MicroRNAs (miRNAs) are a group of small noncoding RNAs that act as regulators of posttranslational gene/protein expression and are known to play a key role in physiological and pathological processes. The objective of our study was to compare expression of miR-21 in renal tissue from dogs affected with chronic kidney disease (CKD) caused by X-linked hereditary nephropathy (XLHN), a disease equivalent to human Alport syndrome, to that from unaffected dogs. Additionally, we sought to characterize changes in relative mRNA expression of various genes associated with miR-21 function. miRNA was isolated from kidney tissue collected from both affected dogs and unaffected, age-matched littermates at defined milestones of disease progression, including end-stage renal disease (ESRD). Additionally, autopsy samples from affected dogs at ESRD and corresponding unaffected dogs were evaluated. Samples were scored based on histological changes, and relative expression of miR-21 and kidney disease-related genes was determined using quantitative real-time polymerase chain reaction. In affected dogs, significant upregulation of kidney miR-21 was first detected at the milestone corresponding with increased serum creatinine. Furthermore, miR-21 expression correlated significantly with urine protein: urine creatinine ratio, serum creatinine concentration, glomerular filtration rate, and histologic lesions (glomerular damage, tubular damage, chronic inflammation, and fibrosis). At end-stage disease, COL1A1, TGFB1 and its receptor, TGFB2, and Serpine1 were upregulated, while PPARA, PPARGC1A, ACADM, SOD1, and EGF were downregulated. In conclusion, miR-21 is abnormally upregulated in the kidneys of dogs with CKD caused by XLHN, which may play an important pathologic role in the progression of disease by dysregulating multiple pathways.

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases.

Keywords: equine; mesenchymal stem cells; musculoskeletal disorders; regenerative medicine; secretome.

Diabetic nephropathy (DN) is a complication of diabetes mellitus that can lead to proteinuria and a progressive decline in renal function. Endoglin, a co-receptor of TGF-β, is known primarily for regulating endothelial cell function; however, endoglin is also associated with hepatic, cardiac, and intestinal fibrosis. This study investigates whether endoglin contributes to the development of interstitial fibrosis in DN. Kidney autopsy material from 80 diabetic patients was stained for endoglin and Sirius Red and scored semi-quantitatively. Interstitial endoglin expression was increased in samples with DN and was correlated with Sirius Red staining (p < 0.001). Endoglin expression was also correlated with reduced eGFR (p = 0.001), increased creatinine (p < 0.01), increased systolic blood pressure (p < 0.05), hypertension (p < 0.05), and higher IFTA scores (p < 0.001). Biopsy samples from DN patients were also co-immunostained for endoglin together with CD31, CD68, vimentin, or α-SMA Endoglin co-localized with both the endothelial marker CD31 and the myofibroblast marker α-SMA. Finally, we used shRNA to knockdown endoglin expression in a human kidney fibroblast cell line. We found that TGF-β1 stimulation upregulated SERPINE1, CTGF, and ACTA2 mRNA and α-SMA protein, and that these effects were significantly reduced in fibroblasts after endoglin knockdown. Taken together, these data suggest that endoglin plays a role in the pathogenesis of interstitial fibrosis in DN.

Keywords: TGF-β; diabetic nephropathy; endoglin; fibroblast; interstitial fibrosis.

Background: Glaucoma is a leading cause of irreversible blindness. Remodeling of the scleral extracellular matrix (ECM) plays an important role in the development of glaucoma. The aim of this study was to identify the key genes and pathways for the ECM remodeling of sclera in glaucoma by bioinformatics analysis and to explore potential therapeutic agents for glaucoma management. Methods: Genes associated with glaucoma, sclera and ECM remodeling were detected using the text mining tool pubmed2ensembl, and assigned Gene Ontology (GO) biological process terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the GeneCodis program. A protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape, module analysis was performed using the Molecular Complex Detection (MCODE) plugin, and GO and KEGG analyses of the gene modules were performed using the Database of Annotation, Visualization and Integrated Discovery (DAVID) platform. The genes that clustered in the significant module were selected as core genes, and functions and pathways of the core genes were visualized using ClueGO and CluePedia. Lastly, the drug-gene interaction database was used to explore drug-gene interactions of the core genes to find drug candidates for glaucoma. Results: We identified 125 genes common to "Glaucoma", "Sclera", and "ECM remodeling" by text mining. Gene functional enrichment analysis yielded 30 enriched GO terms and 20 associated KEGG pathways. A PPI network that included 60 nodes with 249 edges was constructed, and three gene modules were obtained using the MCODE. We selected 13 genes that clustered in module 1 as core candidate genes that were associated mainly with ECM degradation and cell proliferation and division. The HIF-1 signaling pathway, FOXO signaling pathway, PI3K-Akt signaling pathway and TGFB signaling pathway were found to be enriched. We found that 11 of the 13 selected genes could be targeted by 26 existing drugs. Conclusions: The results showed that VEGFA, TGFB1, TGFB2, TGFB3, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1, and VWF were potential key genes involved to scleral ECM remodeling. Furthermore, 26 drugs were identified as potential therapeutic agents for glaucoma treatment and management.

Keywords: drug discovery; extracellular matrix; glaucoma; sclera; text mining.

Gastric cancer affects millions of people each year; it is the fifth deadliest cancer globally. Due to failure to perform routine tests such as endoscopy, it is usually diagnosed in the invasive stages. Therefore, finding diagnostic biomarkers in blood can help to speed up the initial diagnosis of cancer. This study aimed to find appropriate diagnostic biomarkers in the extracellular matrix of noninvasive to invasive stages of gastric cancer patients, using bioinformatics analysis. First, we selected the appropriate datasets from the GEO database. We evaluated the genes' signaling pathways, biological processes, and molecular functions. More accurately, we assessed the genes, in which their protein products are released into the extracellular matrix; we evaluated their protein network. Then, we validated the candidate proteins in the GEPIA and TCGA databases. The extracellular matrix, tyrosine kinase receptors, and immune response pathways are effective factors, which are related to the highly expressed genes and metabolism; cell cycle pathways are also impressive on low-expression genes. 69 highly expressed proteins are released into the extracellular matrix. After drawing the protein network, 5 proteins were selected as more suitable candidates for further studies. These proteins' expression significantly increases in the human samples, and the survival chart showed up to about 80% mortality in the individuals over time. With integrated bioinformatics analysis, BGN, LOX, MMP-9, SERPINE1, and TGFB1 proteins have been selected as suitable diagnostic biomarkers for noninvasive to invasive stages of gastric cancer. Further studies are needed to evaluate more precise mechanisms between these proteins.

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

The improved effectiveness and safety of the combined antiretroviral therapy (cART) has largely diminished mortality and AIDS-defining morbidity of HIV-patients. Nevertheless, chronic age-related diseases in these individuals are more common and their underlying pathogenic mechanisms of these actions seem to involve accelerated aging and enhanced inflammation. The present study explores markers of these processes in a heterogenous Spanish HIV cohort using peripheral blood samples of HIV-patients and matched uninfected controls. We isolated mononuclear cells (PBMCs) and i) compared the expression of a panel of 14 genes related to inflammation and senescence in PBMCs of HIV-patients vs matched uninfected controls, ii) analyzed the expression in HIV-patients in association with a number of demographic, biochemical and immunological parameters and iii) in relation with the current cART they received. PBMCs of HIV-patients displayed significantly increased expression of general inflammatory genes (IL6, IL18 and CXCL10) and this occurs irrespectively of the antiviral therapy they have been receiving. Conversely, levels of senescence-associated genes TP53, SERPINE1, IGFBP3 were slightly but significantly reduced in patients compared to uninfected matched individuals and this effect is related to NNRTI-containing treatments. The expression of the inflammatory markers IL6, IL18, IL1B, TNFA, RELA, CCL2, CCL20 and CXCL10 displayed correlation with certain demographic, morbidity- and HIV infection-related parameters. The levels of TP53 mRNA were positively associated only with plasma LDL. Correlation analysis between the expressions of pairs of genes revealed a different pattern between HIV-patients and controls. The diminished expression of TP53 and SERPINE1 in HIV-patients was also observed at a protein level, and the correlation between the two proteins (p53 and PAI1) in patients and controls showed the opposite trend. In conclusion, HIV-patients show dysregulation of p53 and p53-related mediators, a phenomenon which may be of pathophysiological relevance and could be related to the shorter health- and/or life-span observed in these individuals.

Background: Tyrosine kinase inhibitors that act against epidermal growth factor receptor (EGFR) show strong efficacy against non-small cell lung cancer (NSCLC) involving mutated EGFRs. However, most such patients eventually develop resistance to EGFR-TKIs. Numerous researches have reported that messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) may be involved in EGFR-TKI resistance, but the comprehensive expression profile and competitive endogenous RNA (ceRNA) regulatory network between mRNAs and ncRNAs in EGFR-TKI resistance of NSCLC are incompletely known. We aimed to define a ceRNA regulatory network linking mRNAs and non-coding RNAs that may mediate this resistance. Methods: Using datasets GSE83666, GSE75309 and GSE103352 from the Gene Expression Omnibus, we identified long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs differentially expressed between NSCLC cells that were sensitive or resistant to EGFR-TKIs. The potential biological functions of the corresponding differentially expressed genes were analyzed based KEGG pathways. We combined interactions among lncRNAs, miRNAs and mRNAs in the RNAInter database with KEGG pathways to generate transcriptional regulatory ceRNA networks associated with NSCLC resistance to EGFR-TKIs. Kaplan-Meier analysis was used to assess the ability of core ceRNA regulatory sub-networks to predict the progression-free interval and overall survival of NSCLC. The expression of two core ceRNA regulatory sub-networks in NSCLC was validated by quantitative real-time PCR. Results: We identified 8,989 lncRNAs, 1,083 miRNAs and 3,191 mRNAs that were differentially expressed between patients who were sensitive or resistant to the inhibitors. These DEGs were linked to 968 biological processes and 31 KEGG pathways. Pearson analysis of correlations among the DEGs of lncRNAs, miRNAs and mRNAs identified 12 core ceRNA regulatory sub-networks associated with resistance to EGFR-TKIs. The two lncRNAs ABTB1 and NPTN with the hsa-miR-150-5p and mRNA SERPINE1 were significantly associated with resistance to EGFR-TKIs and survival in NSCLC. These lncRNAs and the miRNA were found to be down-regulated, and the mRNA up-regulated, in a resistant NSCLC cell line relative to the corresponding sensitive cells. Conclusion: In this study, we provide new insights into the pathogenesis of NSCLC and the emergence of resistance to EGFR-TKIs, based on a lncRNA-miRNA-mRNA network.

Keywords: EGFR-TKI; NSCLC; competitive endogenous RNA; network; resistance.

Transforming growth factor-β (TGF-β) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-β signaling can lead to breast cancer. TGF-β elicits its effects on proliferation and differentiation via specific cell surface TGF-β type I and type II receptors (i.e., TβRI and TβRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-β-induced heteromeric complex formation, activated TβRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-β acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-β signaling and EMT responses using MCF10A-Ras and NMuMG breast cancer cell lines as examples. We describe methods to determine TGF-β-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-β-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-β receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-β-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-β-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We aimed to determine: 1) the mechanism(s) that enable glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)‑driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs; and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PD^S188F) in rats promoted transcription of SMC‑restricted genes. G6PD knockdown or inhibition decreased (48.5%) HDAC activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control SD rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PD^S188F rats a high-fat diet for 4 months increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PD^S188F rats. Our findings demonstrate G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.

Keywords: G6PD; Mediterranean variant; Metabolic Syndrome; PPP; smooth muscle cell phenotype.

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

Background: Plasminogen activator inhibitor type-1 (PAI-1, aka SERPINE1) is a moderately heritable glycoprotein that regulates fibrin clot dissolution (fibrinolysis).

Objectives: Test whether the heritabilities (h² ) of PAI-1 and other hemostatic factors are constant throughout their distribution or whether they are quantile-specific, i.e. a larger or smaller h² depending upon whether their concentrations are high or low.

Methods: Quantile regression was applied to 5606 parent-offspring pairs and 5310 full siblings of the Framingham Heart Study. Quantile-specific heritability was estimated from the parent-offspring regression slope (β_PO , h² =2β_PO /(1+r_spouse )) and the full-sib regression slope (β_FS , h² ={(1+8r_spouse β_FS )⁰ .⁵ -1}/(2r_spouse )).

Results: Heritability (h² ±SE) increased significantly with increasing percentiles of the offspring's age- and sex-adjusted PAI-1 distribution when estimated from β_PO (P_{linear trend} =0.0001): 0.09±0.02 at the 10^th , 0.09±0.02 at the 25^th , 0.16±0.02 at the 50^th , 0.29±0.04 at the 75^th , and 0.26±0.08 at the 90^th percentile of the PAI-1 distribution, and when estimated from β_FS (P_{linear trend} =6.5x10^-7 ). There was no significant evidence for quantile-specific heritability for Factor VII (P_{linear trend} =0.35), D-dimer (P_{linear trend} =0.08), tPA (P_{linear trend} =0.74), or von Willebrand factor (P_{linear trend} =0.79).

Conclusion: Higher mean plasma PAI-1 antigen concentrations tend to accentuate genetic effects (quantile-dependent expressivity), which is consistent with the greater reported differences in PAI-1 concentrations between rs1799889 SERPINE1 (4G/5G) genotypes in patients with osteonecrosis, meningococcal sepsis, obesity, prior myocardial infarction, deep vein thrombosis, and polycystic ovarian syndrome than in healthy controls. It is also consistent with the greater increases in PAI-1 concentrations in 4G-allele carriers than 5G/5G homozygotes following fibrinolytic treatment, low-salt intake, and high saturated fat intake.

Keywords: Gene-environment interaction; SERPINE1; factor VII; heritability; plasminogen activator inhibitor type-1; von Willebrand factor.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFβ signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

Keywords: CCND1; SERPINE1; field effect; myometrium; uterine fibroids.

Prognostic molecular subgrouping of glioblastoma is an ongoing effort and the current classification includes IDH-wild-type and IDH-mutant entities, the latter showing significantly better prognosis. We performed a comparative integrated analysis of the FGFR glioblastoma subgroup consisting of 5 cases from a prospective 101-patient-cohort. FGFR alterations included FGFR2-TACC2 and FGFR2 amplifications arising in a multifocal IDH-mutant glioblastoma with unexpected 2.5-month patient survival, novel FGFR3 carboxy-terminal duplication and FGFR3-TLN1 fusion, and two previously described FGFR3-TACC3 fusions. The FGFR2 tumors showed additional mutations in SERPINE1/PAI-1 and MMP16, as part of extensive extracellular matrix remodeling programs. Whole transcriptomic analysis revealed common proliferation but distinct morphogenetic gene expression programs that correlated with tumor histology. The kinase program revealed EPHA3, LTK and ALK receptor tyrosine kinase overexpression in individual FGFR tumors. Paradoxically, all FGFR-fused glioblastomas shared strong PI3K and MAPK pathway suppression effected by SPRY, DUSP and AKAP12 inhibitors, whereas the FGFR2-TACC2 tumor elicited also EGFR suppression by ERRFI1 upregulation. This integrated analysis outlined the proliferation and morphogenetic expression programs in FGFR glioblastoma, and identified four novel, clinically targetable FGFR2 and FGFR3 alterations that confer aggressive phenotype and trigger canonical pathway feedback inhibition, with important therapeutic implications.

Keywords: FGFR2 amplification; FGFR2-TACC2; FGFR3-TACC3; FGFR3-TLN1; Leptomeningeal gliomatosis; MMP; PAI-1; Proteomics; Transcriptomics.

The clinical outcomes of hepatocellular carcinoma (HCC) remain dismal. Elucidating the molecular mechanisms for the progression of aggressive HCC holds the promise for developing novel intervention strategies. The transactivation response element RNA-binding protein (TRBP/TARBP2), a key component of microRNA (miRNA) processing and maturation machinery has been shown to play conflicting roles in tumor development and progression. We sought to investigate the expression of TARBP2 in HCC using well-characterized HCC cell lines, patient-derived tissues and blood samples. Additionally, the potential prognostic and diagnostic value of TARBP2 in HCC were analyzed using Kaplan-Meier plots and ROC curve. Cell counting kit-8 (CCK-8), wound healing and transwell assays examined the ability of TARBP2 to induce cell proliferation, migration, and invasion in HCC cell lines. RNA sequencing was applied to identify the downstream elements of TARBP2. The interaction of potential targets of TARBP2, miR-145 and serpin family E member 1 (SERPINE1), was assessed using luciferase reporter assay. TARBP2 expression was down-regulated in HCC cell lines relative to normal hepatocyte cells, with a similar pattern further confirmed in tissue and blood samples. Notably, the loss of TARBP2 was demonstrated to promote proliferation, migration, and invasion in HCC cell lines. Interestingly, the reduction of TARBP2 was shown to result in the upregulation of SERPINE1, also known as plasminogen activator inhibitor (PAI-1), which is a vital gene of the HIF-1 signaling pathway. Knockdown of SERPINE1 rescued the TARBP2-lost phenotype. Moreover, TARBP2 depletion induced the upregulation of SERPINE1 through reducing the processing of miR-145, which directly targets SERPINE1. Finally, overexpression of miR-145 repressed SERPINE1 and rescued the functions in sh-TARBP2 HCC cells. Our findings underscore a linear TARBP2-miR-145-SERPINE1 pathway that drives HCC progression, with the potential as a novel intervention target for aggressive HCC.

Keywords: HCC; SERPINE1; TARBP2; miR-145; progression.

Cancer-associated fibroblasts (CAFs) contribute to the progression of various cancers. Previously, we reported the significance of CAFs in esophageal squamous cell carcinoma (ESCC); however, the functions of CAFs in the ESCC microenvironment remain unknown. To investigate CAFs' function, we established an indirect coculture assay between human bone marrow-derived mesenchymal stem cells (MSCs) and ESCC cells. Cocultured MSCs expressed more fibroblast activation protein, one of the markers of CAFs, compared with monocultured MSCs. Therefore, we defined cocultured MSCs as CAF-like cells. To identify molecules associated with the ESCC progression in CAFs, we conducted a cDNA microarray analysis on monocultured MSCs and CAF-like cells to compare their gene expression profiles. We found that SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was more abundant in CAF-like cells than in monocultured MSCs, and the PAI-1 derived from CAF-like cells induced the abilities of migration and invasion in both ESCC cells and macrophages by the Akt and Erk1/2 signaling pathways via the low-density lipoprotein receptor-related protein 1 (LRP1), which is a PAI-1 receptor. Based on immunohistochemistry assays of ESCC tissues, higher expression levels of PAI-1 and LRP1 were correlated with poor prognosis in ESCC patients. These results suggest that the PAI-1/LRP1 axis contributes to the progression of ESCC, making it a potential target for ESCC therapy.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Plasminogen activator inhibitor-1 (PAI-1), encoded by SERPINE1, is highly expressed in various types of tumor tissues, which contributes to cancer progression. The present study explored the role and underlying mechanisms of PAI-1 in ESCC. We found that the PAI-1 protein was extracellularly secreted more from ESCC cells with high PAI-1 expression using Western blotting and enzyme linked immunosorbent assay (ELISA). Knockdown of SERPINE1 expression significantly inhibited the invasion and migration of ESCC KYSE150 and KYSE450 cell lines, which could be restored when adding exogenous human recombinant PAI-1 into the culture medium of the cells stably expressing SERPINE1 shRNA. In vivo experiments showed that SERPINE1 knockdown significantly inhibited xenograft growth and lung metastasis of ESCC cells. Molecular analysis demonstrated that PAI-1 activated AKT and ERK signaling pathways. Co-immunoprecipitation (Co-IP) assays identified that PAI-1 may interact with the membrane receptor LDL receptor related protein 1 (LRP1). These results indicated that overexpression of PAI-1, through interacting with LRP1, might enhance invasion and migration of ESCC cells as well as promote ESCC progression.

Clinical investigations have demonstrated that polycystic ovary syndrome (PCOS) is often accompanied by insulin resistance (IR) in more than 70% of women with PCOS. However, the etiology of PCOS with IR remains to be characterized. Growth differentiation factor 8 (GDF8) is an intraovarian factor that plays a vital role in the regulation of follicle development and ovulation. Previous studies have reported that GDF8 is a pathogenic factor in glucose metabolism disorder in IR patients. To date, the role of GDF8 on glucose metabolism of granulosa cell in PCOS patients remains to be determined. In the current study, we demonstrated that the expression and accumulation of GDF8 in human granulosa-lutein (hGL) cells and follicular fluid from PCOS patients were higher compared with those of non-PCOS women. GDF8 treatment caused glucose metabolism defects in hGL cells. Transcriptome sequencing results showed that SERPINE1 mediated GDF8-induced impairment of hGL glucose metabolism defects. Using pharmacological and small interfering RNA (siRNA)-mediated knockdown approaches, we demonstrated that GDF8 upregulated the expression of SERPINE1 via the ALK5-mediated SMAD2/3-SMAD4 signaling pathway. Interestingly, the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway was also activated with GDF8 treatment but did not participate in the effect of GDF8 on SERPINE1 expression. Our results also showed that TP53 was required for the GDF8-stimulated increase in SERPINE1 expression. Importantly, our study demonstrated that SB-431542 treatment significantly improved DHEA-induced PCOS-like ovaries. These findings support a potential role for GDF8 in metabolic disorders in PCOS.

Keywords: glucose metabolism; growth differentiation factor 8; human granulosa-lutein cells; polycystic ovary syndrome.

Aim: Our aim was to evaluate gene expression profiling of fibroblasts from human alveolar mucosa (M), buccal attached gingiva (G) and palatal (P) tissues during early wound healing, correlating it with clinical response.

Materials and methods: M, G and P biopsies were harvested from six patients at baseline and 24 hrs after surgery. Clinical response was evaluated through Early Wound Healing Score (EHS). Fibrotic markers expression and autophagy were assessed on fibroblasts isolated from those tissues by Western blot and qRT-PCR. Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients.

Results: at 24 hrs after surgery, EHS was higher in P and G than in M. In line with our clinical results, no autophagy and myofibroblast differentiation were observed in G and P. We observed significant variations in mRNA expression of key genes: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblasts differentiation; IL6 and CXCL1, involved in inflammation.

Conclusions: We identified some genes involved in periodontal soft tissues clinical outcome, providing novel insights into the molecular mechanisms of oral repair (ClinicalTrial.gov-NCT04202822).

Keywords: gene expression profiling; gingiva; human biopsy; palate; wound healing.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus. Owing to its complicated pathogenesis, no satisfactory treatment strategies for DN are available. Milkvetch Root is a common traditional Chinese medicine (TCM) and has been extensively used to treat DN in clinical practice in China for many years. However, due to the complexity of botanical ingredients, the exact pharmacological mechanism of Milkvetch Root in treating DN has not been completely elucidated. The aim of this study was to explore the active components and potential mechanism of Milkvetch Root by using a systems pharmacology approach. First, the components and targets of Milkvetch Root were analyzed by using the Traditional Chinese Medicine Systems Pharmacology database. We found the common targets of Milkvetch Root and DN constructed a protein-protein interaction (PPI) network using STRING and screened the key targets via topological analysis. Enrichment of Gene Ontology (GO) pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed. Subsequently, major hubs were identified and imported to the Database for Annotation, Visualization and Integrated Discovery for pathway enrichment analysis. The binding activity and targets of the active components of Milkvetch Root were verified by using the molecular docking software SYBYL. Finally, we found 20 active components in Milkvetch Root. Moreover, the enrichment analysis of GO and KEGG pathways suggested that AGE-RAGE signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, and TNF signaling pathway might be the key pathways for the treatment of DN; more importantly, 10 putative targets of Milkvetch Root (AKT1, VEGFA, IL-6, PPARG, CCL2, NOS3, SERPINE1, CRP, ICAM1, and SLC2A) were identified to be of great significance in regulating these biological processes and pathways. This study provides an important scientific basis for further elucidating the mechanism of Milkvetch Root in treating DN.

The present study aimed to identify microRNAs (miRNAs) that may be crucial for the mechanism of mesenchymal stem cell (MSC) treatment in cisplatin‑induced acute kidney injury (AKI) and to investigate other potential drugs that may have a similar function. Transcriptomics (GSE85957) and miRNA expression (GSE66761) datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified using the linear models for microarray data method and mRNA targets of DEMs were predicted using the miRWalk2.0 database. The crucial DEGs were screened by constructing a protein‑protein interaction (PPI) network and module analysis. Functions of target genes were analyzed using the database for annotation, visualization and integrated discovery. Small molecule drugs were predicted using the connectivity map database. As a result, 5 DEMs were identified to be shared and oppositely expressed in comparisons between AKI model and control groups, and between MSC treatment and AKI model groups. The 103 DEGs were overlapped with the target genes of 5 common DEMs, and the resulting list was used for constructing the miRNA‑mRNA regulatory network, including rno‑miR‑210/Serpine1 and rno‑miR‑378/Fos. Serpine1 (degree=17) and Fos (degree=42) were predicted to be hub genes according to the topological characteristic of degree in the PPI network. Function analysis indicated Serpine1 and Fos may be inflammation‑related. Furthermore, gliclazide was suggested to be a potential drug for the treatment of AKI because the enrichment score was the closest to ‑1 (‑0.9). In conclusion, it can be speculated that gliclazide may have a similar mechanism to MSC as a potential therapeutic agent for cisplatin‑induced AKI, by regulating miR‑210/Serpine1 and miR‑378‑/Fos‑mediated inflammation and cell apoptosis.

JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors. We have discovered that JS-K was effective against Hepatitis B virus (HBV)-positive HepG2.2.15 cells. This study used iTRAQ to identify differentially expressed proteins following JS-K treatment of HepG2.2.15 cells. Silenced Transgelin (shTAGLN-2.15) cells were constructed, and the cell viability was analyzed by the CCK8 assay after treatment with JS-K. There were 182 differentially expressed proteins in JS-K treated-HepG2.2.15 cells; 73 proteins were up-regulated and 109 proteins were down-regulated. These proteins were categorized according to GO classification. KEGG enrichment analysis showed that Endocytosis, Phagosome and Proteoglycans were the most significant pathways. RT-PCR confirmed that the expression levels of TAGLN, IGFBP1, SMTN, SERPINE1, ANXA3, TMSB10, LGALS1 and KRT19 were significantly up-regulated, and the expression levels of C5, RBP4, CHKA, SIRT5 and TRIM14 were significantly down-regulated in JS-K treated-HepG2.2.15 cells. Western blotting confirmed the increased levels of USP13 and TAGLN proteins in JS-K treated-HepG2.2.15 cells. Molecular docking revealed the binding of JS-K to TAGLN and shTAGLN-2.15 cells were resistant to JS-K cytotoxicity, suggesting that TAGLN could be an important target in JS-K anti-HBV-positive liver cancer cells. These proteomic findings could shed new insights into mechanisms underlying the effect of JS-K against HBV-related HCC.

Background: To understand the molecular mechanisms of the antitumour response, we analysed the immune landscape of cervical cancer to identify novel immune molecular classes.

Methods: We established a stable immune molecular classification using a nonnegative matrix factorization algorithm and validated the correlation in two validation sets of 249 samples.

Results: Approximately 78% of cervical cancers (CCs) (228/293) were identified to show significant enrichment in immune cells (e.g., CD8 T cells and macrophages), a type I IFN response, enhanced cytolytic activity and high PDCD1, and these CCs were referred to as the "immune class". We further identified two subtypes of the immune class: active immune and exhausted subtypes. Although the active immune subtype was characterized by enrichment of IFN signatures and better survival, the exhausted subtype expressed activated stroma, a wound healing signature, enhanced M2 macrophages and absence of CD8 T cells and the TGF-β response signature. Integrative analysis of multiomics data identified EGFR, JUN, MYC, FN1 and SERPINE1 as key modulators of the tumour immune microenvironment and potential targets for combination therapies which was validated in two validation sets.

Conclusions: Our study introduces a novel immune classification that might predict ideal candidates to receive immunotherapy or specific combination therapies.

Keywords: Cervical cancer; Immune classification; Multiomics.