and sequester it in the cytoplasm, adjusting the feedback AKT pathway. The accumulation of FOXO1 in the cytosol and nuclear exclusion promoted cell proliferation and inhibited apoptosis. CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential prognostic biomarker and therapeutic target for NSCLC. CONCLUSIONS: CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential diagnosis biomarker and therapeutic target for NSCLC. Our findings indicate that circHERC1 facilitates the invasion and metastasis of NSCLC cells by regulating the miR-142-3p/HMGB1 axis and activating the MAPK/ERK and NF-κB pathways. In addition, circHERC1 can promote cell proliferation and inhibit apoptosis by sequestering FOXO1 in the cytoplasm to regulate AKT activity and BIM transcription.

circTAB2 inhibits lung cancer proliferation, migration and invasion by sponging miR-3142 to upregulate GLIS2.

Circular RNAs (circRNAs) are a specialized circular structure, are deregulated in cancers and play essential roles in biological processes involved in tumor progression. However, the mechanism by which circRNAs affect lung tumorigenesis and progression remains largely unexplored. To investigate the role of circRNA in lung cancer, circRNA expression profile was screened by bioinformatics analysis. The levels of circTAB2, miR-3142, and GLIS family zinc finger 2 (GLIS2) were measured by quantitate real-time (qRT-PCR) or western blot. Cell proliferation, apoptosis, migration and invasion were detected by EdU, flow cytometry, and transwell assays, respectively. Bioinformatics, western blot, RIP, pull down, dual luciferase reporter and rescue experiments were used to verify the direct relationship between miR-3142 and circTAB2 or GLIS2. The xenograft assays were used to assess the role of circTAB2 in vivo.CircTAB2 exhibited low expression in cancer tissues. Gain and loss-of-function assays indicated that circTAB2 could inhibit cell proliferation, migration and invasion. Functional studies revealed that circTAB2 acted as a miRNA sponge, directly interacted with miR-3142 and consequently regulated GLIS2 /AKT. Taken together, circTAB2 serves as an inhibitory role in lung cancer through a novel circTAB2 /miR-3142 /GLIS2 /AKT pathway and could be exploited a novel marker in lung cancer.

Human Kinase IGF1R/IR Inhibitor Linsitinib Controls the In Vitro and Intracellular Growth of Mycobacterium tuberculosis.

ATP provides energy in the biosynthesis of cellular metabolites as well as regulates protein functions through phosphorylation. Many ATP-dependent enzymes are antibacterial and anticancer targets including human kinases acted on by most of the successful drugs. In search of new chemotherapeutics for tuberculosis (TB), we screened repurposing compounds against the essential glutamine synthase (GlnA1) of Mycobacterium tuberculosis (Mtb) and identified linsitinib, a clinical-stage drug originally targeting kinase IGF1R/IR as a potent GlnA1 inhibitor. Linsitinib has direct antimycobacterial activity. Biochemical, molecular modeling, and target engagement analyses revealed the inhibition is ATP-competitive and specific in Mtb. Linsitinib also improves autophagy flux in both Mtb-infected and uninfected THP1 macrophages, as demonstrated by the decreased p-mTOR and p62 and the increased lipid-bound LC3B-II and autophagosome forming puncta. Linsitinib-mediated autophagy reduces intracellular growth of wild-type and isoniazid-resistant Mtb alone or in combination with bedaquiline. We have demonstrated that an IGF-IR/IR inhibitor can potentially be used to treat TB. Our study reinforces the concept of targeting ATP-dependent enzymes for novel anti-TB therapy.

Functional analysis of the ScAG and ScAGL11 MADS-box transcription factors for anthocyanin biosynthesis and bicolour pattern formation in Senecio cruentus ray florets.

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

Integrative Analysis of lncRNA-mRNA Coexpression in Human Lung Epithelial Cells Exposed to Dimethyl Selenide-Derived Secondary Organic Aerosols.

Dimethyl selenide (DMSe) is one of the major volatile organoselenium compounds released into the atmosphere through plant metabolism and microbial methylation. DMSe has been recently revealed as a precursor of secondary organic aerosol (SOA), and its resultant SOA possesses strong oxidizing capability toward thiol groups that can perturb several major biological pathways in human airway epithelial cells and is linked to genotoxicity, DNA damage, and p53-mediated stress responses. Mounting evidence has suggested that long noncoding RNAs (lncRNAs) are involved in stress responses to internal and environmental stimuli. However, the underlying molecular interactions remain to be elucidated. In this study, we performed integrative analyses of lncRNA-mRNA coexpression in the transformed human bronchial epithelial BEAS-2B cell line exposed to DMSe-derived SOA. We identified a total of 971 differentially expressed lncRNAs in BEAS-2B cells exposed to SOA derived from O3 and OH oxidation of DMSe. Gene ontology (GO) network analysis of cis-targeted genes showed significant enrichment of DNA damage, apoptosis, and p53-mediated stress response pathways. trans-Acting lncRNAs, including PINCR, PICART1, DLGAP1-AS2, and LINC01629, known to be associated with human carcinogenesis, also showed altered expression in cell treated with DMSe-SOA. Overall, this study highlights the regulatory role of lncRNAs in altered gene expression induced by DMSe-SOA exposure.

Exposure to Dimethyl Selenide (DMSe)-Derived Secondary Organic Aerosol Alters Transcriptomic Profiles in Human Airway Epithelial Cells.

Dimethyl selenide (DMSe) is one of the major volatile organoselenium compounds released from aquatic and terrestrial environments through microbial transformation and plant metabolism. The detailed processes of DMSe leading to secondary organic aerosol (SOA) formation and the pulmonary health effects induced by inhalation of DMSe-derived SOA remain largely unknown. In this study, we characterized the chemical composition and formation yields of SOA produced from the oxidation of DMSe with OH radicals and O3 in controlled chamber experiments. Further, we profiled the transcriptome-wide gene expression changes in human airway epithelial cells (BEAS-2B) after exposure to DMSe-derived SOA. Our analyses indicated a significantly higher SOA yield resulting from the OH-initiated oxidation of DMSe. The oxidative potential of DMSe-derived SOA, as measured by the dithiothreitol (DTT) assay, suggested the presence of oxidizing moieties in DMSe-derived SOA at levels higher than typical ambient aerosols. Utilizing RNA sequencing (RNA-Seq) techniques, gene expression profiling followed by pathway enrichment analysis revealed several major biological pathways perturbed by DMSe-derived SOA, including elevated genotoxicity, DNA damage, and p53-mediated stress responses, as well as downregulated cholesterol biosynthesis, glycolysis, and interleukin IL-4/IL-13 signaling. This study highlights the significance of DMSe-derived SOA as a stressor in human airway epithelial cells.

Promoter hypermethylation of SOX11 promotes the progression of cervical cancer in vitro and in vivo.

The development of cervical cancer (CC) is a multi­gene, multi­step carcinogenic process that involves complex genetic and epigenetic mechanisms. SRY­related HMG­box gene 11 (SOX11) is a member of the SOX family of transcription factors with an emerging crucial role in the development of various tumor types. To elucidate the function of SOX11 in cervical carcinogenesis, the expression level of SOX11 during the development of human CC was analyzed by immunohistochemistry and western blot analysis. Additionally, the methylation status of the SOX11 was examined using bisulfite sequencing and methylation­specific polymerase chain reaction. The SOX11 expression and promoter methylation in human CC cell lines were also determined. The effect of SOX11 expression restoration after 5­aza­2'­deoxycytidine (5­Aza­dC) treatment on the CC cell proliferation ability was evaluated in CC cell lines. SOX11 was highly expressed in normal cervix (NC) and precancerous low­grade squamous intraepithelial lesions, but weakly expressed or virtually absent in precancerous high­grade squamous intraepithelial lesions and CC, which is consistent with the result of the western blot analysis. Hypermethylation of the SOX11 promoter was detected in CC, which was significantly higher than that in NC samples at each CpG site. The expression level of SOX11 in the CC cell lines was downregulated compared with the positive control, Tera­1human teratoma cell line. Upon 5­Aza­dC treatment, SOX11 expression was significantly upregulated in the CC cell lines at the mRNA and protein levels, and cell proliferation was inhibited. The results indicated that the downregulation of SOX11 in CC is due to the hypermethylation of the SOX11 promoter region. Thus, SOX11 methylation may have a role in the growth of CC cells and cervical carcinogenesis.