Integrin Alpha Research Articles

Integrin alpha1 beta1 promotes interstitial fibrosis in a mouse model of polycystic kidney disease.

Fibrosis is the cause of end-stage kidney failure in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD). The molecular and cellular mechanisms involved in fibrosis are complex and anti-fibrotic therapies have so far failed to make an impact on patient welfare. Using unbiased proteomics analysis on the Pkd1 nl/nl mouse, we found that expression of the integrin α1 subunit is increased in this model of ADPKD. In human ADPKD tissue and two single cell RNA kidney disease datasets, ITGA1 was also upregulated. To investigate the functional role of this integrin subunit in ADPKD, we generated a Pkd1 nl/nl Itga1 -/- mouse. We observed a significant reduction in kidney volume and kidney dysfunction in mice lacking the integrin α1 subunit. Kidneys from Pkd1 nl/nl Itga1 -/- mice had smaller cysts and reduced interstitial expansion and tubular atrophy. Picrosirius red staining identified a restriction in collagen staining in the interstitium and the myofibroblast marker α smooth muscle actin was also downregulated. Myofibroblast cell proliferation was reduced in Pkd1 nl/nl Itga1 -/- mice and primary fibroblast cultures demonstrated an abrogated fibrogenic phenotype in integrin α1-depleted fibroblasts. These results highlight a previously unrecognised role for the integrin α1 subunit in kidney fibrosis.

Clinical significance of upregulated Rho GTPase activating protein 12 causing resistance to tyrosine kinase inhibitors in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major health challenge with high incidence and poor survival rates in China. Systemic therapies, particularly tyrosine kinase inhibitors (TKIs), are the first-line treatment for advanced HCC, but resistance is common. The Rho GTPase family member Rho GTPase activating protein 12 (ARHGAP12), which regulates cell adhesion and invasion, is a potential therapeutic target for overcoming TKI resistance in HCC. However, no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported. To unveil the expression of ARHGAP12 in HCC, its role in TKI resistance and its potential associated pathways. This study used single-cell RNA sequencing (scRNA-seq) to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis. CellChat was used to investigate focal adhesion (FA) pathway regulation. We integrated bulk RNA data (RNA-seq and microarray), immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels, correlating with clinical outcomes. We assessed ARHGAP12 expression in TKI-resistant HCC, integrated conventional HCC to explore its mechanism, identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy. ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA. In malignant hepatocytes in high-score FA groups, MDK-[integrin alpha 6 (ITGA6) + integrin β-1 (ITGB1)] showed specificity in ligand-receptor interactions. ARHGAP12 mRNA and protein were upregulated in bulk RNA, immunohistochemistry and proteomics, and higher expression was associated with a worse prognosis. ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway. ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA. High expression of ARHGAP12 was associated with adverse reactions to sorafenib, cabozantinib and regorafenib, but not to immunotherapy. ARHGAP12 expression is elevated in HCC and TKI-resistant HCC, and its regulatory role in FA may underlie the TKI-resistant phenotype.

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P<0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

Long non-coding RNA FAM87A is associated with overall survival and promotes cell migration and invasion in gastric cancer.

The role of long non-coding RNAs (lncRNAs) in the invasion and metastasis of gastric cancer remains largely unclear. Integrating transcriptome data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, differentially expressed genes were identified in gastric cancer. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) database-curated gene set, lncRNAs associated with invasion and metastasis were identified. The Cox analyses were performed to identify prognostic lncRNAs. The competing endogenous RNA (ceRNA) regulation network was constructed to identify hub lncRNAs in gastric cancer. Functional and pathway analyses were used to investigate the function of identified lncRNAs. RT-qPCR and Transwell assays were used to investigate the expression in gastric cancer tissues and functions in gastric cancer cell lines. Based on GEO and TCGA databases, 111 differentially expressed lncRNAs were identified between gastric cancer and normal samples. A total of 43 lncRNAs were significantly correlated with hallmark genes of cancer invasion and metastasis. Among them, as a hub lncRNA in the invasion-related ceRNA regulation network, FAM87A showed potential regulation on MAPK signaling and transforming growth factor (TGF) signaling cascade, such as TGFB2, TGFBR1, and TGFBR2. Furthermore, FAM87A also showed a significant correlation with cell adhesion molecules, such as Integrin alpha 6 (ITGA6) and Contactin-1 (CNTN1). RT-qPCR experiments showed that FAM87A expression was upregulated in gastric cancer tissues compared to normal samples (n = 30). Transwell assays showed that FAM87A knockdown inhibited the migration and invasion abilities of gastric cancer cells in vitro. Notably, clinical data analysis showed that lncRNA FAM87A could be an independent factor for the overall survival of patients with gastric cancer. LncRNA FAM87A may play a pivotal role in regulating migration and invasion of gastric cancer cells. FAM87A could be a potential biomarker for the overall survival of patients with gastric cancer.

Galectin-8 Contributes to Human Trophoblast Cell Invasion.

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.

Integrin Alpha2 and Integrin Beta3 Polymorphisms are Risk Factors for Infertility in Iraqi Infertile Females under In vitro Fertilization Program

صُممت الدراسة الحالية للتحري عن تأثير تعدد الطرز أحادي النوكليوتيدات (SNP) ، للتتابع المرجعي 5918 ،(T&gt; C) لمورث إنتيجرين بيتا 3 (ITGB3) وللتابع المرجعي 1801106 (G&gt; A) إنتجرين ألفا 2 (ITGB3) في عينات دم اناث عراقيات. 71 أنثى مصابة بالعقم خاضعة لبروتوكول أطفال الأنابيب (مقسمة إلى مجموعتين فرعيتين ، 29 عملية زرع ناجحة و 42 عملية زرع فاشلة) و 50 أنثى خصبة كمجموعة سيطرة. تم الحصول على عينات الدم من المرضى ومجموعات السيطرة في المستشفيات العامة في بغداد ، العراق من مارس 2021 إلى أبريل 2021. تم إجراء التنميط الوراثي لتعدد الطرز باستخدام تحليل الذوبان عالي الدقة في الوقت الحقيقي (HRM في الوقت الحقيقي PCR) من الحمض النووي الرايبوزي منقوص الاوكسجين الجينومي المنقى المأخوذ من عينات الدم. أظهرت النتائج أن الطرز الوراثية TT و GG لـلتتابعات المرجعية 5918 في ITGB3 و 1801106 في ITGA2 على التوالي ، كانت أعلى بشكل ملحوظ في الإناث الخصبة من الاناث العقيمات. يشير ذلك إلى أن الطراز الوراثي GG يعمل كعامل وقائي ، بينما يعمل الطراز الوراثي TC لـلتتابع المرجعي 5918 في ITGB3 و AA من الطراز الوراثي للتتابع المرجعي 1801106 في ITGA2 كعوامل خطر. الاستنتاج ، ان لتعدد الطرز في جينات ITGA2 و ITGB3 تأثير على استعداد مجموعة من الاناث ضمن الدر اسة للإصابة بالعقم.

Comparative transcriptome analysis reveals molecular mechanisms of the effects of light intensity and photoperiod on ovarian development in Procambarus clarkii (Girard, 1852)

Procambarus clarkii (Girard, 1852) has important economic value in China and internationally. In this research, the comparative transcriptome analysis was used to reveal molecular mechanisms of influences of photoperiod and light intensity on ovarian development in P. clarkii for the first time. Some genes (such as laminin, collagen, integrin beta, catenin) and pathways (including TGF-beta signaling pathway, focal adhesion, ECM–receptor interaction) associated with ovarian development and oocyte maturation were significantly upregulated. Some genes related to circadian clock (such as CLK, PER) were identified in this research. The results indicated that when light intensity or photoperiod increased, P. clarkii could up-regulate the expression levels of the laminin and collagen, thereby synthesizing related proteins, promoting meiosis of the oocytes, thus increasing the number of oocytes in the ovary. At the same time, P. clarkii could up-regulate the expression levels of integrin beta, integrin alpha 6, and diacylglycerol to synthesize related proteins, thereby promoting the formation of proteins and fats such as triglycerides, these proteins and fats can provide material basis for maturation and development of oocytes, resulting in oocyte maturation and ovarian development. P. clarkii could synthesize related proteins by upregulating expression levels of genes (such as catenin), these proteins or hormones can adhere to other actins (such as integrins), thereby stabilizing the morphology of the oocytes and ensuring normal development. Meantime, the increase in light intensity or photoperiod could cause release GSH and VTG, resulting in oocytes development and maturation. The data in this research can reveal molecular mechanisms of impacts of photoperiod and light intensity on oocyte maturation and ovarian development in P. clarkii, can offer crucial genomic data for studying developmental mechanisms of ovary and oocyte in crustacean.

An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species

Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses. We computationally integrate binding motifs of human integrin alphaV beta6, a skeletal muscle receptor, into a liver-detargeting capsid. Designed AAVs show higher productivity and superior muscle transduction compared to their parent. One variant, LICA1, demonstrates comparable muscle transduction to other myotropic AAVs with reduced liver targeting. LICA1’s myotropic properties are observed across species, including non-human primate. Consequently, LICA1, but not AAV9, effectively delivers therapeutic transgenes and improved muscle functionality in two mouse MD models (male mice) at a low dose (5E12 vg/kg). These results underline the potential of our design method for AAV engineering and LICA1 variant for MD gene therapy.

Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation.

Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.

Abstract Tu018: Integrin alpha 1 plays a critical role in angiotensin II-induced abdominal aortic aneurysm rupture

Background: Abdominal aortic aneurysm (AAA) is a life-threatening condition without an established specific therapy. This study aims to identify a new therapeutic target for preventing AAA rupture. Methods and Results: Angiotensin II (AngII)-infused ApoE-/- mice develop AAA, an established model of AAA. To identify molecular pathways contributing to AAA development, we conducted single-cell RNA sequence analysis using a dataset of aortic tissues harvested from mice infused with AngII for 4 weeks (GSE186865). Gene set enrichment analysis revealed upregulation of integrin- and TGFB-related pathways in AngII-stimulated smooth muscle cells (SMCs). Integrin α1 (Itga1) constitutes half of the α1β1 integrin duplex and serves as a major collagen receptor in SMCs. Male wild-type (n=37) and Itga1 knockout (n=36) mice with an ApoE-/- background received a continuous infusion of AngII for 4 weeks to develop AAA. Kaplan-Meier analysis indicated that ablating Itga1 significantly suppressed AAA rupture (p=0.009), without affecting the maximum diameter of the abdominal aorta. Considering extracellular matrix degradation as a critical hallmark of AAA rupture, we examined the abundance of major matrix metalloproteinases (MMPs) in the aortic wall, specifically MMP2 and MMP9, two major MMPs in the aorta. Gelatin zymography revealed that ablation of Itga1 suppressed AngII-induced MMP9 increase, without affecting MMP2 abundance. The expression of MMP9 is controlled by inflammation and proliferation signals; hence, we focused on these signals. qPCR analysis (n=8 per group) demonstrated that AngII-induced upregulation of Tnfa (p&lt;0.001) and Tgfb1 (p=0.026) was mitigated by Itga1 ablation in aortic tissues. Conclusion: The integrin pathway is activated in the tissue of AAA tissue and deletion of Itga1 suppresses AAA rupture. Here we showed that the Itga1-Tgfb1-MMP9 axis would be a novel molecular target to inhibit AAA rupture.

Identification and functional validation of miR-190b-5p and miR-296-3p as novel therapeutic attenuators of liver fibrosis

Background & AimsLiver fibrosis and its end-stage form known as cirrhosis contributes to millions of deaths annually. The lack of robust anti-fibrotic molecules is in part attributed to absence of any functional screens to identify molecular regulators using patient-derived primary human hepatic myofibroblasts, which are key drivers of fibrosis. MethodsHere, to identify robust regulators of fibrosis, we performed functional microRNA screenings in primary human hepatic myofibroblasts followed by in vivo validation in three independent mouse models of fibrosis (toxin, cholestasis and MASH). ResultsWe identified miR-190b-5p and miR-296-3p as robust anti-fibrotic miRNAs that suppress liver fibrosis. Notably, the expression of miR-190b-5p and miR-296-3p was found significantly reduced in human livers with fibrosis. Mechanistically, we discovered hyaluronan synthase 2 (HAS2) and integrin alpha-6 (ITGA6) as novel targets of miR-190b-5p and miR-296-3p, respectively. Furthermore, we demonstrated that the anti-fibrotic properties of miR-190b-5p and miR-296-3p are, at least in part, dependent on HAS2 and ITGA6. Finally, we showed the anti-fibrotic function of both miRNAs in a human liver bud model, which mimics multiple features of human liver. ConclusionsCollectively, in our study we discovered miR-190b-5p and miR-296-3p as two novel anti-fibrotic miRNAs, and that HAS2 and ITGA6 contribute to miR-190b-5p- and miR-296-3p-mediated inhibition of liver fibrosis. These results provide a foundation for future research to explore the clinical utility of miR-190b-5p and miR-296-3p in liver injuries with fibrosis. Impact and ImplicationsLiver fibrosis and cirrhosis contribute to millions of deaths world-wide and, till date, remain as unmet medical needs. In this study, we discovered two microRNAs, miR-190b-5p and miR-296-3p, which suppress liver fibrosis in preclinical mouse models and a human liver bud model. Our promising results encourage further studies that aim to develop both miRNAs for the treatment of liver fibrosis in patients.

Humanin activates integrin αV–TGFβ axis and leads to glioblastoma progression

The role of mitochondria peptides in the spreading of glioblastoma remains poorly understood. In this study, we investigated the mechanism underlying intracranial glioblastoma progression. Our findings demonstrate that the mitochondria-derived peptide, humanin, plays a significant role in enhancing glioblastoma progression through the intratumoral activation of the integrin alpha V (ITGAV)–TGF beta (TGFβ) signaling axis. In glioblastoma tissues, humanin showed a significant upregulation in the tumor area compared to the corresponding normal region. Utilizing multiple in vitro pharmacological and genetic approaches, we observed that humanin activates the ITGAV pathway, leading to cellular attachment and filopodia formation. This process aids the subsequent migration and invasion of attached glioblastoma cells through intracellular TGFβR signaling activation. In addition, our in vivo orthotopic glioblastoma model provides further support for the pro-tumoral function of humanin. We observed a correlation between poor survival and aggressive invasiveness in the humanin-treated group, with noticeable tumor protrusions and induced angiogenesis compared to the control. Intriguingly, the in vivo effect of humanin on glioblastoma was significantly reduced by the treatment of TGFBR1 inhibitor. To strengthen these findings, public database analysis revealed a significant association between genes in the ITGAV–TGFβR axis and poor prognosis in glioblastoma patients. These results collectively highlight humanin as a pro-tumoral factor, making it a promising biological target for treating glioblastoma.

Dental pulp mesenchymal stem cells-response to fibrin hydrogel reveals ITGA2 and MMPs expression

Regenerative endodontic procedures (REP) aim at reestablishing tooth vitality by replacing the irreversibly damaged dental pulp removed by the dental practitioner with a new functional one. The current treatment of advanced caries relies on the replacement of the inflamed or necrosed dental pulp with an inert filling material. This leads to a functional but non-vital tooth, which lacks the ability to sense dental tissue damage, and to protect from further bacterial attack. Therapeutic strategies inspired by tissue engineering called REP propose to regenerate a fully functional dental pulp directly in the canal space. Promising results were obtained using dental pulp mesenchymal stem cells (DP-MSCs) in combination with bio-inspired artificial and temporary 3D hydrogels made of extracellular matrix molecules such as collagen and fibrin biomacromolecules. However, the uncontrolled mechanisms of DP regeneration from DP-MSCs in 3D biomacromolecules fail to regenerate a fully functional DP and can induce fibrotic scarring or mineralized tissue formation to a non-negligible extent. The lack of knowledge regarding the early molecular mechanisms initiated by DP-MSCs seeded in ECM-made hydrogels is a scientific lock for REP. In this study, we investigated the early DP-MSC–response in a 3D fibrin hydrogel. DP-MSCs isolated from human third molars were cultured for 24 h in the fibrin hydrogel. The differential transcript levels of extracellular and cell surface genes were screened with 84-gene PCR array. Out of the 84 genes screened, 9 were found to be overexpressed, including those coding for the integrin alpha 2 subunit, the collagenase MMP1 and stromelysins MMP3, MMP10 and MMP12. Over-expression of ITGA2 was confirmed by RT-qPCR. The expression of alpha 2 integrin subunit protein was assessed over time by immunoblot and immunofluorescence staining. The increase in the transcript level of MMP1, MMP3, MM10 and MMP12 was confirmed by RT-qPCR. The overexpression of MMP1 and 3 at the protein level was assessed by immunoblot. MMP3 expression by DP-MSCs was observed by immunofluorescence staining. This work demonstrates overexpression of ITGA2 and of MMP1, 3, 10 and 12 by DP-MSCs cultured in a fibrin hydrogel. The main preliminary extracellular and cell surface response of the DP-MSCs to fibrin hydrogel seems to rely on a ITGA2/MMP3 axis. Further investigations are needed to precisely decipher the role of this axis in dental pulp tissue building. Nevertheless, this work identifies extracellular and cell surface molecules that could be potential checkpoints to be targeted to guide proper dental pulp tissue regeneration.

Proteomic analysis of plasma at the preterminal stage of rhesus nonhuman primates exposed to a lethal total-body dose of gamma-radiation

The identification and validation of radiation biomarkers is critical for assessing the radiation dose received in exposed individuals and for developing radiation medical countermeasures that can be used to treat acute radiation syndrome (ARS). Additionally, a fundamental understanding of the effects of radiation injury could further aid in the identification and development of therapeutic targets for mitigating radiation damage. In this study, blood samples were collected from fourteen male nonhuman primates (NHPs) that were exposed to 7.2 Gy ionizing radiation at various time points (seven days prior to irradiation; 1, 13, and 25 days post-irradiation; and immediately prior to the euthanasia of moribund (preterminal) animals). Plasma was isolated from these samples and was analyzed using a liquid chromatography tandem mass spectrometry approach in an effort to determine the effects of radiation on plasma proteomic profiles. The primary objective was to determine if the radiation-induced expression of specific proteins could serve as an early predictor for health decline leading to a preterminal phenotype. Our results suggest that radiation induced a complex temporal response in which some features exhibit upregulation while others trend downward. These statistically significantly altered features varied from pre-irradiation levels by as much as tenfold. Specifically, we found the expression of integrin alpha and thrombospondin correlated in peripheral blood with the preterminal stage. The differential expression of these proteins implicates dysregulation of biological processes such as hemostasis, inflammation, and immune response that could be leveraged for mitigating radiation-induced adverse effects.

Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

BackgroundNon-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20–30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression.MethodsThe mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined.ResultsThe protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect.ConclusionsThe Integrin αVβ1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

Hsa_circ_0000518 stimulates the malignant progression of hepatocellular carcinoma via regulating ITGA5 to activate the Warburg effect

Studies have shown that the abnormal expression of circular RNA (circRNA) is inextricably linked to hepatocellular carcinoma (HCC). Recently, hsa_circ_0000518 (circ_0000518) was discovered in many cancer progressions. However, its function in HCC is still unclear. Through GEO database analysis combined with gene expression detection of HCC related clinical samples and cell lines, we identified that circ_0000518 was abnormally overexpressed in HCC. Cell and animal model experiments jointly indicated that circ_0000518 can stimulate HCC cell proliferation, migration, invasion and suppress apoptosis. Furthermore, we also found that knocking down the circ_0000518 could inhibit the Warburg effect in HCC cells. Mechanistically, circ_0000518 was found to be primarily localized in the cytoplasm, and sponge hsa-miR-326 (miR-326) promoted integrin alpha 5 (ITGA5) expression. In addition, circ_0000518 could enhance the stability of HuR-mediated ITGA5 mRNA, thereby activating the Warburg effect. In conclusion, this study elucidated that circ_0000518 was a cancer-promoting circRNA, which could enhance ITGA5 expression through competing endogenous RNAs (ceRNA) and RNA Binding Protein (RBP) mechanisms, thus facilitating the development of HCC. It provides a meaningful diagnostic and therapeutic target for HCC.

Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice.

Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-β1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-β1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-β1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications.

The effect of folic acid supplementation and folate deficiency on embryo implantation

Implantation is a critical stage of pregnancy, which occurs in a short period of interaction between the receptive endometrium and the embryo. Folic acid (FA) is a synthetic derivative of folate and is recommended as a pre-conceptional supplement. However, the impact of different doses of FA supplementation and folate deficiency during the early stages of pregnancy requires further investigation. The aim of this study was to investigate the possible effects of FA supplementation and folate deficiency on expression of Estrogen Receptor Alpha (ER-α), Vascular Endothelial Growth Factor-A (VEGFA), and Integrin alpha V and beta3 (Integrin αVβ3). A total of 32, 6–8-week old Wistar albino rats were divided into four groups of control, folate-deficiency, low-dose, and high-dose FA supplement groups. After five weeks of FA supplementation and folate deficiency model formation, mated rats were sacrificed on the 5th gestational day (GD), and implantation sites were collected. The expression of ER- α, VEGFA, and Integrin αVβ3 in the implantation sites were examined with immunohistochemistry and real-time PCR. The results revealed that the mRNA levels of ESR1, VEGFA, and Integrin αV and β3 were significantly increased in the high-dose FA group and significantly decreased in the folate deficiency group compared to the control group (p < 0.05). Based on these results, it can be concluded that FA supplementation before pregnancy has positive effects on the maintenance of pregnancy, and folate deficiency may lead to implantation disorders.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Landscape of extrachromosomal circular DNAs, transcriptome, and proteome analysis reveals insights into alcoholic liver cirrhosis

Alcoholic liver cirrhosis (ALC) is a result of excessive and chronic alcohol consumption. Because alchol can cause DNA damage, extrachromosomal circular DNA (eccDNA) was investigated in ALC liver due to it can be a result of DNA damage. Considering eccDNA has ability to lead to genomic instability as an enhancer of gene transcription, we utilized Circle-Seq to identify differences in eccDNA profiles and gene expression patterns in liver samples obtained from ALC patients (n = 3) and healthy controls (n = 3) to investigate the role of eccDNA in the development of ALC. The abundance of eccDNA in ALC (mean = 13,349) were higher than the healthy control (mean = 11,557) without significant difference (pvalue = 0.6530). We observed 1,032 eccDNA containing genes showed higher expression in ALC patients compared to healthy controls (p < 0.05, log2FC > 1). Notably, we discovered seven genes that exhibited a significant positive correlation between eccDNA abundance and gene expression levels. These genes include A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2), Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C), Protein TANC1 (TANC1), Integrin alpha-2 (ITGA2), EH domain-containing protein 4 (EHD4), Phosphofurin acidic cluster sorting protein 1 (PACS1), and Neuron navigator 2 (NAV2). Through mass spectrometry proteomics, ITGA2 were found to have significantly higher abbudance in ALC. Integrins are a family of proteins plays key roles in the fibrosis development of liver. Thus, our study opens a new perspective for liver fibrosis development.