Integrin Alpha Research Articles

Tumor Endothelial Cells-Associated Integrin Alpha-6 as a Promising Biomarker for Early Detection and Prognosis of Hepatocellular Carcinoma.

HCC remains a lethal cancer type, with early detection being critical for improved patient outcomes. This study introduces a comprehensive methodological approach to identify the ITGA6 gene as a potential blood marker for early HCC (eHCC) detection. We initially analyzed the GSE114564 dataset encompassing various stages of liver disease, identifying 972 differentially expressed genes in HCC. A refined analysis yielded 59 genes specifically differentially expressed in early HCC, including ITGA6. Subsequent validation in multiple datasets confirmed the consistent upregulation of ITGA6 in HCC. In addition, when analyzing progression-free survival (PFS) within the entire patient cohort and overall survival (OS) specifically among patients classified as tumor grade G1, the group of patients characterized by high expression levels of ITGA6 displayed an elevated risk ratio in relation to prognosis. Further analyses demonstrated the predominant expression of ITGA6 in TECs and its enrichment in angiogenesis-related pathways. Additionally, positive correlations were found between ITGA6 expression and pro-tumorigenic immune cells, but not with anti-tumorigenic immune cells. Our study elucidates the potential of ITGA6 as a blood-based marker for HCC early detection and diagnosis and its complex interplay with the tumor microenvironment. Further research may lead to novel strategies for HCC management and patient care.

Phenotypic and proteomic analysis of plasma extracellular vesicles highlights them as potential biomarkers of primary Sjögren syndrome.

Sjögren syndrome (SjS) is an autoimmune disease characterized by the destruction of the exocrine gland epithelia, causing a dryness of mucosa called sicca symptoms, and whose main life-threatening complication is lymphoma. There is a need for new biomarkers in this disease, notably diagnostic biomarkers for patients with genuine sicca symptoms that do not meet current criteria, and prognostic biomarkers for patients at risk of lymphoma. Plasma extracellular vesicles (EVs) are promising biomarker candidates in several diseases, but their potential has not yet been explored in SjS. In this proof-of-concept study, we characterized EVs from primary SjS patients (pSS, n=12) at the phenotypic and proteomic levels, compared to EVs from healthy donor (HD, n=8) and systemic lupus erythematosus patients (SLE, n=12). Specific plasma EVs subpopulations, derived from neutrophils, endothelial, and epithelial cells, were found increased in pSS. We also identified a pSS proteomic signature in plasma EVs, including neutrophil-, epithelial-, and endothelial-related proteins, such as integrin alpha M (ITGAM), olfactomedin-4 (OLFM4), Ras-related protein RAB10, and CD36. Overall, our results support the relevance of plasma EVs as biomarkers in SjS.

Integrin alpha-4 gene polymorphism in relation to natalizumab response in multiple sclerosis patients

Objectives: The aim of this study was to assess the possible the association between +3061 (G>A, rs1143676) missense mutation in exon 24 of the integrin α-4 subunit (ITGA-4) gene and the response to natalizumab in a sample of Iraqi multiple sclerosis patients. Methods: A sample of 59 patients with multiple sclerosis (16 males and 43 females; mean age of 32 years; age range of 15 to 52 years) receiving natalizumab for at least 12 consecutive months were involved in the study between March and August/ 2022. The sample was categorized into two groups according to their response to natalizumab treatment (responders and non-responders). Polymerase chain reaction and Sanger’s sequencing for the extracted deoxyribonucleic acid was performed to identify the polymorphism at ITGA-4 gene promoter region. Results: The 3061 AA and AG genotypes were present in both groups (responders and non-responders to natalizumab treatment) with the lack of the wild form GG genotype. The AG genotype was significantly present in the non-responders’ group and appeared to have a significant impact on the responsiveness to natalizumab by increasing the propensity of being non-responder with a positive correlation (Phi-coefficient of 0.294) on the contrary of AA genotype. Conclusion: The +3061 (G.A) missense mutation is related to the response to natalizumab in multiple sclerosis patients with the AG genotype, thereby increasing the likelihood of non-response significantly.

LncRNA PCBP1-AS1 mediated downregulation of ITGAL as a prognostic biomarker in lung adenocarcinoma.

Integrin alpha L (ITGAL) seemed to play a critical role in carcinogenesis and immune regulation. Nevertheless, the effects of ITGAL on non-small cell lung cancer (NSCLC) remain elusive. The present paper intended to determine the effects of ITGAL in NSCLC via the integration of bioinformatic analyses. In this study, we found that the mRNA and protein levels of ITGAL were downregulated in NSCLC tissues. Significantly, low ITGAL expression was related to poorer prognosis and increased malignancy of NSCLC. In addition, GO analysis and KEGG pathway analysis revealed that the coexpressed genes of ITGAL were predominantly associated with various immune-associated signaling pathways, like the T cell receptor signaling pathway, Th17 cell differentiation, chemokine signaling pathway, and NF-κB signaling pathway. Our result indicated that lncRNA-mediated downregulation of integrin alpha L expression was tightly related to immunocyte infiltration, immune modulators, and chemotactic factors in NSCLC, which potentially serves as a biomarker for clinical prognosis prediction and immunotherapy of NSCLC. This is the first comprehensive analysis of ITGAL in the prognosis, immune microenvironment, and immunotherapy of lung adenocarcinoma. ITGAL are promising biomarkers for predicting clinical outcomes and immunotherapy responses in patients with NSCLC.

Transcriptomic analysis of regenerated skins reveals key genes involved in skin immune response and air-breathing of loach (Paramisgurnus dabryanus)

Fish skin acts as the first immune barrier against pathogens from external environments. Recently, fish skin immunity research has gradually become a hot topic in aquaculture disease control. Interestingly, the skin can do air-breathing in some bimodal respiration fish. However, there is no comprehensive understanding of these two functions. In this study, the skin regeneration of loach (Paramisgurnus dabryanus) was investigated through morphological and histological observations. Then, original skins (OS) and the regenerated skins (RS) when their capillaries were the most abundant during healing, were collected for transcriptomic analysis. 285,899,386 clean reads with a total length of 42.34 Gb were obtained. A total of 1282 differentially expressed genes (DEGs) were detected, including 1030 DEGs up-regulated and 252 DEGs down-regulated in the comparison of RS vs. OS. Fc gamma R-mediated phagocytosis (containing gelsolin (Gsn)), chemokine signaling pathway (containing C–C motif chemokine ligand 28 (Ccl28)), and B cell receptor signaling pathway (containing CD80 molecule (Cd81)) were closely related to skin immune response of the loach. PI3K-Akt signaling pathway [containing fibronectin 1 (Fn1) and lysyl oxidase-like 2b (Loxl2b)], TGF-beta signaling pathway [containing thrombospondin 1 (Thbs1)], ECM-receptor interaction [containing integrin alpha 7 (Itga7), Itgb8, Itgb10 and Itgb5], and dilated cardiomyopathy [containing laminin subunit alpha1 (Lama1)] were closely associated with skin air-breathing of the loach. This study is conducive to explore the molecular mechanisms of skin immune response during its regeneration and air-breathing of bimodal respiration fish. This study will benefit for the aquaculture of P. dabryanus and its related species.

Enhanced reparatory effect of EI1 on dental pulp via extracellular matrix remodeling by miR-181b-2-3p inhibitor

Background/purposeExtracellular matrix (ECM) is crucial for dental pulp repair. The aim of this paper is to investigate the ECM remodeling effect of miR-181b-2-3p (a microRNA) and to verify the reparatory effect of EI1 (an epigenetic drug) and miR-181b-2-3p inhibitor on dental pulp. Materials and methodsLevels of ECM-related factors in EI1-treated human dental pulp cells (hDPCs) were measured by qRT-PCR and Western blot. The anti-inflammation effect of EI1 was examined in Lipopolysaccharide-stimulated hDPCs. miR-181b-2-3p mimics or inhibitors were transfected into hDPCs and then the cells’ functions were detected. A dual luciferase reporter assay was used to identify the targets of miR-181b-2-3p. Pulpotomy using miR-181b-2-3p antagomirs and EI1 as pulp capping materials was performed in male six-week-old Sprague-Dawley rats. ResultsEI1 upregulated ECM-related genes expression in hDPCs, but failed to upregulate the collagen1A1 (COL1A1) protein level. Pro-inflammatory factors were downregulated by EI1 in Lipopolysaccharide-stimulated hDPCs. Overexpression of miR-181b-2-3p downregulated the expression of transforming growth factor-β2 (TGF-β2) and fibronectin type III domain-containing protein 5 precursor (FNDC5), while the inhibition had the opposite effect. Dual luciferase reporter assays demonstrated that miR-181b-2-3p targets TGF-β2, FNDC5 and integrin alpha 4 protein (ITGA4). Compared to EI1 was used alone, EI1 combined with the inhibitor upregulated the protein levels of COL1A1, fibronectin (FN1) and TGF-β2 in hDPCs, promoted hDPCs migration, and exhibited reparatory effects on inflamed rat pulp tissue. ConclusionmiR-181b-2-3p inhibitor could enhance the reparatory effect of EI1 via ECM remodeling in dental pulp both in vitro and in vivo.

Developmentally regulated expression of integrin alpha-6 distinguishes neural crest derivatives in the skin.

Neural crest-derived cells play essential roles in skin function and homeostasis. However, how they interact with environmental cues and differentiate into functional skin cells remains unclear. Using a combination of single-cell data analysis, neural crest lineage tracing, and flow cytometry, we found that the expression of integrin α6 (ITGA6) in neural crest and its derivatives was developmentally regulated and that ITGA6 could serve as a functional surface marker for distinguishing neural crest derivatives in the skin. Based on the expression of ITGA6, Wnt1-Cre lineage neural crest derivatives in the skin could be categorized into three subpopulations, namely, ITGA6bright, ITGA6dim, and ITGA6neg, which were found to be Schwann cells, melanocytes, and fibroblasts, respectively. We further analyzed the signature genes and transcription factors that specifically enriched in each cell subpopulation, as well as the ligand or receptor molecules, mediating the potential interaction with other cells of the skin. Additionally, we found that Hmx1 and Lhx8 are specifically expressed in neural crest-derived fibroblasts, while Zic1 and homeobox family genes are expressed in mesoderm-derived fibroblasts, indicating the distinct development pathways of fibroblasts of different origins. Our study provides insights into the regulatory landscape of neural crest cell development and identifies potential markers that facilitate the isolation of different neural crest derivatives in the skin.

Serum exosomal miR-192 serves as a potential detective biomarker for early pregnancy screening in sows.

The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

IL-17A-mediated JMJD3 regulation of integrin alpha 3 gene expression alters migration of diabetic keratinocytes and impairs wound repair.

Abstract Keratinocytes are key structural cells in cell migration during wound repair. Keratinocyte migration is dysfunctional in diabetes and results in non-healing, however, the reason for this is unclear. Using bulk and single cell sequencing, we identified that Th17 CD4+ T-cells predominate as does an IL-17A signature in human diabetic wound tissue compared to non-diabetic controls. The effects of increased IL-17A in diabetic skin on keratinocyte function is unknown. To examine this, we subjected isolated keratinocytes to scratch injury and a 24hr rIL-17A (20ng) stimulation. We observed a significant decrease in migration rate in IL-17A stimulated keratinocytes compared to their controls. Next, we examined migration related genes in keratinocytes from mice fed with a normal (ND) or a high fat (HFD) diet. Integrin alpha 3 subunit, Itga3, (a protein known to delay keratinocyte migration during wound re-epithelialization) was higher in diabetic (HFD) keratinocytes compared to ND controls at baseline and with rIL-17A. We analyzed epigenetic enzymes known to increase gene expression and identified that JMJD3, a histone demethylase that relaxes chromatin, was increased both in diabetic keratinocytes from our human scRNA-seq and in murine diabetic keratinocytes with rIL17A. Further, treatment with a JMJD3-specific inhibitor (GSK-J4) decreased Itga3 in diabetic keratinocytes, suggesting that JMJD3 may be relevant to IL-17A-mediated regulation of integrin alpha 3 and other keratinocyte migration genes. Continued investigation into upstream IL-17A signaling in normal and diabetic keratinocytes that alters downstream migration is crucial for our understanding of impaired keratinocyte functions associated with diabetic wound repair. NIH T32 AI 007413, Research Training in Experimental Immunology Rackham Pre-Candidate Graduate Student Research Grant, University of Michigan

Molecular Pathology of Micropapillary Carcinomas: Is Characteristic Morphology Related to Molecular Mechanisms?

Micropapillary carcinoma is an entity defined histologically in many organs. It is associated with lymph node metastasis and poor prognosis. The main mechanism for its histopathologic appearance is reverse polarization. Although the studies on this subject are limited, carcinomas with micropapillary morphology observed in different organs are examined by immunohistochemical and molecular methods. Differences are shown in these tumors compared with conventional carcinomas regarding the rate of somatic mutations, mRNA and miRNA expressions, and protein expression levels. TP53, PIK3CA, TERT, KRAS, EGFR, MYC, FGFR1, BRAF, AKT1, HER2/ERBB2, CCND1, and APC mutations, which genes frequently detected in solid tumors, have also been detected in invasive micropapillary carcinoma (IMPC) in various organs. 6q chromosome loss, DNAH9, FOXO3, SEC. 63, and FMN2 gene mutations associated with cell polarity or cell structure and skeleton have also been detected in IMPCs. Among the proteins that affect cell polarity, RAC1, placoglobin, as well as CLDNs, LIN7A, ZEB1, CLDN1, DLG1, CDH1 (E-cadherin), OCLN, AFDN/AF6, ZEB1, SNAI2, ITGA1 (integrin alpha 1), ITGB1 (integrin beta 1), RHOA, Jagged-1 (JAG1) mRNAs differentially express between IMPC and conventional carcinomas. Prediction of prognosis and targeted therapy may benefit from the understanding of molecular mechanisms of micropapillary morphology. This review describes the molecular pathologic mechanisms underlying the micropapillary changes of cancers in various organs in a cell polarity-related dimension.

Exploring the pharmacological and adverse reaction mechanism of a drug by network pharmacology strategy: Using colchicine to treat Behcet syndrome as an example

Colchicine (COLC) is a natural alkaloid used to treat Behcet syndrome (BS), but its adverse reactions limit its clinical application in treating BS. However, the adverse reaction mechanism of COLC in the treatment of BS remains unclear. Herein, a network pharmacology-based strategy was designed to analyze the pharmacological and adverse reaction mechanism of COLC in treating BS. The biological functions of COLC and BS pathogenesis were analyzed through a series of network construction and analysis. The data above predicted the pharmacological and adverse reaction mechanism of COLC in BS treatment. The pharmacological mechanism of COLC against BS was predicted to control inflammatory responses. Interleukin-8, interleukin-18, integrin alpha-4, integrin beta-2, and tubulin targets are crucial in treating BS. The adverse reactions of COLC in BS treatment were predicted as neurotoxicity and hepatotoxicity. The mechanism of hepatotoxicity may be related to the decrease of cytochrome P450 family 3 subfamily A activity caused by various factors, such as poor hepatic function, the dosage of COLC, and combination with inhibitors. The mechanism of neurotoxicity may be related to the disruption of microtubules in the nervous system by COLC transport across the blood-brain barrier. This study provided basic evidence for the medication safety management of COLC used in treating BS. Moreover, this study demonstrated that it is feasible to analyze the adverse reaction mechanisms of drugs using a network pharmacology strategy, which facilitates systematic drug safety management and evaluation.

Abstract 6234: Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression

Abstract Integrins have long been sought as therapeutic targets in cancer, but there are no FDA approved drugs to target this gene family. Since integrins require the dimerization between an alpha and beta subunit to function, we developed a CRISPR library targeting all 26 alpha and beta subunits to identify which pair is the most important. We found a strong correlation between ITGAV and ITGB5 (Integrin Beta 5), which form a known heterodimer, in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. As the more essential partner, we designed a CRISPR tiling scan targeting the entire coding region of ITGAV and determine the essential nature of each amino acid. We found a sub-domain within the beta propeller to be important for ITGAV function, the CRISPR identified pocket (CIP). Genetic targeting of this pocket disrupted the ability to detect the heterodimer signal of ITGAV-ITGB5 by a specific antibody. Furthermore, we confirmed that essential amino acids within the CIP responsible for the heterodimer stability by the NanoBRET energy transfer system. Knowing the role of the CIP in heterodimer stability we searched for a small molecule to bind in the CIP to phenocopy our genetic findings. A combined in-silico and in-vitro high throughput drug screen started tested compounds for cell killing and disruption of the ITGAV-ITGB5 heterodimer. “Compound 2” (cpd_2) showed both strong cell killing and dissociation of the heterodimer. Disruption was measured by detection by flow cytometry and through the NanoBRET system. Cpd_2’s binding was confirmed with thermal shift studies with purified ITGAV beta propeller, validating that this compound is behaving and binding as we expect at the CIP. Next, we sought to understand how the ITGAV-ITGB5 heterodimer controls cancer cell survival. From the Dependency Map Public database (DepMap) we found that Rac1 dependency is significantly correlated only with ITGAV and ITGB5. We hypothesized that this meant that ITGAV-ITGB5 regulated Rac1 activity. Loss of Rac1 phenocopies knock out of ITGAV or ITGB5 and loss of either Rac1 or ITGAV led to dysregulation of the actin cytoskeleton, a known downstream feature of Rac1. A constitutively Rac1 (CA_Rac1) rescues the effects of targeting of ITGAV or treatment with cpd_2. Altogether this data suggests that ITGAV-ITGB5 signals to Rac1 and that attenuation of their function can be reversed by an active Rac1 In summary, our study highlights a novel strategy of targeting the integrin heterodimer ITGAV-ITGB5 with a small molecule binding to a novel subdomain, CIP, of ITGAV. The CIP is responsible for the heterodimer stability of ITGAV and ITGB5. Disruption of this heterodimer led to the dysregulation of Rac1 and destabilization of the actin cytoskeleton. This mechanism highlights a novel strategy for targeting integrins as a therapeutic target in cancer. Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Liu, Ming li, Qiao Liu, Xiaobao Xu, Priyanka Singh, Leisi Zhang, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6234.

ITGA5 promotes tumor angiogenesis in cervical cancer.

Integrins are critical to cancer progression. Integrin alpha 5 (ITGA5) is correlated with the prognosis of cervical cancer patients. However, whether ITGA5 plays an active role in cervical cancer progression or not remains unknown. ITGA5 protein expression was detected in 155 human cervical cancer tissues by immunohistochemistry. Data from The Cancer Genome Atlas were utilized to identify risk factors for the overall survival of cervical cancer patients and ITGA5-associated differentially expressed genes. Analyses of single-cell RNA-seq based on Gene Expression Omnibus datasets were performed to show the coexpression of ITGA5 and angiogenesis factors. Tube formation assay, 3D spheroid sprout assay, qRT-PCR, Western Blotting, ELISA, and immunofluorescence were conducted to explore the angiogenic function of ITGA5 in vitro and underlying mechanisms. High ITGA5 level was significantly correlated with increased risk in terms of overall survival and advanced disease stage in cervical cancer patients. ITGA5-associated differentially expressed genes linked ITGA5 to angiogenesis, and immunohistochemistry showed a positive correlation between ITGA5 and microvascular density in cervical cancer tissues. Moreover, tumor cells transfected with ITGA5-targeting siRNA decreased ability to promote endothelial tube formation in vitro. ITGA5/VEGFA coexpression was observed in a tumor cell subpopulation and the decreased endothelial angiogenesis by downregulating ITGA5 could be reversed by VEGFA. Bioinformatics analysis highlighted the PI3K-Akt signaling pathway as downstream of ITGA5. Downregulation of ITGA5 in tumor cells significantly decreased p-AKT and VEGFA levels. Fibronectin (FN1) coated cells or transfected with FN1-targeting siRNA showed fibronectin may play a critical role on ITGA5-mediated angiogenesis. ITGA5 promotes angiogenesis and possibly be a potential predictive biomarker for poor survival of patients in cervical cancer.

Transcriptome analyses reveal differentially expressed genes associated with development of the palatal organ in bighead carp (Hypophthalmichthys nobilis)

The palatal organ is a filter-feeding related organ and occupies a considerable proportion of the head of bighead carp (Hypophthalmichthys nobilis), a large cyprinid fish intensive aquaculture in Asia. In this study, we performed RNA-seq of the palatal organ during growth periods of two (M2), six (M6) and 15 (M15) months of age after hatching. The numbers of differentially expressed genes (DEGs) were 1384, 481 and 1837 for M2 VS M6, M6 VS M15 and M2 VS M15 respectively. The following signaling pathways of energy metabolism and cytoskeleton function were enriched, including ECM-receptor interaction, Cardiac muscle contraction, Steroid biosynthesis and PPAR signaling pathway. Several members of collagen family (col1a1, col2a1, col6a2, col6a3, col9a2), Laminin gamma 1 (lamc1), integrin alpha 1 (itga1), Fatty acid binding protein 2 (fads2) and lipoprotein lipase (lpl), and Protein tyrosine kinase 7 (Ptk7) are candidate genes for growth and development of basic tissues of the palatal organ. Furthermore, taste-related genes such as fgfrl1, fgf8a, fsta and notch1a were also identified, which may be involved in the development of taste buds of the palatal organ. The transcriptome data obtained in this study provide insights into the understanding functions and development mechanisms of palatal organ, and potential candidate genes that may be related to the genetic modulation of head size of bighead carp.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

Evaluation of the in vitro and in vivo effect of liposomal doxorubicin along with oncolytic Newcastle disease virus on 4T1 cell line: Animal preclinical research.

Breast cancer is one of the most common malignancies in women, with one in 20 globally. Oncolytic viruses have recently been the first step in the biological treatment of cancer, either genetically engineered or naturally occurring. They increase specifically inside cancer cells and destroy them without damaging normal tissues or producing a host immune response against tumour cells or expressing transgenes. One of the most known members of this family is the Newcastle disease virus (NDV), a natural oncolytic virus that selectively induces apoptosis and DNA fragmentation in human cancer cells. This study performed biochemical and molecular investigations with variable doses of NDV (32, 64, 128 HAU) and liposomal doxorubicin (9mg/kg) on mouse triple-negative mammary carcinoma cell line 4T1 and BALB/c models tumours for the first time. Real-time quantitative PCR analysis in NDV-treated animal tumours showed increased expression of P21, P27 and P53 genes and decreased expression of CD34, integrin Alpha 5, VEGF and VEGF-R genes. Additional assessments in treated mouse models also showed that NDV increased ROS production, induced apoptosis, reduced tumour size and significantly improved prognosis, with no adverse effect on normal tissues. These findings all together might indicate that NDV in combination with chemotherapy drugs could improve prognosis in cancer patients although many more conditions should be considered.

Systematic Analysis of the Prognostic Significance and Roles of the Integrin Alpha Family in Non-Small Cell Lung Cancers.

Lung cancer is one of the most common cancer malignancies and the principal cause of cancer-associated deaths worldwide. Non-small cell lung cancers (NSCLCs) account for more than 80% of all lung cancer cases. Recent studies showed that the genes of the integrin alpha (α) (ITGA) subfamily play a fundamental role in various cancers. However, little is known about the expression and roles of distinct ITGA proteins in NSCLCs. Gene Expression Profiling Interactive Analysis and UALCAN (University of ALabama at Birmingham CANcer) web resources and The Cancer Genome Atlas (TCGA), ONCOMINE, cBioPortal, GeneMANIA, and Tumor Immune Estimation Resource databases were used to evaluate differential expression, correlations between the expression levels of individual genes, the prognostic value of overall survival (OS) and stage, genetic alterations, protein-protein interactions, and the immune cell infiltration of ITGAs in NSCLCs. We used R (v.4.0.3) software to conduct gene correlation, gene enrichment, and clinical correlation of RNA sequencing data of 1016 NSCLCs from TCGA. To evaluate the expression of ITGA5/8/9/L at the expression and protein levels, qRT-PCR, immunohistochemistry (IHC), and hematoxylin and eosin (H&E) were performed, respectively. Upregulated levels of ITGA11 messenger RNA and downregulated levels of ITGA1/3/5/7/8/9/L/M/X were observed in the NSCLC tissues. Lower expression of ITGA5/6/8/9/10/D/L was discovered to be expressively associated with advanced tumor stage or poor patient prognosis in patients with NSCLC. A high mutation rate (44%) of the ITGA family was observed in the NSCLCs. Gene Ontology functional enrichment analyses results revealed that the differentially expressed ITGAs could be involved in roles related to extracellular matrix (ECM) organization, collagen-containing ECM cellular components, and ECM structural constituent molecular functions. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that ITGAs may be involved in focal adhesion, ECM-receptor interaction, and amoebiasis; the expression of ITGAs was significantly correlated with the infiltration of diverse immune cells in NSCLCs. ITGA5/8/9/L was also highly correlated with PD-L1 expression. The validation results for marker gene expression in NSCLC tissues by qRT-PCR, IHC, and H&E staining indicated that the expression of ITGA5/8/9/L decreased compared with that in normal tissues. As potential prognostic biomarkers in NSCLCs, ITGA5/8/9/L may fulfill important roles in regulating tumor progression and immune cell infiltration.

The expression of SERPINE1 in colon cancer and its regulatory network and prognostic value

BackgroundSerpin Peptidase Inhibitor 1 (SERPINE1) promotes cancer progression by making it easier for cancer cells to spread to surrounding normal tissue. We expect to understand the prognostic value and regulatory network of SERPINE1 in colon cancer using bioinformatics methods.MethodsThe expression of target gene SERPINE1 in varying cancers was analyzed by the Tumor Immune Estimation Resource (TIMER) database. SERPINE1 expression in Colon Adenocarcinoma and normal tissue samples was assessed by starBase and UALCAN databases. SERPINE1 expression in clinical tissues was assayed using quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR). SERPINE1 expression was detected in colon cancer patients with various clinical features (age, gender, nodal metastasis status, race, stages, and subtype) using analysis of variance. Survival curve was used to analyze the effect of high and low expression of SERPINE1 on the survival time of patients with different clinical phenotypes. Gene Set Enrichment Analysis (GSEA) was conducted on the results of LinkFinder calculation using LinkInterpreter module, which was combined with Pearson correlation analysis to obtain the kinase targets and miRNA targets, transcription factor targets, and corresponding signaling pathways associated with SERPINE1. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on GSEA result. Finally, Gene Multiple Association Network Integration Algorithm (GeneMANIA) was utilized to establish a network of genes related to the kinases MAPK1, miR-18a, and SRF_Q, and biological functions were analyzed.ResultsBased on TIMER, starBase, and UALCAN databases, SERPINE1 was found to be remarkably highly expressed in colon cancer patients, which was further verified by clinical tissue. It was also associated with different clinical features (nodal metastasis status, stages, subtypes). Additionally, survival analysis showed that patients with low expression of SERPINE1 had a longer survival time, suggesting that SERPINE1 was a prognostic risk factor for colon cancer. Pearson correlation analysis revealed that the expression of Integrin Alpha 5 (ITGA5), Matrix Metallopeptidase 19 (MMP19), and ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 4 (ADAMTS4) had the highest correlation with that of SERPINE1. The GSEA results indicated that these genes were mainly enriched in the pathways of RNA expression and kinases. Finally, GeneMANIA analysis was introduced to construct the molecular network of SERPINE1.ConclusionOverall, our bioinformatics analyses comprehensively described the networks involved SERPINE1 in colon cancer and the potentially associated molecular mechanisms.

Macrophages promote anti-androgen resistance in prostate cancer bone disease.

Metastatic castration-resistant prostate cancer (PC) is the final stage of PC that acquires resistance to androgen deprivation therapies (ADT). Despite progresses in understanding of disease mechanisms, the specific contribution of the metastatic microenvironment to ADT resistance remains largely unknown. The current study identified that the macrophage is the major microenvironmental component of bone-metastatic PC in patients. Using a novel in vivo model, we demonstrated that macrophages were critical for enzalutamide resistance through induction of a wound-healing-like response of ECM-receptor gene expression. Mechanistically, macrophages drove resistance through cytokine activin A that induced fibronectin (FN1)-integrin alpha 5 (ITGA5)-tyrosine kinase Src (SRC) signaling cascade in PC cells. This novel mechanism was strongly supported by bioinformatics analysis of patient transcriptomics datasets. Furthermore, macrophage depletion or SRC inhibition using a novel specific inhibitor significantly inhibited resistant growth. Together, our findings elucidated a novel mechanism of macrophage-induced anti-androgen resistance of metastatic PC and a promising therapeutic approach to treat this deadly disease.

Abstract 39: Targeting Neutrophil-specific Integrin Alpha9 In Obesity-induced Hyperglycemia Mice Improves Stroke Outcome

Background: Obesity-induced hyperglycemia is one of the significant risk factors for stroke. Integrin α9β1 is highly expressed on activated neutrophils and stabilizes adhesion to the endothelium via ligands, including tenascin C. While myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. Purpose: To determine the role of neutrophil-specific α9 in stroke outcome in mice with obesity-induced hyperglycemia. Methods: α9 Neu-KO (α9 fl/fl MRP8Cre + ) and littermate control α9 WT (α9 fl/fl MRP8 Cre - ) mice were fed a 60% high fat diet for 20 weeks. Functional outcomes were evaluated up to 28 days after stroke in mice of both sexes using a transient (30 min) middle cerebral artery ischemia. Infarct volume (MRI) and post-reperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, p-NFκB, NETosis, TNFα, and IL1β levels) were measured post 6 or 48 h of reperfusion. Functional outcomes (mNSS, rotarod, corner, and wire-hanging test) were measured at week 1, 2, 3, and 4. Results: Stroke upregulated neutrophil α9 expression in obese mice (P&lt;.05 vs. lean mice). Genetic ablation of neutrophil-specific α9 improved functional outcomes up to 4 weeks that was concomitant with reduced infarct size, improved CBF, decreased post-reperfusion thrombo-inflammation, and reduced NETosis (P&lt;.05 vs. α9 WT obese mice) irrespective of sex. Intravital microscopy revealed that obese α9 Neu-KO mice were less susceptible to experimental carotid thrombosis (P&lt;.05 vs. α9 WT obese mice). Infusion of tenascin C increased stroke severity in both α9 Neu-KO and α9 WT compared to vehicle, suggesting that neutrophil-specific α9 does not exacerbate stroke via tenascin C. Obese WT mice infused with a blocking anti-integrin α9 IgG exhibited improved functional outcomes up to 4 weeks (P&lt;.05 vs. control IgG). Functional outcomes after stroke were comparable in α9 Neu-KO mice infused with anti-integrin α9 IgG or control IgG, suggesting no off-target effects of the antibody. Conclusion: Both genetic ablation of neutrophil-specific α9 and pharmacological inhibition improve long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation and NETosis.