Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5 min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20 × 106 to 1.75 × 107  CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

DUSP5 regulated by YTHDF1-mediated m6A modification promotes epithelial-mesenchymal transition and EGFR-TKI resistance via the TGF-ß/Smad signaling pathway in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) patients have a dismal survival rate because of cancer metastasis and drug resistance. The study aims to identify the genes that concurrently modulate EMT, metastasis and EGFR-TKI resistance, and to investigate the underlying regulatory mechanisms. METHODS: Cox regression and Kaplan-Meier analyses were applied to identify prognostic oncogenes in LUAD. Gene set enrichment analysis (GSEA) was used to indicate the biological functions of the gene. Wound-healing and Transwell assays were used to detect migratory and invasive ability. EGFR-TKI sensitivity was evaluated by assessing the proliferation, clonogenic survival and metastatic capability of cancer cells with treatment with gefitinib. Methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) analyses established the level of m6A modification present on the target gene and the protein's capability to interact with RNA, respectively. Single-sample gene set enrichment (ssGSEA) algorithm used to investigate levels of immune cell infiltration. RESULTS: Our study identified dual-specificity phosphatase 5 (DUSP5) as a novel and powerful predictor of adverse outcomes for LUAD by using public datasets. Functional enrichment analysis found that DUSP5 was positively enriched in EMT and transforming growth factor-beta (TGF-ß) signaling pathway, a prevailing pathway involved in the induction of EMT. As expected, DUSP5 knockdown suppressed EMT via inhibiting the canonical TGF-ß/Smad signaling pathway in in vitro experiments. Consistently, knockdown of DUSP5 was first found to inhibit migratory ability and invasiveness of LUAD cells in in vitro and prevent lung metastasis in in vivo. DUSP5 knockdown re-sensitized gefitinib-resistant LUAD cells to gefitinib, accompanying reversion of EMT progress. In LUAD tissue samples, we found 14 cytosine-phosphate-guanine (CpG) sites of DUSP5 that were negatively associated with DUSP5 gene expression. Importantly, 5'Azacytidine (AZA), an FDA-approved DNA methyltransferase inhibitor, restored DUSP5 expression. Moreover, RIP experiments confirmed that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a m6A reader protein, could bind DUSP5 mRNA. YTHDF1 promoted DUSP5 expression and the malignant phenotype of LUAD cells. In addition, the DUSP5-derived genomic model revealed the two clusters with distinguishable immune features and tumor mutational burden (TMB). CONCLUSIONS: Briefly, our study discovered DUSP5 which was regulated by epigenetic modification, might be a potential therapeutic target, especially in LUAD patients with acquired EGFR-TKI resistance.

Surgical Trauma-induced CCL2 Upregulation Mediates Lung Cancer Progression by Promoting Treg Recruitment in Mice and Patients.

Surgical removal of the tumor is currently the first-line treatment for lung cancer, but the procedure may accelerate cancer progression through immunosuppression. However, whether CCL2 (C-C motif chemokine ligand 2) enhances cancer progression by affecting regulatory T cells (Tregs) remains unknown. We found that the volume and weight of tumors were larger in the surgical trauma group than in the control group. CCL2 expression and Treg abundance were increased in tumor tissues after surgical trauma, and CCL2 expression was positively associated with Treg abundance. These results demonstrated that surgical trauma contributes to lung cancer progression by increasing CCL2 expression, thus promoting Treg recruitment.

Changes of N6-methyladenosine modulators promote breast cancer progression.

BACKGROUND: Breast cancer (BC) displays striking genetic, epigenetic and phenotypic diversity. N6-methyladenosine (m6A) in mRNA has emerged as a crucial epitranscriptomic modification that controls cancer self-renewal and cell fate. However, the key enzymes of m6A expression and function in human breast carcinogenesis remain unclear. METHODS: The expression of m6A methylases (METTL3, METTL14 and WTAP) and demethylases (FTO and ALKBH5) were analyzed by using ONCOMINE and The Cancer Genome Atlas databases and in 36 pairs of BC and adjacent non-cancerous tissue. The level of m6A in BC patients was detected by ELISA, and the function of m6A was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The database of bc-GenExMiner v4.0, Kaplan-Meier Plotter and cBioPortal were queried for correlation, mutation and prognosis analysis of BC. RESULTS: The m6A methylases and demethylases were dysregulated in several major malignant tumors. Specifically, the expression of all m6A methylases was reduced in BC as compared with normal controls, but the demethylase ALKBH5 was induced in ONCOMINE databases and confirmed in clinical patients. METTL14 expression was positively correlated with METTL3 expression, and both showed high expression in normal breast-like and luminal-A and -B BC. Functionally, reducing m6A expression by overexpressing METTL14 and/or knockdown of ALKBH5 could inhibit breast cell viability, colony formation and cell migration. Furthermore, Kaplan-Meier, meta-analysis and univariate Cox assay showed that the expression of m6A members including METTL3, METTL14, WTAP and FTO but not their gene mutation and amplification, was tightly associated with cancer progression and poor survival. CONCLUSIONS: Changes of m6A modulators reduced m6A may promote tumorigenesis and predict poor prognosis in BC.

Placental Growth Factor Mediates Crosstalk Between Lung Cancer Cells and Tumor-Associated Macrophages in Controlling Cancer Vascularization and Growth.

BACKGROUND/AIMS: Assistance with tumor-associated vascularization is needed for the growth and invasion of non-small cell lung cancer (NSCLC). Recently, it was shown that placental growth factor (PLGF) expressed by NSCLC cells had a critical role in promoting the metastasis of NSCLC cells. However, the underlying molecular mechanisms remain elusive. METHODS: Here, we first established a NSCLC model in mice that allows us not only to isolate tumor cells from non-tumor cells in the tumor, but also to trace tumor cells in living animals. Levels of PLGF, its unique receptor Flt-1, as well as transforming growth factor ß1 (TGFß1) was examined in tumor cells and tumor-associated macrophages (TAM) by RT-qPCR. A transwell well co-culture system and HUVEC assay were applied to study the crosstalk between NSCLC cells and TAM. RESULTS: NSCLC cells produced and secreted PLGF to signal to tumor-associated macrophages (TAM) through surface expression of Flt-1 on macrophages. In a transwell co-culture system, PLGF secreted by NSCLC cells triggered macrophage polarization to a TAM subtype that promote growth of NSCLC cells. Moreover, polarized TAM seemed to secrete TGFß1 to enhance the growth of endothelial cells in a HUVEC assay. CONCLUSION: The cross-talk between TAM and NSCLC cells via PLGF/Flt-1 and TGFß receptor signaling may promote the growth and vascularization of NSCLC.

Musashi1 Promotes Non-Small Cell Lung Carcinoma Malignancy and Chemoresistance via Activating the Akt Signaling Pathway.

BACKGROUND/AIMS: Lung cancer is one of the leading causes for cancer mortality. The poor therapeutic outcome of non-small cell lung carcinoma (NSCLC) is mainly due to late diagnosis and chemoresistance. In this study, we investigated the role of Musashi1 (MSI1) in NSCLC malignancy and chemoresistance. METHODS: Colony formation, MTT, glucose uptake and lactate production assays were employed to study lung cancer cell malignancy and chemoresistance. RT-PCR and Western blotting were performed to detect mRNA and protein expressions of genes. We used immunohistochemistry and Pearson correlation analysis to study the relationship of gene expression. RESULTS: We demonstrated that MSI1 was able to promote the proliferation and glucose metabolism of NSCLC cells, and to mediate the sensitivity to chemotherapy drugs in NSCLC cells. Importantly, we found that MSI1 could regulate the activity of Akt signaling. The regulation of NSCLC proliferation, glucose metabolism and chemoresistance by MSI1 was dependent on the modulation of the activity of the Akt signaling pathway. We also found that MSI1 was a target of miR-181a-5p, a microRNA involved in the regulation of cancer development. The expression levels of MSI1 and miR-181a-5p were negatively correlated in NSCLC. CONCLUSION: MSI1 promotes non-small cell lung carcinoma malignancy and chemoresistance via activating the Akt signaling pathway, which provides a new strategy for the therapy of NSCLC.

Overexpression of Sphk2 is associated with gefitinib resistance in non-small cell lung cancer.

Lung cancer is the major cause of cancer-related death worldwide, and 80 % of them are non-small cell lung cancer (NSCLC) cases. Recent studies have shown that sphingosine kinase 2 (SphK2) could promote tumor progression; however, whether SphK2 could affect the chemoresistance of NSCLC to chemotherapy remains unclear. To determine whether SphK2 serves as a potential therapeutic target of NSCLC, we utilized small interference RNA (siRNA) to knock down SphK2 expression in human NSCLC cells and analyzed their phenotypic changes. The data demonstrated that knockdown of SphK2 led to decreased proliferation and enhanced chemosensitivity and apoptosis to gefitinib in NSCLC cells. In this study, we describe the findings that overexpression of SphK2 promotes chemoresistance in NSCLC cells. Inhibition of SphK2 might be considered as a strategy in NSCLC treatment with gefitinib.

Fast-track surgery improves postoperative clinical recovery and cellular and humoral immunity after esophagectomy for esophageal cancer.

BACKGROUND: Our aim was to investigate the influence of FTS on human cellular and humoral immunity using a randomized controlled clinical study in esophageal cancer patients. METHODS: Between October 2013 and December 2014, 276 patients with esophageal cancer in our department were enrolled in the study. The patients were randomized into two groups: FTS pathway group and conventional pathway group. The postoperative hospital stay, hospitalization expenditure, and postoperative complications were recorded. The markers of inflammatory and immune function were measured before operation as well as on the 1st, 3rd, and 7th postoperative days (POD), including serum level of interleukin-6 (IL-6), C-reactive protein (CRP), serum globulin, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) and lymphocyte subpopulations (CD3 lymphocytes, CD4 lymphocytes, CD8 lymphocytes and the CD4/CD8 ratio) in the patients between the two groups. RESULTS: In all, 260 patients completed the study: 128 in the FTS group and 132 in the conventional group. We found implementation of FTS pathway decreases postoperative length of stay and hospital charges (P < 0.05). In addition, inflammatory reactions, based on IL-6 and CRP levels, were less intense following FTS pathway compared to conventional pathway on POD1 and POD3 (P < 0.05). On POD1 and POD3, the levels of IgG, IgA, CD3 lymphocytes, CD4 lymphocytes and the CD4/CD8 ratio in FTS group were significantly higher than those in control group (All P < 0.05). However, there were no differences in the level of IgM and CD8 lymphocytes between the two groups. CONCLUSIONS: FTS improves postoperative clinical recovery and effectively inhibited release of inflammatory factors via the immune system after esophagectomy for esophageal cancer. TRIAL REGISTRATION: ChiCTR-TRC-13003562 , the date of registration: August 29, 2013.

Fluorescence enhancement of flavonoids and its application in ingredient determination for some traditional Chinese medicines by CE-LIF.

Flavonoids are widely used in the treatment of various diseases due to their antioxidant, anti-inflammatory, anticancer and antiviral properties. Fluorescence detection is rarely applied for the determination of flavonoids because of their weak fluorescence. In this work, a method of fluorescence enhancement of flavonoids was firstly introduced by using sodium acetate for flavonoid derivatization. The study discovered that flavonoids, with a hydroxyl at the C3 position, had the ability to emit strong fluorescence after derivatization. Five flavonoids, kaempferide, galangin, isorhamnetin, kaempferol and quercetin, having a special structure, were selected, derivatized and analyzed by capillary electrophoresis with laser-induced fluorescence detection. Under the optimal conditions, the five flavonoids could be completely separated within 3 minutes. Good linear relationships were obtained for all analytes and the limits of detection for the five flavonoids were in the range of 1.18-4.67 × 10-7 mol L-1. Finally, the method was applied to the determination of flavonoids in five traditional Chinese medicines: aster, chamomile, galangal, tangerine peel and cacumen biotae. Flavonoids were successfully found in all these medicines by the developed method. The recoveries were in the range of 84.2-111%. The method developed in this study was fast, sensitive and reliable for the determination of flavonoids.

Down-regulated m6A reader FTO destabilizes PHF1 that triggers enhanced stemness capacity and tumor progression in lung adenocarcinoma.

Aberrant epigenetic drivers or suppressors contribute to LUAD progression and drug resistance, including KRAS, PTEN, Keap1. Human Plant Homeodomain (PHD) finger protein 1 (PHF1) coordinates with H3K36me3 to increase nucleosomal DNA accessibility. Previous studies revealed that PHF1 is markedly upregulated in various tumors and enhances cell proliferation, migration and tumorigenesis. However, its roles in LUAD are still unknown. We aimed to depict the biological roles of PHF1 and identify useful targets for clinical treatment of LUAD. Based on the bioinformatic analysis, we found that PHF1 was down-regulated in LUAD samples and low PHF1 expressions correlated with unfavorable clinical characteristics. Patients with low PHF1 had poorer survival outcomes relative to those with high PHF1. Targeting PHF1 potentiated cell growth, migration and in vivo proliferation. Mechanistically, FTO mediated the stabilization of PHF1 mRNA by demethylating m6A, which particularly prevented YTHDF2 from degrading PHF1 transcripts. Of note, FTO also expressed lowly in LUAD that predicts poor prognosis of patients. FTO inhibition promoted LUAD progression, and PHF1 overexpression could reverse the effect. Lastly, down-regulated FTO/PHF1 axis could mainly elevate FOXM1 expression to potentiate the self-renewal capacity. Targeting FOXM1 was effective to suppress PHF1low/- LUAD growth. Collectively, our findings revealed that FTO positively regulates PHF1 expression and determined the tumor-suppressive role of FTO/PHF1 axis, thereby highlighting insights into its epigenetic remodeling mechanisms in LUAD progression and treatment.

Discovery of First-in-Class TAK1-MKK3 Protein-Protein Interaction (PPI) Inhibitor (R)-STU104 for the Treatment of Ulcerative Colitis through Modulating TNF-α Production.

Tumor necrosis factor α (TNF-α) has been demonstrated to be a therapeutic target for autoimmune diseases. However, this biological therapy exhibits some inevitable disadvantages, such as risk of infection. Thus, small-molecule alternatives by targeting TNF-α production signaling pathway are still in demand. Herein, we describe the design, synthesis, and structure-activity relationships of 3-aryindanone compounds regarding their modulation of TNF-α production. Among them, (R)-STU104 exhibited the most potent inhibitory activity on TNF-α production, which suppressed the TAK1/MKK3/p38/MnK1/MK2/elF4E signal pathways through binding with MKK3 and disrupting the TAK1 phosphorylating MKK3. As a result, (R)-STU104 demonstrated remarkable dose-effect relationships on both acute and chronic mouse UC models. In addition to its good pharmacokinetic (PK) and safety profile, (R)-STU104 showed better anti-UC efficacy in vivo at 10 mg/kg/d than mesalazine at the dose of 50 mg/kg/d. These results suggested that TAK1-MKK3 interaction inhibitors could be potentially utilized for the treatment of UC.

A Pyroptosis-Related Signature Predicts Overall Survival and Immunotherapy Responses in Lung Adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is a highly malignant cancer with a bleak prognosis. Pyroptosis is crucial in LUAD. The present study investigated the prognostic value of a pyroptosis-related signature in LUAD. Methods: LUAD's genomic data were downloaded from TCGA and GEO databases. K-means clustering was used to classify the data based on pyroptosis-related genes (PRGs). The features of tumor microenvironment were compared between the two subtypes. Differentially expressed genes (DEGs) were identified between the two subtypes, and functional enrichment and module analysis were carried out. LASSO Cox regression was used to build a prognostic model. Its prognostic value was assessed. Results: In LUAD, genetic and transcriptional changes in PRGs were found. A total of 30 PRGs were found to be differentially expressed in LUAD tissues. Based on PRGs, LUAD patients were divided into two subgroups. Subtype 1 has a higher overall survival rate than subtype 2. The tumor microenvironment characteristics of the two subtypes differed significantly. Compared to subtype 1, subtype 2 had strong immunological infiltration. Between the two groups, 719 DEGs were discovered. WGCNA used these DEGs to build a co-expression network. The network modules were analyzed. A prognostic model based on seven genes was developed, including FOSL1, KRT6A, GPR133, TMPRSS2, PRDM16, SFTPB, and SFTA3. The developed model was linked to overall survival and response to immunotherapy in patients with LUAD. Conclusion: In LUAD, a pyroptosis-related signature was developed to predict overall survival and treatment responses to immunotherapy.

Reducing N6AMT1-mediated 6mA DNA modification promotes breast tumor progression via transcriptional repressing cell cycle inhibitors.

DNA N6-methyladenosine (6mA) is a novel epigenetic signaling modification in humans and has been implicated in the progression and tumorigenesis of several cancers. However, the function and mechanism of 6mA in breast cancer (BC), the most common cancer among women, are unclear. Here, we found that decreases in N6AMT1 correlated with the extent of 6mA in clinical BC tissues and predicted a worse survival of BC patients. Functionally, knockdown of N6AMT1 markedly reduced 6mA in DNA and promoted colony formation and migration of BC cells, whereas overexpression of N6AMT1 had the opposite effect. Moreover, silencing of N6AMT1 reduced 6mA modification and enhanced the growth of BC cells in vitro and tumors in vivo. 6mA immunoprecipitation sequencing (6mA-IP-seq), RNA-seq, 6mA-IP-PCR, and bioinformatics analysis indicated that N6AMT1 was a functional methyltransferase for genomic 6mA DNA modifications and related to gene transcriptional activity. Critical negative regulators of the cell cycle, such as RB1, P21, REST, and TP53 were identified as targets of N6AMT1 in BC. These results suggest N6AMT1 enhances DNA 6mA levels to repress tumor progression via transcriptional regulation of cell cycle inhibitors.

A novel epithelial-mesenchymal transition (EMT)-related gene signature of predictive value for the survival outcomes in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a remarkably heterogeneous and aggressive disease with dismal prognosis of patients. The identification of promising prognostic biomarkers might enable effective diagnosis and treatment of LUAD. Aberrant activation of epithelial-mesenchymal transition (EMT) is required for LUAD initiation, progression and metastasis. With the purpose of identifying a robust EMT-related gene signature (E-signature) to monitor the survival outcomes of LUAD patients. In The Cancer Genome Atlas (TCGA) database, least absolute shrinkage and selection operator (LASSO) analysis and cox regression analysis were conducted to acquire prognostic and EMT-related genes. A 4 EMT-related and prognostic gene signature, comprising dickkopf-like protein 1 (DKK1), lysyl oxidase-like 2 (LOXL2), matrix Gla protein (MGP) and slit guidance ligand 3 (SLIT3), was identified. By the usage of datum derived from TCGA database and Western blotting analysis, compared with adjacent tissue samples, DKK1 and LOXL2 protein expression in LUAD tissue samples were significantly higher, whereas the trend of MGP and SLIT3 expression were opposite. Concurrent with upregulation of epithelial markers and downregulation of mesenchymal markers, knockdown of DKK1 and LOXL2 impeded the migration and invasion of LUAD cells. Simultaneously, MGP and SLIT3 silencing promoted metastasis and induce EMT of LUAD cells. In the TCGA-LUAD set, receiver operating characteristic (ROC) analysis indicated that our risk model based on the identified E-signature was superior to those reported in literatures. Additionally, the E-signature carried robust prognostic significance. The validity of prediction in the E-signature was validated by the three independent datasets obtained from Gene Expression Omnibus (GEO) database. The probabilistic nomogram including the E-signature, pathological T stage and N stage was constructed and the nomogram demonstrated satisfactory discrimination and calibration. In LUAD patients, the E-signature risk score was associated with T stage, N stage, M stage and TNM stage. GSEA (gene set enrichment analysis) analysis indicated that the E-signature might be linked to the pathways including GLYCOLYSIS, MYC TARGETS, DNA REPAIR and so on. In conclusion, our study explored an innovative EMT based prognostic signature that might serve as a potential target for personalized and precision medicine.

Kynurenine promotes neonatal heart regeneration by stimulating cardiomyocyte proliferation and cardiac angiogenesis.

Indoleamine 2,3 dioxygenase-1 (IDO1) catalyzes tryptophan-kynurenine metabolism in many inflammatory and cancer diseases. Of note, acute inflammation that occurs immediately after heart injury is essential for neonatal cardiomyocyte proliferation and heart regeneration. However, the IDO1-catalyzed tryptophan metabolism during heart regeneration is largely unexplored. Here, we find that apical neonatal mouse heart resection surgery led to rapid and consistent increases in cardiac IDO1 expression and kynurenine accumulation. Cardiac deletion of Ido1 gene or chemical inhibition of IDO1 impairs heart regeneration. Mechanistically, elevated kynurenine triggers cardiomyocyte proliferation by activating the cytoplasmic aryl hydrocarbon receptor-SRC-YAP/ERK pathway. In addition, cardiomyocyte-derived kynurenine transports to endothelial cells and stimulates cardiac angiogenesis by promoting aryl hydrocarbon receptor nuclear translocation and enhancing vascular endothelial growth factor A expression. Notably, Ahr deletion prevents indoleamine 2,3 dioxygenase -kynurenine-associated heart regeneration. In summary, increasing indoleamine 2,3 dioxygenase-derived kynurenine level promotes cardiac regeneration by functioning as an endogenous regulator of cardiomyocyte proliferation and cardiac angiogenesis.

Speckle patterning of soft tissues for strain field measurement using digital image correlation: preliminary quality assessment of patterns.

Methods for creating speckle patterns on mouse arteries for use in deformation and strain field measurements in a stereomicroscope digital image correlation (DIC) system are described. Both fluorescent microsphere binding and ethidium bromide (EB) nuclear staining were used to generate high contrast, random patterns on mouse carotid arteries. To quantify the quality of each pattern, several metrics are used including (a) histogram distribution for each intensity pattern and (b) pixel-level variance in intensity pattern noise. Results demonstrate that both approaches provide sufficient pattern contrast for use in image-based methods to measure deformations in soft tissue. While fluorescent nuclear staining generates higher pixel-level intensity noise, this method provides better overall pattern quality (greater spatial uniformity and broader histogram) for automated DIC analysis when used at the appropriate magnification. Using recently developed theoretical predictions, estimates for the standard deviation in image-correlation-based displacements due to the measured intensity pattern variance are presented for fluorescent microsphere binding and EB nuclear staining patterns. Results confirm that both patterning approaches provide relatively small standard deviation in displacement measurements and hence are appropriate for measurement of deformations in small artery specimens.

Characterizing the Copy Number Variation of Non-Coding RNAs Reveals Potential Therapeutic Targets and Prognostic Markers of LUSC.

Lung squamous cell carcinoma (LUSC) has a poor clinical prognosis and a lack of available targeted therapies. Therefore, there is an urgent need to identify novel prognostic markers and therapeutic targets to assist in the diagnosis and treatment of LUSC. With the development of high-throughput sequencing technology, integrated analysis of multi-omics data will provide annotation of pathogenic non-coding variants and the role of non-coding sequence variants in cancers. Here, we integrated RNA-seq profiles and copy number variation (CNV) data to study the effects of non-coding variations on gene regulatory network. Furthermore, the 372 long non-coding RNAs (lncRNA) regulated by CNV were used as candidate genes, which could be used as biomarkers for clinical application. Nine lncRNAs including LINC00896, MCM8-AS1, LINC01251, LNX1-AS1, GPRC5D-AS1, CTD-2350J17.1, LINC01133, LINC01121, and AC073130.1 were recognized as prognostic markers for LUSC. By exploring the association of the prognosis-related lncRNAs (pr-lncRNAs) with immune cell infiltration, GPRC5D-AS1 and LINC01133 were highlighted as markers of the immunosuppressive microenvironment. Additionally, the cascade response of pr-lncRNA-CNV-mRNA-physiological functions was revealed. Taken together, the identification of prognostic markers and carcinogenic regulatory mechanisms will contribute to the individualized treatment for LUSC and promote the development of precision medicine.

YTHDF1 Promotes Cyclin B1 Translation through m6A Modulation and Contributes to the Poor Prognosis of Lung Adenocarcinoma with KRAS/TP53 Co-Mutation.

KRAS and TP53 mutations are the two most common driver mutations in patients with lung adenocarcinoma (LUAD), and they appear to reduce latency and increase metastatic proclivity when a KRAS and TP53 co-mutation (KRAS/TP53-mut) occurs. However, the molecular mechanism involved is unclear. N6-methyladenosine (m6A), the most abundant RNA modification in mammal mRNAs, plays a critical role in tumorigenesis. Here, we used genomic and transcriptomic data and found that only LUAD patients with KRAS/TP53-mut, but not an individual mutation, appeared to exhibit poor overall survival when compared with patients without KRAS and TP53 mutation (wildtype). Subsequently, we analyzed the differential expression of the 15-m6A-related genes in LUAD with different mutations and found that YTHDF1 was the most upregulated in KRAS/TP53-mut patients and associated with their adverse prognosis. Bioinformatics and experimental evidence indicated that elevated YTHDF1 functionally promoted the translation of cyclin B1 mRNA in an m6A-dependent manner, thereby facilitating the tumor proliferation and poor prognosis of LUAD with KRAS/TP53-mut. Furthermore, the concurrent increase in YTHDF1 and cyclin B1 was confirmed by immunohistochemistry staining in patients with co-occurring KRAS/TP53 mutations. YTHDF1 was correlated with an unfavorable clinical stage and tumor size. Collectively, we identified and confirmed a novel "YTHDF1-m6A-cyclin B1 translation" axis as an essential molecular pathway for the prognosis of KRAS/TP53-mut LUAD.

Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

Oncogenic KRAS mutations combined with the loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the regulation and role of m6A modification in this cancer are unclear. We found that decreased m6A levels correlated with the disease progression and poor survival for KL patients. The correlation was mediated by a special increase in ALKBH5 (AlkB family member 5) levels, an m6A demethylase. ALKBH5 gain- or loss-of function could effectively reverse LKB1 regulated cell proliferation, colony formation, and migration of KRAS-mutated lung cancer cells. Mechanistically, LKB1 loss upregulated ALKBH5 expression by DNA hypermethylation of the CTCF-binding motif on the ALKBH5 promoter, which inhibited CTCF binding but enhanced histone modifications, including H3K4me3, H3K9ac, and H3K27ac. This effect could successfully be rescued by LKB1 expression. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. The above findings were confirmed in clinical KRAS-mutated lung cancer patients. We conclude that loss of LKB1 promotes ALKBH5 transcription by a DNA methylation mechanism, reduces m6A modification, and increases the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS-mutated lung cancer.

Deformation measurements and material property estimation of mouse carotid artery using a microstructure-based constitutive model.

A series of pressurization and tensile loading experiments on mouse carotid arteries is performed with deformation measurements acquired during each experiment using three-dimensional digital image correlation. Using a combination of finite element analysis and a microstructure-based constitutive model to describe the response of biological tissue, the measured surface strains during pressurization, and the average axial strains during tensile loading, an inverse procedure is used to identify the optimal constitutive parameters for the mouse carotid artery. The results demonstrate that surface strain measurements can be combined with computational methods to identify material properties in a vascular tissue. Additional computational studies using the optimal material parameters for the mouse carotid artery are discussed with emphasis on the significance of the qualitative trends observed.