Background: Immunohistochemical quantification of the immune response is prognostic for colorectal cancer (CRC). Here, we evaluate the suitability of alternative immune classifiers on prognosis and assess whether they relate to biological features amenable to targeted therapy.

Methods: Overall survival by immune (CD3, CD4, CD8, CD20 and FOXP3) and immune-checkpoint (ICOS, IDO-1 and PD-L1) biomarkers in independent CRC cohorts was evaluated. Matched mutational and transcriptomic data were interrogated to identify associated biology.

Results: Determination of immune-cold tumours by combined low-density cell counts of CD3, CD4 and CD8 immunohistochemistry constituted the best prognosticator across stage II-IV CRC, particularly in patients with stage IV disease (HR 1.98 [95% CI: 1.47-2.67]). These immune-cold CRCs were associated with tumour hypoxia, confirmed using CAIX immunohistochemistry (P = 0.0009), which may mediate disease progression through common biology (KRAS mutations, CRIS-B subtype and SPP1 mRNA overexpression).

Conclusions: Given the significantly poorer survival of immune-cold CRC patients, these data illustrate that assessment of CD4-expressing cells complements low CD3 and CD8 immunohistochemical quantification in the tumour bulk, potentially facilitating immunophenotyping of patient biopsies to predict prognosis. In addition, we found immune-cold CRCs to associate with a difficult-to-treat, poor prognosis hypoxia signature, indicating that these patients may benefit from hypoxia-targeting clinical trials.

The bone mineral deficiency in osteoporosis poses a threat to the long-term outcomes of endosseous implants. The inhibitors of cathepsin K (CatK) significantly affect bone turnover, bone mineral density (BMD) and bone strength in the patients with osteoporosis. Therefore, we hypothesised that the application of a CatK inhibitor (CatKI) could increase the osseointegration of endosseous implants under osteoporotic conditions. Odanacatib (ODN), a highly selective CatKI, was chosen as the experimental drug. Sixteen rats were randomised into 4 groups: sham, ovariectomy (OVX) with vehicle, OVX with low-dose ODN (5 mg/kg) and OVX with high-dose ODN (30 mg/kg). Titanium implants were placed into the distal metaphysis of bilateral femurs of each OVX rat. After 8 weeks of gavaging, CatKI treatment increased the removal torque, BMD and bone-to-implant contact (BIC). Moreover, high-dose CatKI exerted a better influence than low-dose CatKI. Furthermore, CatKI treatment not only robustly suppressed CatK gene (CTSK) expression, but also moderately reduced expression of the osteoblast-related genes Runx2, Collagen-1, BSP, Osterix, OPN, SPP1 and ALP. Thus, CatKI could affect the osteoblast-related genes, although the balance of bone turnover was achieved mainly by CatK inhibition. In conclusion, CatKI prevented bone loss and aided endosseous implantation in osteoporotic conditions.

Glucose filtered in the glomerulus is actively reabsorbed by sodium-glucose co-transporter 2 (SGLT2) in proximal tubular epithelial cells (PTEC) and passively returned to the blood via glucose transporter 2 (GLUT2). Healthy PTEC rely primarily on fatty acid beta-oxidation (FAO) for energy. In phase III trials, SGLT2 inhibitors improved outcomes in diabetic kidney disease (DKD). Tubulointerstitial renal fibrosis due to altered metabolic reprogramming of PTEC might be at the root of the pathogenesis of DKD. Here, we investigated the molecular mechanism of SGLT2 inhibitors' renoprotective effect by examining transcriptional activity of Spp1, which encodes osteopontin, a key mediator of tubulointerstitial renal fibrosis. With primary cultured PTEC from Spp1-enhanced green fluorescent protein knock-in mice, we proved that in high-glucose conditions, increased SGLT2- and GLUT-mediated glucose uptake is causatively involved in aberrant activation of the glycolytic pathway in PTEC, thereby increasing mitochondrial reactive oxygen species (ROS) formation and transcriptional activation of Spp1. FAO activation did not play a direct role in these processes, but elevated expression of a tubular-specific enzyme, myo-inositol oxygenase, was at least partly involved. Notably, canagliflozin blocked overexpression of myo-inositol oxygenase. In conclusion, SGLT2 inhibitors exerted renoprotective effects by inhibiting aberrant glycolytic metabolism and mitochondrial ROS formation in PTEC in high-glucose conditions.

Keywords: 2-deoxy-D-glucose; GLUT2; SGLT2; myo-inositol oxygenase; osteopontin; proximal tubular epithelial cells.

Vitamin A (VA) is an important nutrient for weaning piglets. It plays a significant role in the normal formation, development, and maintenance of epithelial cells. Previous studies have shown that VA supplements could improve the host's intestinal barrier function. Therefore, we hypothesized that VA supplements can affect intestinal function in weaned piglets by regulating intestinal stem cells. Thirty-two 21-day-old weaned [(Yorkshire × Landrace) × Duroc] piglets with an average weight of 8.34 ± 0.13 kg were randomly divided into four treatment groups, with (i) 2 mg/kg (control), (ii) 4 mg/kg, (iii) 8 mg/kg and (iv) 16 mg/kg doses of VA, respectively. The experiment lasted for 14 days. Weaned piglets were given ad libitum access to food and water during the test. The ADG (Linear, P = 0.020) and G: F (Linear, P = 0.005) of the piglets were found to increase significantly from day 8 to 14. The Lgr5+ gene expression (P = 0.012) in the jejunum mucosa of the 16 mg/kg VA group was increased. The jejunum villus height (P = 0.027) and villi surface area (P = 0.035) were significantly increased in the 4 mg/kg VA treatment group. The crypt depth increased significantly in the 4 and 8 mg/kg VA treatment groups (Quadratic, P = 0.043), and the ratios of villus height to crypt depth significantly increased in the 16 mg/kg VA group (Quadratic, P = 0.015). The maltase (P = 0.032), sucrose (P = 0.041) and alkaline phosphatase activity (Linear, P = 0.024) were significantly increased when further supplemented with 4 mg/kg VA. Slc2a2 mRNA abundance was significantly increased in the 2 mg/kg VA group (Linear, P = 0.024). Moreover, the budding rates, buddings number per organoid and Chromogranin A and Muc2 expression of piglet intestinal organoids were significantly reduced (P < 0.05) by VA and its metabolites (retinoic acid). Compared with the control group, the expression of Spp1 and Trop2 increased. These results indicated that VA may increase the stemness of intestinal stem cell in vitro. This study suggested that VA could affect growth performance and intestinal function by regulating intestinal stem cells in the jejunum of weaned piglets.

To investigate the impact of the androgen precursor dehydroepiandrosterone (DHEA) on the decidualization of human endometrial stromal cells isolated from women of advanced reproductive age.

In vitro study.

University research institute.

Proliferative phase primary human endometrial stromal fibroblasts (hESFs) were isolated from women of advanced reproductive age (n = 16; mean age, 44.7 ± 2.3). None of the women were receiving hormone therapy or had endometriosis.

Isolated hESFs were decidualized in vitro by incubation with P (1 μM) and cAMP (0.1 mg/mL) in the presence, or absence, of DHEA (10 nM, 100 nM).

Secretion of androgens was assessed by ELISA. Expression of decidualization markers and endometrial receptivity markers was assessed by quantitative polymerase chain reaction and ELISA.

Decidualization responses were retained in hESF isolated from women of advanced reproductive age. Supplementation with DHEA increased androgen biosynthesis and concentrations of T and dihydrotestosterone were ∼3× greater after coincubation with DHEA compared with hESF stimulated with decidualization alone. Addition of DHEA to decidualized hESF increased expression of the decidualization markers IGFBP1 and PRL and the endometrial receptivity marker SPP1. DHEA enhanced secretion of IGFBP1, PRL, and SPP1 proteins maximally by day 8 of the decidualization time course concomitant with peak androgen concentrations.

These novel results demonstrate DHEA can enhance in vitro decidualization responses of hESF from women of advanced reproductive age. Supplementation with DHEA during the receptive phase may augment endometrial function and improve pregnancy rates in natural or assisted reproductive cycles.

The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622-treated mice that corresponded with elevated expression of transcripts encoding disease-associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.

Keywords: coronavirus; demyelination; host defense; microglia; remyelination.

Improving lactation persistency (LP) in dairy cattle has a beneficial effect on animal health and fertility and herd productivity. A complex trait, LP not only reflects the cow's ability to maintain milk secretion activity after the lactation peak but is also a function of the postcalving development of the mammary gland and, later on, of tissue remodeling as lactation declines. This decline is a consequence of an imbalance between cell proliferation and cell removal. In a previous study, single nucleotide polymorphisms were identified in the osteopontin (OPN) gene, SPP1. Osteopontin is a multifaceted protein that plays an important role in immune regulation and tissue remodeling. Because OPN is involved in involution, it might also have an effect on LP. The objective of the present study was to evaluate whether LP could be influenced by genetic variations in the SPP1 gene. This association with LP was analyzed in the population of 578 bulls characterized in a previous study. The population mean of estimated breeding value (EBV) for LP was 100.95 ± 5.06 units. Allele and genotype association analyses were performed by comparing the frequencies of the different genotypes and alleles with EBV for LP for the respective lactation using logistic regression. The EBV for LP at the first lactation (LP1), second lactation (LP2), and third lactation (LP3) and for overall lactation (OLP) are reported for the genotypes SPP1c.-1301G>A, SPP1c.-1251C>T, SPP1c.-430G>A, and SPP1c.*40A>C. The first single nucleotide polymorphism, SPP1c.-1301G>A, affected LP1, LP2, LP3, and OLP. Analysis of the estimated average allele substitution effects also confirmed that G is a favorable allele for LP, given the gain observed over LP1, LP2, LP3, and OLP. Differences in EBV for LP were observed between animals with different haplotypes at LP1, LP2, LP3, and OLP. Contrast analysis for OLP revealed that mean EBV is greater for block H1 (101.34 ± 0.30) than for animals that do not have H1 (98.20 ± 0.77). The gain with block H1 (GCGA) suggests the presence of the favorable allele G (first position in the block: SPP1c.-1301G). The pleiotropic roles of OPN position it at the crossroads of immune regulation, tissue remodeling, and involution. From a genetic perspective, data from the present study suggest OPN as a candidate gene associated with LP for dairy cows.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

Oral squamous cell carcinoma (OSCC) is one of the leading cancers with poor disease survival rate. Herein, we explored molecular basis, in silico identification and in vitro verification of genes associated with OSCC. Five gene expression series including, GSE30784, GSE13601, GSE9844, GSE23558 and GSE37991 were screened for differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched by cluster Profiler. Further, protein-protein interaction network was analysed and hub genes were verified. A total of 6476 (up-regulated: 2848; down-regulated: 3628) DEGs were identified among OSCC patients and healthy controls. Gene Ontology analysis indicated DEGs enrichment in cellular motility, invasion and adhesion processes. KEGG analysis revealed enrichment of PI3K-Akt signalling, focal adhesion and regulation of actin cytoskeleton pathways. Subsequently, nine DEGs including APP, EHMT1, ACACB, PCNA, PLAU, FST, HMGA2, LAMC2 and SPP1 were correlated with TCGA expression data along with significant association towards patient's survival, recognized as hub genes. This dysregulated mRNA signature of genes was validated in two OSCC cell lines with an anti-cancer agent, fisetin. Fisetin inhibited the expression of APP, EHMT1, PCNA, PLAU, FST, HMGA2, LAMC2, SPP1 and upregulated the expression of ACACB gene which were associated with growth inhibition of both the OSCC cell lines. The regulatory effect of fisetin supported crucial role of nine hub genes identified in OSCC. This study signified that hub genes and pathways might influence the aggressiveness of OSCC. Thus, the proposed hub genes could be potential diagnostic biomarker and drug targets for OSCC.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-02737-4.

Keywords: Biomarkers; Cytoscape; Gene expression profiling; Hub genes; Oral squamous cell carcinoma; Survival analysis.

BACKGROUND Esophageal cancer is a malignant tumor with a complex pathogenesis and a poor 5-year survival rate, which encourages researchers to explore its molecular mechanisms deeper to improve the prognosis. MATERIAL AND METHODS DEGs were from 4 Gene Expression Omnibus (GEO) databases (GSE92396, GSE20347, GSE23400, and GSE45168) including 87 esophageal tumor samples and 84 normal samples. We performed Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Protein-Protein interaction (PPI) analysis, and GeneMANIA to identify the DEGs. Gene set enrichment analysis (GSEA) and Kaplan-Meier survival analyses were performed. RESULTS There was an overlapping subset consisting of 120 DEGs that was present in all esophageal tumor samples. The DEGs were enriched in extracellular matrix (ECM)-receptor interaction, as well as focal adhesion and transcriptional mis-regulation in cancer. The 2 most crucial regulatory pathways in esophageal cancer were the amebiasis pathway and the PI3K-Akt signaling pathway. Secreted phosphoprotein 1 (SPP1) and fibronectin 1 (FN1) were selected and verified in an independent cohort and samples using the TCGA and GTEx projects. Gene set enrichment analysis (GSEA) showed that proteasome and nucleotide excision repair were 2 most differentially enriched pathways in the SPP1 high-expression phenotype, and ECM-receptor interaction and focal adhesion in FN1 high-expression phenotype. Kaplan-Meier survival analysis showed that SPP1 and FN1 were significantly positively related to overall survival and had the potential to predict patient relapse. CONCLUSIONS Our analysis is the first to show that SPP1 and FN1 might work as biological markers of progression and prognosis in esophageal carcinoma (ESCA).

Coating characteristics of dental implants such as composition and topography regulate cell response during implant healing. The aim of this study was to assess how surface topography can affect osteogenic differentiation of mesenchymal stem cells (MSCs) by analyzing the expression levels of bone-related genes and MSCs marker. Thirty disk-shaped, commercially pure Grade 2 titanium samples (10 × 2 mm) with 3 different surface topographies (DENTSPLY-Friadent GmbH, Mannheim, Germany) were used in the present study: 10 Ti machined disks (control), 10 Ti sandblasted and acid-etched disks (DPS(®)) and 10 sandblasted and acid-etched disks at high temperature (Plus(®)). Samples were processed for real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. By comparing machined and Plus(®) disks, quantitative real-time RT-PCR showed a significant reduction of the bone-related genes osteocalcin (BGLAP) and osteoblast transcriptional factor (RUNX2). The comparison between DPS(®) and Plus(®) disks showed a slight induction of all the genes examined (RUNX2, ALPL, COL1A1, COL3A1, ENG, FOSL1, SPP1, and SP7); only the expression of BGLAP remained stable. The present study, demonstrated that implant surface topography affects osteoblast gene expression. Indeed, Plus(®) surface produces an effect on MSCs in the late differentiation stages.

BACKGROUND

Our study was designed to test a possible association between polymorphisms of the SPP1 gene (rs4754, rs28357094) and markers of carotid atherosclerosis (CIMT, number of affected segments of carotid arteries, sum of plaque thickness, presence of carotid plaques, and presence of unstable carotid plaques) in subjects with T2DM. The second aim was to test the possible association between polymorphisms of the SPP1 gene (rs4754, rs28357094) and the progression of carotid atherosclerosis (CIMT progression, change in total plaque thickness, change in the number of sites with plaques) in subjects with T2DM.

METHODS

In the prospective study 595 T2DM subjects were enrolled. Markers of carotid atherosclerosis were assessed ultrasonographically. rs4754 and rs28357094 polymorphisms of the phosphoprotein 1 (SPP1) gene were determined with real-time PCR.

RESULTS

In our study we found an association between SPP1 rs4754 and the presence of plaques at the time of recruitment, whereas we did not find any association between SPP1 rs28357094 and subclinical markers of carotid atherosclerosis at the time of recruitment. Moreover, we did not find any statistically significant effect of either rs4754 or rs28357094 on subclinical markers of carotid atherosclerosis progression (CIMT progression, change in total plaque thickness, change in the number of sites with plaques). As shown by the multiple linear regression analysis, genotypes of either rs4754 or rs28357094 did not have a statistically significant effect on the progression of subclinical markers of carotid atherosclerosis (CIMT progression, change in total plaque thickness, change in the number of sites with plaques) after the adjustment for confounding variables.

CONCLUSIONS

We demonstrated an important effect of the SPP1 rs4754 on subclinical markers of carotid atherosclerosis in subjects with T2DM; however, as demonstrated by the multiple linear regression analysis, neither rs4754 nor rs28357094 had an important impact on the progression of subclinical markers of carotid atherosclerosis in subjects with T2DM.

Hepatocellular carcinoma (HCC) is a heterogeneous disease with various genetic and epigenetic abnormalities. Previous studies of HCC driver genes were primarily based on frequency of mutations and copy number alterations. Here, we performed an integrative analysis of genomic and epigenomic data from 377 HCC patients to identify driver genes that regulate gene expression in HCC. This integrative approach has significant advantages over single-platform analyses for identifying cancer drivers. Using this approach, HCC tissues were divided into four subgroups, based on expression of the transcription factor E2F and the mutation status of TP53. HCC tissues with E2F overexpression and TP53 mutation had the highest cell cycle activity, indicating a synergistic effect of E2F and TP53. We found that overexpression of the identified driver genes, stratifin (SFN) and SPP1, correlates with tumor grade and poor survival in HCC and promotes HCC cell proliferation. These findings indicate SFN and SPP1 function as oncogenes in HCC and highlight the important role of enhancers in the regulation of gene expression in HCC.

Keywords: SPP1; epigenome; liver cancer; stratifin; systematic integration.

Caspases, well-known players in apoptosis or inflammation, appear to have roles also in other processes such as cell differentiation. Caspase-3, in particular, was recently demonstrated to have non-apoptotic functions in osteogenesis. However, the molecular pathways involved are not yet known. Therefore, we used osteogenic PCR arrays to provide a comprehensive screening of possible interactions of caspases in general and specifically of caspase-3 in osteogenic networks. Embryonic micromass cultures derived from mouse forelimbs were established and pharmacological fluoromethylketone (FMK) inhibitors applied. Alterations were observed in expression of several genes after caspase inhibition (Bmp1, Bmp5, Bmp6, Col10a1, Col2a1, Comp, Egf, Fgfr2, Gli1, Igf1, Nog, Phex, Sox9, Spp1). The list suggests molecular interactions of caspases and osteogenic molecules and creates a background for further temporospatial and functional studies.

In monogastric animals, phosphorus (P) homeostasis is maintained by regulating intestinal absorption, bone mobilization, and renal excretion. Since P is a non-renewable resource, a shortage is imminent due to widespread over-usage in the farming and animal husbandry industries. As a consequence, P efficiency should be improved in pig production. We sought to characterize the transcriptional response in re-/absorbing and excreting tissues in pigs to diets varying in calcium: phosphorus ratios. Weaned piglets were assigned to one of three groups fed diets varying in digestible P content for a period of five weeks. Gene expression profiles were analyzed in jejunum, colon, and kidney.

Transcriptome analysis revealed that reduced dietary P intake affects gene expression in jejunum and kidney, but not in colon. The regulation of mineral homeostasis was reflected via altered mRNA abundances of CYP24A1, CYP27A1, TRPM6, SPP1, and VDR in jejunum and kidney. Moreover, lowered abundances of transcripts associated with the classical complement system pathway were observed in the jejunum. In kidney, shifted transcripts were involved in phospholipase C, calcium signaling, and NFAT signaling, which may have immunomodulatory implications.

Our results revealed local transcriptional consequences of variable P intake in intestinal and renal tissues. The adaptive responses are the result of organismal efforts to maintain systemic mineral homeostasis while modulating immune features at local tissue sites. Therefore, the deviation from the currently recommended dietary P supply must be carefully considered, as the endogenous mechanisms that respond to low P diets may impact important adaptive immune responses.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. However, the molecular mechanism underlying its development and progression is yet unclear. Genes that are differentially expressed, that is, differentially expressed genes (DEGs), between normal and diseased tissues are believed to be involved in disease development and progression. To identify the DEGs in OSCC and explore their role in occurrence and progression, we established a Chinese hamster OSCC model, determined the DEG, screened the identified DEGs, and performed Gene Ontology (GO) and KEGG enrichment analyses. A protein-protein interaction (PPI) network was generated to screen potential candidate genes. We then analyzed the expression, tumor stage and prognosis of candidate genes using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Finally, we verified the candidate DEGs by quantitative real-time PCR and Gene Expression Omnibus analysis. The results showed 194 significantly DEGs, 140 enriched GO terms, and 8 KEGG pathways, which suggested that OSCC was closely related to the immune system, cell migration, and extracellular matrix. GEPIA and PPI network analysis revealed that SPP1, TNC, and ACTA1 were significantly related to tumor staging; SPP1, tissue inhibitors of matrix metallopeptidases (MMPs) 1 (TIMP1), and ACTA1 were closely related to prognosis. The scores for the top five highest degree genes were close, and the TIMP1/MMP9 axis appeared to be at the center of the PPI network, indicating that expression changes in the TIMP1/MMP9 axis and related genes may be involved in tumor invasion and metastasis. These findings provide novel insights into the mechanism of oral cancer.

Keywords: Chinese hamster; animal model; differentially expressed genes (DEGs); high-throughput sequencing; oral squamous cell carcinoma.

Colorectal cancer (CRC) is the third most common cancer worldwide and liver metastases are the leading cause of death in patients with CRC. In this study, we performed next-generation sequencing profiling on primary colorectal tumor tissues obtained from three CRC patients with liver metastases and three CRC patients without liver metastases to identify differentially expressed genes (DEGs) that might be responsible for the metastases process. After filtering 2690 DEGs, comprising 996 upregulated and 1694 downregulated RNAs, 22 upregulated and 73 downregulated DEGs were identified. Gene ontology (GO) and pathway analyses were performed to determine the underlying mechanisms. Single-organism process (biological process), cell (cellular component), and binding (molecular function) were the most related terms in the GO analysis. We selected the top 13 upregulated and top 12 downregulated genes by fold change to verify their differential expression using quantitative real-time reverse transcription PCR (qRT-PCR) and immunohistochemistry (IHC). The validation showed that three most significantly upregulated DEGs were HOXD10, UGT2A3, and SLC13A2, whereas the five most significantly downregulated DEGs were SPP1, CXCL8, MMP3, OSM, and CXCL6, respectively. These aberrantly expressed genes may play pivotal roles in promoting or inhibiting metastases. Further studies are required to determine the functions of DEGs to promote the diagnosis of metastases and provide novel chemotherapy targets.

Acquisition of endometrial receptivity for embryo implantation is one of the crucial processes during pregnancy and is induced mainly by progesterone and enhanced by conceptus signals. Prokineticin 1 (PROK1) is characterized as a secretory protein with diverse functions in various tissues, including the reproductive tract. PROK1, with its receptor PROKR1, are up-regulated in the porcine endometrium during implantation and in women's receptive endometrium and decidua. However, the function of PROK1 in embryo-maternal communication has still not been fully elucidated. Hence, we hypothesize that PROK1 is involved in endometrial receptivity development and implantation in pigs. In this study, using the porcine in vivo model of intrauterine infusions of estradiol-17β (E2) and prostaglandin E2 (PGE2), we revealed that these hormones elevated endometrial expression of PROK1 and PROKR1 mRNA, respectively. Moreover, E2, acting synergistically with PGE2, increased PROKR1 protein expression. We also evidenced that PROK1-PROKR1 signaling induced expression of following genes and/or proteins CCN2, CDH13, FGF2, NFATC2, ANGPT1, ANGPT2, CDH1, MUC4, SPP1, IFNG, IL6, LIF, LIFR, TNF, TGFB3, and FGF9, as well as phosphorylation of PTK2 and secretion of IL6 and IL11 by endometrial explants in vitro. Ingenuity Pathway Analysis revealed that functions associated with the PROK1-regulated genes/proteins include cell-to-cell contact, cell attachment, migration and viability, differentiation of epithelial tissue, leukocyte migration, inflammatory response, angiogenesis, and vasculogenesis. Summarizing, our study suggests that PROK1 acts pleiotropically as an embryonic signal mediator that regulates endometrial receptivity by increasing the expression of the genes and proteins involved in implantation and pregnancy establishment in pigs.

Keywords: Prokineticin 1; endometrial receptivity; implantation; pregnancy; prokineticin receptor 1; the pig.

Secreted phosphoprotein 1 (SPP1) in the bovine corpus luteum (CL) regulates cell function during the transitional periods of luteinization and luteal regression. The objectives were to i) characterize SPP1 expression in the CL throughout the estrous cycle, ii) determine factors that regulate SPP1 expression in luteal cells, and iii) examine the role of SPP1 in lymphocyte chemotaxis, proliferation, and function. SPP1 mRNA was greater in fully functional (d10) CL and late cycle (d18) CL compared with developing (d4) CL. Additionally, SPP1 mRNA increased within 1 h and remained elevated 4 and 8 h following induction of luteolysis with prostaglandin (PG)F2α. Expression of the SPP1 receptor, β3 integrin, was not different throughout the estrous cycle but decreased following induction of luteolysis. Expression of CD44 increased during the estrous cycle but did not change during luteal regression. In cultured luteal cells, SPP1 mRNA was upregulated by PGF2α and/or tumor necrosis factor α. Western blots revealed the presence of both full-length SPP1 and multiple cleavage products in cultured luteal cells and luteal tissue. Depletion of endogenous SPP1 did not hinder luteal cell-induced lymphocyte proliferation or lymphocyte phenotype but did inhibit lymphocyte migration toward luteal cells. Based on these data, it is concluded that SPP1 is initially activated to establish and maintain cellular interactions between steroidogenic and nonsteroidogenic cells during the development of the CL. Upon induction of luteolysis, SPP1 serves as a signaling molecule to recruit or activate immune cells to facilitate luteal regression and tissue degradation.

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvβ3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvβ3 integrin. Interestingly, we found the αvβ3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvβ3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvβ3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvβ3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvβ3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT-PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvβ3 integrin is one of the downstream factors regulated by BSP in the breast cancer-bone metastatic cascade.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) and their derivative enhanced green fluorescent protein (eGFP)-hBMSCs were employed to evaluate an innovative hybrid scaffold composed of granular hydroxylapatite and collagen hemostat (Coll/HA). The cellular morphology/cytoskeleton organization and cell viability were investigated by immunohistochemistry (IHC) and AlamarBlue metabolic assay, respectively. The expression of osteopontin and osteocalcin proteins was analyzed by IHC and ELISA, whereas osteogenic genes were investigated by quantitative PCR (Q-PCR). Cell morphology of eGFP-hBMSCs was indistinguishable from that of parental hBMSCs. The cytoskeleton architecture of hBMSCs grown on the scaffold appeared to be well organized, whereas its integrity remained uninfluenced by the scaffold during the time course. Metabolic activity measured in hBMSCs grown on a biomaterial was increased during the experiments, up to day 21 (p < 0.05). The biomaterial induced the matrix mineralization in hBMSCs. The scaffold favored the expression of osteogenic proteins, such as osteocalcin and osteopontin. In hBMSC cultures, the scaffold induced up-regulation in specific genes that are involved in ossification process (BMP2/3, SPP1, SMAD3, and SP7), whereas they showed an up-regulation of MMP9 and MMP10, which play a central role during the skeletal development. hBMSCs were induced to chondrogenic differentiation through up-regulation of COL2A1 gene. Our experiments suggest that the innovative scaffold tested herein provides a good microenvironment for hBMSC adhesion, viability, and osteoinduction. hBMSCs are an excellent in vitro cellular model to assay scaffolds, which can be employed for bone repair and bone tissue engineering.

Keywords: bone; expression; gene; osteogenesis; scaffold.

Cardiac dysfunction with progressive inflammation and fibrosis is a hallmark of Chagas disease caused by persistent Trypanosoma cruzi infection. Osteopontin (OPN) is a pro-inflammatory cytokine that orchestrates mechanisms controlling cell recruitment and cardiac architecture. Our main goal was to study the role of endogenous OPN as a modulator of myocardial CCL5 chemokine and MMP-2 metalloproteinase, and its pathological impact in a murine model of Chagas heart disease. Wild-type (WT) and OPN-deficient (spp1 -/-) mice were parasite-infected (Brazil strain) for 100days. Both groups developed chronic myocarditis with similar parasite burden and survival rates. However, spp1 -/- infection showed lower heart-to-body ratio (P<0.01) as well as reduced inflammatory pathology (P<0.05), CCL5 expression (P<0.05), myocyte size (P<0.05) and fibrosis (P<0.01) in cardiac tissues. Intense OPN labeling was observed in inflammatory cells recruited to infected heart (P<0.05). Plasma concentration of MMP-2 was higher (P<0.05) in infected WT than in spp1 -/- mice. Coincidently, specific immunostaining revealed increased gelatinase expression (P<0.01) and activity (P<0.05) in the inflamed hearts from T. cruzi WT mice, but not in their spp1 -/- littermates. CCL5 and MMP-2 induction occurred preferentially (P<0.01) in WT heart-invading CD8+ T cells and was mediated via phospho-JNK MAPK signaling. Heart levels of OPN, CCL5 and MMP-2 correlated (P<0.01) with collagen accumulation in the infected WT group only. Endogenous OPN emerges as a key player in the pathogenesis of chronic Chagas heart disease, through the upregulation of myocardial CCL5/MMP-2 expression and activities resulting in pro-inflammatory and pro-hypertrophic events, cardiac remodeling and interstitial fibrosis.

Malignant tumor represents a major reason for death in the world and its incidence is growing rapidly. Developing the tools for early diagnosis is possibly a promising way to offer diverse therapeutic options and promote the survival chance. Secreted phosphoprotein 1 (SPP1), also called Osteopontin (OPN), has been demonstrated overexpressed in many cancers. However, the specific role of SPP1 in prognosis, gene mutations, and changes in gene and miRNA expression in human cancers is unclear. In this report, we found SPP1 expression was higher in most of the human cancers. Based on Kaplan-Meier plotter and the PrognoScan database, we found high SPP1 expression was significantly correlated with poor survival in various cancers. Using a large dataset of colon adenocarcinoma (COAD), head and neck cancer (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) patients from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, this study identified 22 common genes and 2 common miRNAs. GO, and KEGG paths analyses suggested that SPP1 correlated genes were mainly involved in positive regulation of immune cell activation and infiltration. SPP1-associated genes and miRNAs regulatory networks suggested that their interactions may play a role in the progression of four selected cancers. SPP1 showed significant positive correlation with the immunocyte and immune marker sets infiltrating degrees. All of these data provide strong evidence that SPP1 may promote tumor progress through interacting with carcinogenic genes and facilitating immune cells' infiltration in COAD, HNSC, LUAD, and LUSC.

Keywords: SPP1; biomarker; gene expression; immune infiltration; miRNA; multiple human cancers; tumor prognosis.