Focal Adhesion Signaling Research Articles

RNA-seq analysis of the biological process and regulatory signal of TGF-β1-mediated changes in ovarian granulosa cells in small-tail Han sheep

Transforming growth factor beta-1 (TGF-β1) regulates the proliferation of ovarian granulosa cells and participates in follicular development in small-tail Han sheep via the SMAD pathway. However, which additional biological processes and regulatory mechanisms are involved in TGF-β1-mediated regulation of granulosa cell changes remains unknown. In this study, TGF-β1-treated (10 ng/mL) ovarian granulosa cells of small-tail Han sheep were used as the model, RNA-Seq was employed to screen differentially expressed genes (DEGs), and rescue experiments were used to verify selected key pathways. In total, 1179 upregulated and 873 downregulated DEGs were screened using RNA-Seq. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly involved in the biological processes of cell adhesion, cell migration, cell cycle, cell proliferation and apoptosis, and endocrine regulation. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the DEGs were primarily associated with pathways relating to ECM-receptor interaction, PI3K-AKT, focal adhesion, MAPK, TNF, and FOXO signaling, among others. The addition of doramapimod confirmed that the p38 pathway participates in the TGF-β1-induced proliferation and apoptosis of ovarian granulosa cells as well as the regulation of steroid hormone secretion. These results revealed a novel TGF-β1/p38 pathway-mediated mechanism that induces both the proliferation and apoptosis of ovarian granulosa cells. Our findings provide a basis for better understanding the genetic mechanism of TGF-β1 action in follicle development.

Transcriptomic response of overexpression ZNF32 in breast cancer cells

Breast cancer is one of the deadliest malignancies in women worldwide. Zinc finger protein 32 (ZNF32) has been reported to be involved in autophagy and stem cell like properties of breast cancer cells. However, the effects, mechanisms, target genes and pathways of ZNF32 in breast cancer development have not been fully explored. In this study, stable ZNF32 overexpression breast cancer cell line was generated, and we used RNA-seq and RT-qPCR to quantify and verify the changes in transcription levels in breast cancer cells under ZNF32 overexpression. Transcriptome analysis showed that high expression of ZNF32 is accompanied by changes in downstream focal adhesion, ECM-receptor interaction, PI3K-AKT, HIPPO and TNF signaling pathways, which are critical for the occurrence and development of cancer. Multiple differentially expressed genes (DEGs) were significantly involved in cell proliferation, adhesion and migration, including 11 DEGs such as CA9, CRLF1 and ENPP2P with fundamental change of regulation modes. All the 11 DEGs were validated by RT-qPCR, and 9 of them contained potential transcriptional binding sequences of ZNF32 in their promoter region. This study provides a holistic perspective on the role and molecular mechanism of ZNF32 in breast cancer progression.

Macrophage-Specific Lactate Dehydrogenase Expression Modulates Inflammatory Function In Vitro.

In acute kidney injury, macrophages play a major role in regulating inflammation. Classically activated macrophages (M1) undergo drastic metabolic reprogramming during their differentiation and upregulate the aerobic glycolysis pathway to fulfill their pro-inflammatory functions. NAD+ regeneration is crucial for the maintenance of glycolysis and the most direct pathway by which this occurs is via the fermentation of pyruvate to lactate, catalyzed by lactate dehydrogenase A (LDHA). Our previous study determined that LDHA is predominantly expressed in the proximal segments of the nephron in the mouse kidney and increases with hypoxia. This study investigates the potential of LDHA as a therapeutic target for inflammation by exploring its role in macrophage function in vitro. Bone-marrow-derived macrophages (BMDMs) were isolated from myeloid-specific LDHA knockout mice derived from crossbreeding LysM-Cre transgenic mice and LDHA floxed mice. RNA sequencing and LC-MS/MS metabolomics analyses were used in this study to determine the effect of LDHA deletion on BMDM following stimulation with IFN-γ. LDHA deletion in IFN-γ BMDMs resulted in a significant alteration of the macrophage activation and functional pathways, and change in glycolytic, cytokine, and chemokine gene expression. Metabolite concentrations associated with pro-inflammatory macrophage profiles were diminished while anti-inflammatory-associated ones were increased in LDHA KO BMDMs. Glutamate and amino sugars metabolic pathways were significantly affected by the LDHA deletion. A combined muti-omics analysis highlighted changes in Rap1 signaling, cytokine-cytokine receptor interaction, focal adhesion, and MAPK signaling metabolism pathways. Deletion of LDHA in macrophages results in a notable reduction in the pro-inflammatory profile and concurrent upregulation of anti-inflammatory pathways. These findings suggest that LDHA could serve as a promising therapeutic target for inflammation, a key contributor to the pathogenesis of acute kidney injury.

Dan’e fukang decoction reduces hemorrhage in a rat model of mifepristone induced incomplete abortion and may correlate with cell adhesion molecule signaling interference

Ethnopharmacological relevanceDan’e fukang decoction (DFD) is a traditional Chinese medicine formula. DFD obtains 10 herbs, including Salvia yunnanensis C.H.Wright (Zidanshen), Curcuma zedoaria (Christm.) Roscoe (Ezhu), Angelica sinensis (Oliv.) Diels (Danggui), Cyperus rotundus L. (Xiangfuzi), Corydalis yanhusuo (Y.H.Chou & Chun C. Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu, Bupleurum marginatum Wall. ex DC. (Yanhusuo), Sparganium stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz. (Sanleng), Panax notoginseng (Burkill) F.H.Chen (Sanqi), Paeonia lactiflora Pall. (Shaoyao) and Glycyrrhiza uralensis Fisch. (Gancao). DFD is now clinically used for the treatment of menstrual irregularities, dysmenorrhea and menstrual discomfort caused by blood stasis and easing of endometriosis. Based on this, it is reasonable to presume that DFD may be effective in treating incomplete abortion and reducing postpartum bleeding, but no specific studies have been reported so far. Aim of the studyTo investigate the efficacy of Dan’e fukang decoction (DFD) in reducing prolonged vaginal bleeding followed by mifepristone induced incomplete abortion and explore the mechanisms of action of DFD in treating incomplete abortion. MethodsAn incomplete abortion model of rat was established by single intragastrically administered 8.5 mg/kg mifepristone on the 7th day of pregnancy. From the 8th day of pregnancy, the abortive rats were administered solvent, a positive control drug, or different doses of DFD, respectively for seven consecutive days. The efficacy of DFD was assessed by measuring the vaginal bleeding volume of the rats. Four coagulation parameters and platelet counts were measured. Hematoxylin and eosin (HE) staining was performed to evaluate pathological changes in the uterine embryos. Serum levels of progesterone and estrogen were measured using ELISA. Network pharmacology and transcriptomics were used to predict potential targets and pathways for DFD to reduce hemorrhage. The levels of mRNA related to cell adhesion molecules (CAMs) were detected by RT-qPCR. The levels of progesterone and estrogen receptors and the proteins associated with CAMs pathway in uterine tissues were detected by Western Blot. ResultsDFD significantly reduced the volume of vaginal bleeding of the abortive rats and significantly downgraded the pathological scores of uterine embryos. DFD significantly increased serum levels of E2, and had no impact on serum levels of P4 and the protein expression of ER and PR in the uteri of the abortive rats. Pathways in cancer, lipid, focal adhesion and immune-related signaling were predicted to be influenced by DFD via the analysis of network pharmacology. The CAMs signaling was found the most critical pathway regulated by both mifepristone and DFD via RNA-seq assay, followed by axon guidance, basal cell carcinoma, hippo signaling pathway and neuroactive ligand-receptor interaction. Combining the two analytical methods, ICAM-1 was predicted likely the key targeted gene by DFD. Finally, DFD was validated to decrease the protein expression of ICAM-1, ITGB2, ITGB7 and RASSF5 in the uterine tissues, which correlated to suppress the CAMs signaling pathway. ConclusionDFD significantly reduced hemorrhage. DFD significantly increased the serum levels of E2 and inhibited CAMs signaling pathway, which was likely to be involved in the mechanism of action of DFD facilitating residual uterine embryo expulsion in the rat model of incomplete abortion.

MYO6 contributes to tumor progression and enzalutamide resistance in castration-resistant prostate cancer by activating the focal adhesion signaling pathway

BackgroundEnzalutamide (Enz) resistance is a poor prognostic factor for patients with castration-resistant prostate cancer (CRPC), which often involves aberrant expression of the androgen receptor (AR). Myosin VI (MYO6), one member of the myosin family, plays an important role in regulating cell survival and is highly expressed in prostate cancer (PCa). However, whether MYO6 is involved in Enz resistance in CRPC and its mechanism remain unclear.MethodsMultiple open-access databases were utilized to examine the relationship between MYO6 expression and PCa progression, and to screen differentially expressed genes (DEGs) and potential signaling pathways associated with the MYO6-regulated Enz resistance. Both in vitro and in vivo tumorigenesis assays were employed to examine the impact of MYO6 on the growth and Enz resistance of PCa cells. Human PCa tissues and related clinical biochemical data were utilized to identify the role of MYO6 in promoting PCa progression and Enz resistance. The molecular mechanisms underlying the regulation of gene expression, PCa progression, and Enz resistance in CRPC by MYO6 were investigated.ResultsMYO6 expression increases in patients with PCa and is positively correlated with AR expression in PCa cell lines and tissues. Overexpression of AR increases MYO6 expression to promote PCa cell proliferation, migration and invasion, and to inhibit PCa cell apoptosis; whereas knockdown of MYO6 expression reverses these outcomes and enhances Enz function in suppressing the proliferation of the Enz- sensitive and resistant PCa cells both in vitro and in vivo. Mechanistically, AR binds directly to the promoter region (residues − 503 to − 283 base pairs) of MYO6 gene and promotes its transcription. Furthermore, MYO6 activates focal adhesion kinase (FAK) phosphorylation at tyrosine-397 through integrin beta 8 (ITGB8) modulation to promote PCa progression and Enz resistance. Notably, inhibition of FAK activity by Y15, an inhibitor of FAK, can resensitize CRPC cells to Enz treatment in cell lines and mouse xenograft models.ConclusionsMYO6 has pro-tumor and Enz-resistant effects in CRPC, suggesting that targeting MYO6 may be beneficial for ENZ-resistant CRPC therapy through the AR/MYO6/FAK signaling pathway.

Single Nuclei Transcriptomics Reveals Obesity-Induced Endothelial and Neurovascular Dysfunction: Implications for Cognitive Decline.

Obesity confers risk for cardiovascular disease and vascular dementia. However, genomic alterations modulated by obesity in endothelial cells in the brain and their relationship to other neurovascular unit (NVU) cells are unknown. We performed single nuclei RNA sequencing (snRNAseq) of the NVU (endothelial cells, astrocytes, microglia, and neurons) from the hippocampus of obese (ob/ob) and wild-type (WT) male mice to characterize obesity-induced transcriptomic changes in a key brain memory center and assessed blood-brain barrier permeability (BBB) by gadolinium-enhanced magnetic resonance imaging (MRI). Ob/ob mice displayed obesity, hyperinsulinemia, and impaired glucose tolerance. snRNAseq profiled 14 distinct cell types and 32 clusters within the hippocampus of ob/ob and WT mice and uncovered differentially expressed genes (DEGs) in all NVU cell types, namely, 4462 in neurons, 1386 in astrocytes, 125 in endothelial cells, and 154 in microglia. Gene ontology analysis identified important biological processes such as angiogenesis in endothelial cells and synaptic trafficking in neurons. Cellular pathway analysis included focal adhesion and insulin signaling, which were common to all NVU cell types. Correlation analysis revealed significant positive correlations between endothelial cells and other NVU cell types. Differentially expressed long non-coding RNAs (lncRNAs) were observed in cells of the NVU-affecting pathways such as TNF and mTOR. BBB permeability showed a trend toward increased signal intensity in ob/ob mice. Taken together, our study provides in-depth insight into the molecular mechanisms underlying cognitive dysfunction in obesity and may have implications for therapeutic gene targeting.

Prognostic signature and immunotherapeutic relevance of Focal adhesion signaling pathway-related genes in osteosarcoma

BackgroundAs the most common primary malignant bone tumor in children and adolescents, osteosarcoma currently lacks an effective clinical cure. Focal adhesion plays a crucial role in tumor invasion, migration, and drug resistance by mediating communication between the extracellular matrix and tumor cells. This study investigated the prognostic features and immunotherapeutic relevance of focal adhesion pathway-related genes in osteosarcoma to aid in the development of new therapeutic options. MethodsWe obtained mutational, transcriptomic, gene expression, and clinical data of osteosarcoma patients from the Gene Expression Omnibus (GEO) and Therapeutically Applicable Research to Generate Effective (TARGET) databases. Differentially expressed genes were screened, followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses. Kaplan-Meier survival analysis was performed for genes related to the focal adhesion pathway, and multivariate Cox regression analysis was employed to construct a prognostic signature model. Genes such as SIGLEC15, TIGIT, CD274, HAVCR2, PDCD1, CTLA4, and LAG3 were extracted from the TARGET and CCLE databases for osteosarcoma patients and osteosarcoma cell lines, respectively，to observe the expression of immune checkpoint-related genes. Finally, qRT-PCR was used to verify the expression of these immune checkpoint-related genes in osteosarcoma cell lines. ResultsIn our study, 376 samples were analyzed, including 369 osteosarcoma samples and 7 normal tissue samples. We identified 50 up-regulated and 28 down-regulated differentially expressed genes. Among these, 10 Candidate genes relative to focal Adhesion were selected， and CAV1, ZYX, and ITGA5 were found to have a significant prognostic role based on survival analysis of osteosarcoma samples from the TARGET database. A predictive signature model related to the focal adhesion signaling pathway was constructed using these genes, and the AUCs of the 1-year, 3-year, and 5-year ROC curves were 0. 647, 0. 712, and 0. 717, respectively. The overall survival (OS) rate of osteosarcoma patients with high-risk scores was poorer than those with low-risk scores. Then, samples were divided into two subgroups based on the expression of the three genes, revealing significant differences in the expression of certain immune checkpoint-related genes between the subgroups. Additionally, above three genes and immune checkpoint-related genes in osteosarcoma cell lines were extracted from the CCLE database, showing high expression levels in eight osteosarcoma cell lines. We observed that CD274 and PDCD1LG2 were highly expressed in some osteosarcoma cell lines. Finally, the expression of CAV1, ZYX, ITGA5, CD80, CD274, and PDCD1LG2 in osteosarcoma cell lines was verified by qRT-PCR. ConclusionsOur study validated the prognostic role of three focal adhesion pathway-related genes (ZYX, CAV1, and ITGA5) in patients with osteosarcoma and constructed a prognostic signature model associated with the focal adhesion signaling pathway. We identified significant differences in the expression of multiple immune checkpoint-related genes among subgroups defined by the three genes. Additionally, CD274 and PDCD1LG2 showed higher expression in osteosarcoma cell lines characterized by these genes. These findings may aid in the selection of effective immunotherapy for specific osteosarcoma patients.

Maslinic acid prevented lipopolysaccharide‐induced injury of IPEC‐J2 cells through regulating PTEN‐FAK signaling pathway

AbstractIntestinal epithelial injury is one of the typical symptoms associated with intestinal inflammation and diarrhea, and the repair of the intestinal epithelium intricately linked to cell migration. Here, we test the hypothesis that maslinic acid (MA) regulates porcine intestinal epithelial cell migration by inhibiting focal adhesion kinase (FAK)/AKT signaling pathway. In this experiment, the optimal concentration of MA (0.5 μg/mL) on IPEC‐J2 cell viability was selected to investigate the effect under low‐dose lipopolysaccharide (LPS) (1 μg/mL) conditions. Transcriptome sequencing and polymerase chain reaction array results revealed that MA could alleviate LPS‐induced the gene expressions decreasing in focal adhesion signaling pathway. From the pathway map analysis and western blot analysis results, MA alleviated the LPS‐induced decrease in FAK protein expression mainly by promoting FAK protein phosphorylation, which in turn alleviated the decrease in cell migration and formation of cytoskeleton protein Vinculin and F‐actin, the above results were verified by FAK phosphorylation inhibitors Defactinib. The molecular docking and immunoprecipitation further verified that MA could bind to PTEN protein and significantly inhibit its interaction with FAK protein, blocking the function of PTEN to inhibit FAK phosphorylation finally shown to promote the level of FAK phosphorylation, meanwhile LPS inhibited FAK protein expression and its binding to PKC and PTEN proteins. Our study revealed the role of MA and LPS in FAK protein, and increased understanding of MA anti‐inflammatory mechanism.

Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and inter-tumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury-response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K targeted gRNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury-response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury-response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of β1 integrin, FAK, MEK and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity.

Cooperation between SIX1 and DHX9 transcriptionally regulates integrin-focal adhesion signaling mediated metastasis and sunitinib resistance in KIRC.

High invasive capacity and acquired tyrosine kinase inhibitors (TKI) resistance of kidney renal clear cell carcinoma (KIRC) cells remain obstacles to prolonging the survival time of patients with advanced KIRC. In the present study, we reported that sine oculis homeobox 1 (SIX1) was upregulated in sunitinib-resistant KIRC cells and metastatic KIRC tissues. Subsequently, we found that SIX1 mediated metastasis and sunitinib resistance via Focal adhesion (FA) signaling, and knockdown of SIX1 enhanced the antitumor efficiency of sunitinib in KIRC. Mechanistically, Integrin subunit beta 1 (ITGB1), an upstream gene of FA signaling, was a direct transcriptional target of SIX1. In addition, we showed that DExH-box helicase 9 (DHX9) was an important mediator for SIX1-induced ITGB1 transcription, and silencing the subunits of SIX1/DHX9 complex significantly reduced transcription of ITGB1. Downregulation of SIX1 attenuated nuclear translocation of DHX9 and abrogated the binding of DHX9 to ITGB1 promoter. Collectively, our results unveiled a new signal axis SIX1/ITGB1/FAK in KIRC and identified a novel therapeutic strategy for metastatic KIRC patients.

Hydroxyapatite nanoparticle improves ovine oocyte developmental capacity by alleviating oxidative stress in response to vitrification stimuli

The wide application of ovine oocyte vitrification is limited by its relatively low efficiency. Nanoparticle is potentially to be used in cryopreservation technology for its unique characteristics with high biocompatibility, potent antioxidant property as well as superiority in membrane permeation and heat transduction. However, the effect of nanoparticle on ovine oocyte cryopreservation as well as the underlying mechanism has not been systematically evaluated. The objective of this study was to investigate the impact of nanoparticles on ovine oocytes cryopreservation and further identify the underlying mechanism. Firstly, the effects of Hydroxyapatite (HA) and Fe3O4 nanoparticles on the developmental potential of vitrified ovine oocytes were determined, and the results showed that neither HA (VC = 85.95 ± 6.23 % vs. VH = 92.47 ± 8.11 %, P > 0.05) nor Fe3O4 (VC = 85.95 ± 6.23 % vs. VF = 89.39 ± 6.32 %, P > 0.05) had adverse effect on the survival rate of vitrified-thawed oocytes. Notably, both HA (VC = 77.78 ± 0.09 % vs. VH = 44.00 ± 0.09 %, P＜0.01) and Fe3O4 (VC = 77.78 ± 0.09 % vs. VF = 51.67 ± 0.15 %, P＜0.01) nanoparticles effectively reduced the level of oocyte apoptosis after freezing and thawing. What's more, HA could significantly improve the cleavage rate of frozen oocytes (VC = 33.79 ± 2.83 % vs. VH = 59.54 ± 4.13 %, P＜0.05). Moreover, reduced reactive oxygen species (ROS) level (VC = 13.66 ± 0.47 vs. VH = 12.61 ± 0.53, P < 0.05), increased glutathione (GSH) content (VC = 60.69 ± 7.89 vs. VH = 87.92 ± 1.05, P < 0.05) and elevated mitochondrial membrane potential (MMP) level (VC = 1.43 ± 0.04 vs. VH = 1.63 ± 0.01，P＜0.01) were observed in oocytes treated with HA nanoparticles when compared with that of the control group. Furthermore, Smart-RNA sequence technology was utilized to identify differentially expressed mRNAs (DEMs) induced by nanoparticles during cryopreservation. When compared with the control counterparts, a total of 721 DEMs (309 up-regulated and 412 down-regulated mRNAs) were identified in oocytes treated with HA, while 702 DEMs (480 up-regulated and 222 down-regulated mRNAs) were identified in oocytes treated with Fe3O4. A comparison of DEMs showed that total 692 mRNAs were expressed in oocytes treated with HA and Fe3O4. Notably, we discovered that 15 mRNAs were specially highly expressed in oocytes treated with HA, and Focal adhesion signaling pathway mainly contributed to the improved ovine oocyte quality after vitrification by alleviating oxidative stress.

Lipidomic and Proteomic Insights from Extracellular Vesicles in Postmortem Dorsolateral Prefrontal Cortex Reveal Substance Use Disorder-Induced Brain Changes.

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to glial activation and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmann's area 9 (BA9) in postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n=4, SUD: n=4). The EVs were analyzed for physical properties (concentration, size, zeta potential, morphology) and subjected to integrative multi-omics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells as well as their effects on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between SUD and control groups. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD EVs. Proteomic analysis indicates downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB, and upregulation of C4A, C3, and ALB in SUD EVs. Gene ontology and protein-protein interactome analyses highlight functions such as cell motility, focal adhesion, and acute phase response signaling that is associated with the identified proteins. Both control and SUD EVs increased C3 and C4 mRNA expression in microglia, but only SUD EVs upregulated these genes in astrocytes. SUD EVs also significantly enhanced microglial migration in a wound healing assay.This study successfully isolated EVs from postmortem brains and used a multi-omics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD EVs and the distinct effects of EVs on glial cells suggest a crucial role in acute phase response signaling and neuroinflammation.

Genomic landscape of hepatocellular carcinoma in Egyptian patients by whole exome sequencing

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver cancer. Chronic hepatitis and liver cirrhosis lead to accumulation of genetic alterations driving HCC pathogenesis. This study is designed to explore genomic landscape of HCC in Egyptian patients by whole exome sequencing.MethodsWhole exome sequencing using Ion Torrent was done on 13 HCC patients, who underwent surgical intervention (7 patients underwent living donor liver transplantation (LDLT) and 6 patients had surgical resection}.ResultsMutational signature was mostly S1, S5, S6, and S12 in HCC. Analysis of highly mutated genes in both HCC and Non-HCC revealed the presence of highly mutated genes in HCC (AHNAK2, MUC6, MUC16, TTN, ZNF17, FLG, MUC12, OBSCN, PDE4DIP, MUC5b, and HYDIN). Among the 26 significantly mutated HCC genes—identified across 10 genome sequencing studies—in addition to TCGA, APOB and RP1L1 showed the highest number of mutations in both HCC and Non-HCC tissues. Tier 1, Tier 2 variants in TCGA SMGs in HCC and Non-HCC (TP53, PIK3CA, CDKN2A, and BAP1). Cancer Genome Landscape analysis revealed Tier 1 and Tier 2 variants in HCC (MSH2) and in Non-HCC (KMT2D and ATM). For KEGG analysis, the significantly annotated clusters in HCC were Notch signaling, Wnt signaling, PI3K-AKT pathway, Hippo signaling, Apelin signaling, Hedgehog (Hh) signaling, and MAPK signaling, in addition to ECM-receptor interaction, focal adhesion, and calcium signaling. Tier 1 and Tier 2 variants KIT, KMT2D, NOTCH1, KMT2C, PIK3CA, KIT, SMARCA4, ATM, PTEN, MSH2, and PTCH1 were low frequency variants in both HCC and Non-HCC.ConclusionOur results are in accordance with previous studies in HCC regarding highly mutated genes, TCGA and specifically enriched pathways in HCC. Analysis for clinical interpretation of variants revealed the presence of Tier 1 and Tier 2 variants that represent potential clinically actionable targets. The use of sequencing techniques to detect structural variants and novel techniques as single cell sequencing together with multiomics transcriptomics, metagenomics will integrate the molecular pathogenesis of HCC in Egyptian patients.

Dachsous cadherin related 1 (DCHS1) is a novel biomarker for immune infiltration and epithelial-mesenchymal transition in endometrial cancer via pan-cancer analysis

BackgroundDachsous cadherin related 1 (DCHS1) is one of calcium-dependent adhesion membrane proteins and is mainly involved in the development of mammalian tissues. There is a lack of more detailed research on the biological function of DCHS1 in pan-cancer.Materials and methodsWe evaluated the expression, the prognostic value, the diagnostic value and genomic alterations of DCHS1 by using the databases, including TCGA, UALCAN, HPA, GEPIA2.0 and GSCA. We employed the databases of UCSC, TIMER2.0, TISIDB, GSCA to analyze the association between DCHS1 expression and the immune microenvironment, stemness, TMB, MSI and anticancer drug sensitivity. BioGRID, STRING and GEPIA2.0 were used to perform protein interaction and functional enrichment analysis. Real-time quantitative PCR, CCK8, Transwell assay and Western blot were performed to determine the function of DCHS1 in UCEC.ResultsDCHS1 is differentially expressed in many cancers and its expression is significantly associated with tumor prognosis and diagnosis. DCHS1 expression was significantly correlated with the infiltration of cancer-associated fibroblasts (CAFs), Endothelial cell (ECs), and Hematopoietic stem cell in most cancers. In addition, DCHS1 was significantly associated with sensitivity to many antitumor drugs. Functional enrichment analysis revealed that DCHS1-related proteins were involved in Focal adhesion, Endometrial cancer and Wnt signaling pathway. GSEA results showed that DCHS1 was related to epithelial-mesenchymal transition (EMT) in many cancers. In vitro experiments in UCEC showed that DCHS1 regulated cell proliferation, migration and EMT.ConclusionsOur findings indicated that DCHS1 might be a novel prognostic and diagnostic biomarker and immunotherapy target, and plays an important role in the proliferation, migration and EMT in UCEC.

Targeted multiplex validation of CSF proteomic biomarkers: implications for differentiation of PCNSL from tumor-free controls and other brain tumors.

Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin's lymphoma that affects brain parenchyma, eyes, cerebrospinal fluid, and spinal cord. Diagnosing PCNSL can be challenging because imaging studies often show similar patterns as other brain tumors, and stereotactic brain lesion biopsy conformation is invasive and not always possible. This study aimed to validate a previous proteomic profiling (PMID: 32610669) of cerebrospinal fluid (CSF) and develop a CSF-based proteomic panel for accurate PCNSL diagnosis and differentiation. CSF samples were collected from patients of 30 PCNSL, 30 other brain tumors, and 31 tumor-free/benign controls. Liquid chromatography tandem-mass spectrometry targeted proteomics analysis was used to establish CSF-based proteomic panels. Final proteomic panels were selected and optimized to diagnose PCNSL from tumor-free controls or other brain tumor lesions with an area under the curve (AUC) of 0.873 (95%CI: 0.723-0.948) and 0.937 (95%CI: 0.807- 0.985), respectively. Pathways analysis showed diagnosis panel features were significantly enriched in pathways related to extracellular matrices-receptor interaction, focal adhesion, and PI3K-Akt signaling, while prion disease, mineral absorption and HIF-1 signaling were significantly enriched with differentiation panel features. This study suggests an accurate clinical test panel for PCNSL diagnosis and differentiation with CSF-based proteomic signatures, which may help overcome the challenges of current diagnostic methods and improve patient outcomes.

MicroRNA-330-5p Mediates the TDRG1-Regulated Myocardial Inflammation and Apoptosis after Myocardial Infarction by Inhibiting MAPK1.

Acute myocardial infarction (AMI) is a cardiovascular illness with the highest disability and mortality rates worldwide. This study aimed to estimate the mechanism of TDRG1 in myocardial damage.qRT-PCR was used to study the levels of TDRG1. After establishing hypoxia/reoxygenation (H/R) model, the inflammation was assessed by qRT-PCR, oxidation was detected by commercial kits, and apoptosis was estimated by qRT-PCR and flow cytometry. The luciferase intensity and RNA immunoprecipitation assay were detected for the identification of target relationship. The functional enrichment was unveiled by GO and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein interaction was conducted for screening key genes.The expression of TDRG1 was elevated and negatively correlated with miR-330-5p in the serum AMI patients. TDRG1/miR-330-5p axis regulated inflammation, oxidation, and viability and apoptosis of HL-1 cells induced by H/R. GO and KEGG analyses indicate that 76 overlapping targets of miR-330-5p were primarily involved in focal adhesion, calmodulin binding, and ErbB and Rap1 signaling pathways. MAPK1 was the top key gene and was a target gene of miR-330-5p.TDRG1/miR-330-5p axis could participate in the regulation of apoptosis and inflammation of H/R-induced cardiomyocytes.

Identification of key genes involved in collagen hydrogel-induced chondrogenic differentiation of mesenchymal stem cells through transcriptome analysis: the role of m6A modification

Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration.Graphical

C-Src-induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Identification of diagnose related therapeutic targets of Danggui buxue decoction in Parkinson’s disease

BackgroundParkinson’s disease (PD) is the fastest growing neurological disease. Currently, there is no disease-modifying therapy to slow the progression of the disease. Danggui buxue decoction (DBD) is widely used in the clinic because of its therapeutic effect. However, little is known about the molecular mechanism of DBD against PD. This study intends to explore the possible molecular mechanisms involved in DBD treatment of PD based on network pharmacology, and provide potential research directions for future research. MethodsFirstly, the active components and target genes of DBD were screened from the traditional Chinese medicine systems pharmacology (TCMSP), DrugBank and UniProt database. Secondly, target genes of PD were identified from the (GEO) dataset, followed by identification of common target genes of DBD and PD. Thirdly, analysis of protein–protein interaction (PPI), functional enrichment and diagnosis was performed on common target genes, followed by correlation analysis between core target genes, immune cell, miRNAs, and transcription factors (TFs). Finally, molecular docking between core target genes and active components, and real-time PCR were performed. ResultsA total of 72 common target genes were identified between target genes of DBD and target genes of PD. Among which, 11 target genes with potential diagnostic value were further identified, including TP53, AKT1, IL1B, MMP9, NOS3, RELA, MAPK14, HMOX1, TGFB1, NOS2, and ERBB2. The combinations with the best docking binding were identified, including kaempferol-AKT1/HMOX1/NOS2/NOS3, quercetin-AKT1/ERBB2/IL1B/HMOX1/MMP9/TP53/NOS3/TGFB1. Moreover, IL1B and NOS2 respectively positively and negatively correlated with neutrophil and Type 1 T helper cell. Some miRNA-core target gene regulatory pairs were identified, such as hsa-miR-185-5p-TP53/TGFB1/RELA/MAPK14/IL1B/ERBB2/AKT1 and hsa-miR-214-3p-NOS3. These core target genes were significantly enriched in focal adhesion, TNF, HIF-1, and ErbB signaling pathway. ConclusionDiagnostic TP53, AKT1, IL1B, MMP9, NOS3, RELA, MAPK14, HMOX1, TGFB1, NOS2, and ERBB2 may be considered as potential therapeutic targets of DBD in the treatment of PD.

Integrin αM promotes macrophage alternative M2 polarization in hyperuricemia-related chronic kidney disease.

Hyperuricemia is an essential risk factor in chronic kidney disease (CKD), while urate-lowering therapy to prevent or delay CKD is controversial. Alternatively activated macrophages in response to local microenvironment play diverse roles in kidney diseases. Here, we aim to investigate whether and how macrophage integrin αM (ITGAM) contributes to hyperuricemia-related CKD. In vivo, we explored dynamic characteristics of renal tissue in hyperuricemia-related CKD mice. By incorporating transcriptomics and phosphoproteomics data, we analyzed gene expression profile, hub genes and potential pathways. In vitro, we validated bioinformatic findings under different conditions with interventions corresponding to core nodes. We found that hyperuricemia-related CKD was characterized by elevated serum uric acid levels, impaired renal function, activation of macrophage alternative (M2) polarization, and kidney fibrosis. Integrated bioinformatic analyses revealed Itgam as the potential core gene, which was associated with focal adhesion signaling. Notably, we confirmed the upregulated expression of macrophage ITGAM, activated pathway, and macrophage M2 polarization in injured kidneys. In vitro, through silencing Itgam, inhibiting p-FAK or p-AKT1 phosphorylation, and concurrent inhibiting of p-FAK while activating p-AKT1 all contributed to the modulation of macrophage M2 polarization. Our results indicated targeting macrophage ITGAM might be a promising therapeutic approach for preventing CKD.