Promoter methylation plays a vital role in tumorigenesis through transcriptional silencing of tumor suppressive genes. Using genome-wide methylation array, we first identified PBX/Knotted Homeobox 2 (PKNOX2) as a candidate tumor suppressor in gastric cancer. PKNOX2 mRNA expression is largely silenced in gastric cancer cell lines and primary gastric cancer via promoter methylation. Promoter methylation of PKNOX2 was associated with poor survival in gastric cancer patients. A series of in vitro and in vivo functional studies revealed that PKNOX2 functions as a tumor suppressor. Ectopic PKNOX2 expression inhibited cell proliferation in GC cell lines and suppressed growth of tumor xenografts in mice via induction of apoptosis and cell cycle arrest; and suppressed cell migration and invasion by blocking epithelial-to-mesenchymal transition. On the other hand, knockdown PKNOX2 in normal gastric epithelial cells triggered diverse malignant phenotypes. Mechanistically, PKNOX2 exerts its tumor suppressive effect by promoting the up-regulation of Insulin like Growth Factor Binding Protein 5 (IGFBP5) and TP53. PKNOX2 binds to the promoter regions of IGFBP5 and TP53 and transcriptionally activated their expression by chromatin immunoprecipitation (ChIP)-PCR assay. IGFBP5 knockdown partly abrogated tumor suppressive effect of PKNOX2, indicating that the function(s) of PKNOX2 are dependent on IGFBP5. IGFBP5 promoted PKNOX2-mediated up-regulation of p53. As a consequence, p53 transcription target genes were coordinately up-regulated in PKNOX2-expressing GC cells, leading to tumor suppression. In summary, our results identified PKNOX2 as a tumor suppressor in gastric cancer by activation of IGFBP5 and p53 signaling pathways. PKNOX2 promoter hypermethylation might be a biomarker for the poor survival of gastric cancer patients.

Metastasis of breast cancer represents the major reason for its poor prognosis, leading to high mortality. In breast cancer, a tumor suppressor gene TP53 is commonly mutated. TP53 mutation leads to an altered expression of various genes, an event that is associated with aggressive tumor and is a strong independent marker for survival. In this study, we identified a novel p53 target gene, immunoglobulin superfamily 9 (IGSF9). IGSF9 is generally down-regulated in breast cancer tissues. Loss of IGSF9 is associated with frequent metastasis and poor prognosis of breast cancer patients. Wild-type p53, but not R175H mutant, trans-activates the transcription of IGSF9 via binding to its promoter (-137 to -131 bp), inhibits epithelial-mesenchymal transition (EMT), consequently the inhibition of breast cancer cells migration and invasion. IGSF9 interacts with focal adhesion kinase (FAK) and inhibits FAK/AKT signaling activity. PND1186, FAK inhibitor, inhibits breast cancer metastasis induced by IGSF9 knockdown in vitro and in vivo. Taken together, IGSF9 is trans-activated by p53 and inhibits breast cancer metastasis by modulating FAK/AKT signaling pathway. IGSF9 could serve as a prognostic marker and potential therapeutic target for breast cancer.

Cell-division cycle 20 (CDC20) is overexpressed in a variety of tumor cells and is negatively regulated by wild-type p53 (wtp53). Our previous study uncovered that CDC20 was upregulated and associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) based on bioinformatics analysis. Dysregulation of the MDM2-p53 is a major mechanism to promote DLBCL. Thus, we hypothesized that CDC20 could be a downstream gene of the MDM2-p53 signaling pathway. However, the clinical significance and mechanistic role of a novel MDM2-p53-CDC20 signaling pathway in DLBCL have still remained unclear.

Brain arteriovenous malformations (AVMs) which associate with angiogenesis due to local hypertension, chronic cerebral ischaemia and tissue hypoxia usually lead to haemorrhage, however, the therapeutic medicine for the disease is still lacking. 2-methoxyestradiol (2-ME) has been shown effective in the anti-angiogenic treatment. This study was conducted to examine whether and how 2-ME could improve the vascular malformations. Intracranial venous hypertension (VH) model produced in adult male Sprague-Dawley rats and culture of human umbilical vein endothelial cells (HUVECs) at the anoxia condition were used to induce in vivo and in vitro angiogenesis, respectively. Lentiviral vectors of ID-1 and p53 genes and of their siRNA were intracranially injected into rats and transfected into HUVECs to overexpress and down-regulate these molecules. 2-ME treatment not only reduced the in vivo progression of brain tissue angiogenesis in the intracranial VH rats and the in vitro increases in microvasculature formation, cellular migration and HIF-1α expression induced by anoxia in HUVECs but also reversed the up-regulation of ID-1 and down-regulation of p53 in both the in vivo and in vitro angiogenesis models. All of the anti-angiogenesis effects of 2-ME observed in VH rats and anoxic HUVECs were abrogated by ID-1 overexpression and p53 knockdown. Our data collectively suggest that 2-ME treatment inhibits hypoxia/anoxia-induced angiogenesis dependently on ID-1 down-regulation and p53 up-regulation, providing a potential alternative medical treatment for un-ruptured AVM patients.

Myocardial ischemia-reperfusion (I/R) is a severe disease，but its underlying mechanism is not fully elucidated and no effective clinical treatment is available. Utilizing intracellular peptidomics, we identified a novel native peptide PDRL23A (Peptide Derived from RPL23A), that is intimately related to hypoxic stress. We further show that PDRL23A effectively alleviates hypoxia-induced cardiomyocyte injury in vitro, along with improvements in mitochondrial function and redox homeostasis, including ROS accumulation, oxidative phosphorylation, and mitochondrial membrane potential. Strikingly, the in vivo results indicate that, short-term pretreatment with PDRL23A could effectively inhibit I/R-induced cardiomyocyte death, myocardial fibrosis and decreased cardiac function. Interestingly, PDRL23A was found to interact with 60 S ribosomal protein L26 (RPL26), hampering RPL26-governed p53 translation, and resulting in a reduction in the level of p53 protein, which in turn reduced p53-mediated apoptosis under hypoxic conditions. Collectively, a native peptide, PDRL23A, which translationally regulates p53 to protect against myocardial I/R injury, has been identified for the first time. Our findings provide insight into the adaptive mechanisms of hypoxia and present a potential new treatment for myocardial I/R.

Atopic dermatitis (AD) is a common, chronic inflammatory dermatosis with relapsing eruptions. Our study used bioinformatics to find novel candidate differentially expressed genes (DEGs) and predicted miRNAs between AD patients and healthy controls. The Mesh term "atopic dermatitis" was retrieved to obtain DEGs in GEO datasets. DEGs between AD patients and healthy controls were analyzed using GEO2R. Overlapping DEGs between different datasets were obtained with use of Draw Venn software. GO and KEGG enrichment analyses were conducted by the use of DAVID. STRING and miRWalk were used to individually analyze PPI networks, interactions of candidate genes and predicted miRNAs. A total of 571 skin samples, as retrieved from 9 databases were assessed. There were 225 overlapping DEGs between lesioned skin samples of AD patients and that of healthy controls. Nineteen nodes and 160 edges were found in the largest PPI cluster, consisting of 17 up-regulated and 2 down-regulated nodes. Two KEGG pathways were identified, including the cell cycle (CCNB1, CHEK1, BUB1B, MCM5) and p53 (CCNB1, CHEK1, GTSE1) pathways. There were 56 nodes and 100 edges obtained in the miRNA-target gene network, with has-miR-17-5p targeted to 4 genes and has-miR-106b-5p targeted to 3 genes. While these findings will require further verification as achieved with experiments involving in vivo and in vitro modles, these results provided some initial insights into dysfunctional inflammatory and immune responses associated with AD. Such information offers the potential to develop novel therapeutic targets for use in preventing and treating AD.

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.

Genome-wide physical protein-protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.

Curcumin, a natural polyphenol antioxidant extracted from the root of turmeric (Curcuma longa), can induce apoptosis and DNA demethylation in several types of cancer cells. However, the mechanism of its anticancer potentials and DNA demethylation effects and the potential relationships between these outcomes have not been clearly elucidated. In the present study, the effects of curcumin on the proliferation, colony formation, and migration of human gastric cancer cells (hGCCs) were explored. Reactive oxygen species (ROS) levels, mitochondrial damage, DNA damage, and apoptosis of curcumin-treated hGCCs were analyzed. Changes in the expression of several genes related to DNA damage repair, the p53 pathway, cell cycle, and DNA methylation following curcumin treatment were also evaluated. We observed that curcumin inhibited the proliferation, colony formation, and migration of hGCCs in a dose- and time-dependent fashion. A high concentration of curcumin elevated ROS levels and triggered mitochondrial damage, DNA damage, and apoptosis of hGCCs. Further, curcumin-induced DNA demethylation of hGCCs was mediated by the damaged DNA repair-p53-p21/GADD45A-cyclin/CDK-Rb/E2F-DNMT1 axis. We propose that the anticancer effect of curcumin could largely be attributed to its prooxidative effect at high concentrations and ROS elevation in cancer cells. Moreover, we present a novel mechanism by which curcumin induces DNA demethylation of hGCCs, suggesting the need to further investigate the demethylation mechanisms of other DNA hypomethylating drugs.

Circular RNA (circRNA), a kind of special endogenous RNA, has been shown to be implicated in crucial biological processes of multiple cancers as a gene regulator. However, the functional roles of circRNAs in breast cancer (BC) remain to be poorly explored, and relatively incomplete knowledge of circRNAs handles the identification and prediction of BC-related circRNAs. Towards this end, we developed a systematic approach to identify circRNA modules in the BC context through integrating circRNA, mRNA, miRNA, and pathway data based on a non-negative matrix factorization (NMF) algorithm. Thirteen circRNA modules were uncovered by our approach, containing 4164 nodes (80 circRNAs, 2703 genes, 63 miRNAs and 1318 pathways) and 67,959 edges in total. GO (Gene Ontology) function screening identified nine circRNA functional modules with 44 circRNAs. Within them, 31 circRNAs in eight modules having direct relationships with known BC-related genes, miRNAs or disease-related pathways were selected as BC candidate circRNAs. Functional enrichment results showed that they were closely related with BC-associated pathways, such as 'KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAYS IN CANCER', 'REACTOME IMMUNE SYSTEM' and 'KEGG MAPK SIGNALING PATHWAY', 'KEGG P53 SIGNALING PATHWAY' or 'KEGG WNT SIGNALING PATHWAY', and could sever as potential circRNA biomarkers in BC. Comparison results showed that our approach could identify more BC-related functional circRNA modules in performance. In summary, we proposed a novel systematic approach dependent on the known disease information of mRNA, miRNA and pathway to identify BC-related circRNA modules, which could help identify BC-related circRNAs and benefits treatment and prognosis for BC patients.

This study aims to analyze the targets of the effective active ingredients of Scutellariae radix-Coptidis rhizoma drug pair (SCDP) in ulcerative colitis (UC) by network pharmacology and molecular docking and to explore the associated therapeutic mechanism. The effective active ingredients and targets of SCDP were determined from the TCMSP database, and the drug ingredient-target network was constructed using the Cytoscape software. The disease targets related to UC were searched in GeneCards, DisGeNET, OMIM, and DrugBank databases. Then, the drug ingredient and disease targets were intersected to construct a protein-protein interaction network through the STRING database. The Metascape database was used for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the predicted targets of SCDP for UC. The Autodock software was used for molecular docking between the main active ingredient and the core target to evaluate the binding ability. SCDP has 43 effective active ingredients and 134 intersection targets. Core targets included AKT1, TP53, IL-6, VEGFA, CASP3, JUN, TNF, MYC, EGFR, and PTGS2. GO functional enrichment analysis showed that biological process was mainly associated with a cytokine-mediated signaling pathway, response to an inorganic substance, response to a toxic substance, response to lipopolysaccharide, reactive oxygen species metabolic process, positive regulation of cell death, apoptotic signaling pathway, and response to wounding. KEGG enrichment analysis showed main pathway concentrations were related to pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, bladder cancer, IL-17 signaling pathway, apoptosis, p53 signaling pathway, and PI3K-Akt signaling pathway. The drug active ingredient-core target-key pathway network contains 41 nodes and 108 edges, of which quercetin, wogonin, baicalein, acacetin, oroxylin A, and beta-sitosterol are important active ingredients; PTGS2, CASP3, TP53, IL-6, TNF, and AKT1 are important targets; and the pathways involved in UC treatment include pathways in cancer, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic, apoptosis, IL-17 signaling pathway and herpes simplex infection. The active ingredient has a good binding capacity to the core target. SCDP key active ingredients are mainly quercetin, wogonin, baicalein, acacetin, oroxylin A, and beta-sitosterol, which function mainly by regulating targets, such as PTGS2, CASP3, TP53, IL-6, TNF, and AKT1, and are associated with multiple signaling pathways as pathways in cancer, PI3K-Akt signaling pathway, apoptosis, IL-17 signaling pathways.

Macrophages are increased in human benign prostatic hyperplasia and prostate cancer. We generate a Pb-Csf1 mouse model with prostate-specific overexpression of macrophage colony-stimulating factor (M-Csf/Csf1). Csf1 overexpression promotes immune cell infiltration into the prostate, modulates the macrophage polarity in a lobe-specific manner, and induces senescence and low-grade prostatic intraepithelial neoplasia (PIN). The Pb-Csf1 prostate luminal cells exhibit increased stem cell features and undergo an epithelial-to-mesenchymal transition. Human prostate cancer patients with high CSF-1 expression display similar transcriptional alterations with the Pb-Csf1 model. P53 knockout alleviates senescence but fails to progress PIN lesions. Ablating epithelial Gp130 but not Il1r1 substantially blocks PIN lesion formation. The androgen receptor (AR) is downregulated in Pb-Csf1 mice. ChIP-Seq analysis reveals altered AR binding in 2482 genes although there is no significant widespread change in global AR transcriptional activity. Collectively, our study demonstrates that increased macrophage infiltration causes PIN formation but fails to transform prostate cells.

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

The present study aimed to identify the association between microRNA (miRNA/miR)‑31a‑5p and the development of hypertension, and its potential molecular mechanism. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses were performed to validate the candidate miRNA and genes involved in hypertension, following which an online miRNA database search, luciferase assay, and RT‑qPCR and western blot analyses were performed to confirm the interaction between miR‑31a‑5p and TP53. A MTT assay and flow cytometric analysis were utilized to determine the effect of miR‑31a‑5p on cell growth and apoptosis. The results revealed that miR‑31a‑5p and TP53 were the candidate miRNA and gene regulating hypertension, and that TP53 was the virtual target gene of miR‑31a‑5p with a binding site located in the TP53 3' untranslated region (3'UTR). It was confirmed by luciferase activity that miR‑31a‑5p markedly reduced the luciferase activity of the Luc‑wild‑type‑TP53‑3'UTR, whereas the mutated putative miR‑31a‑5p binding located on the TP53‑3'UTR was found to eliminate such an inhibitory effect. miR‑31a‑5p had no effect on specificity protein 1, E2F transcription factor 2 or forkhead box P3 luciferase activity. Smooth muscle cells collected from spontaneously hypertensive rats treated with gold nano‑particles containing anti‑rno‑miR‑31a‑5p exhibited a lower growth rate and a higher apoptotic rate. The results of the RT‑qPCR and western blot analyses showed that miR‑31a‑5p negatively regulated the expression of TP53, and transfection with the hsa‑miR‑31a‑5p mimic significantly promoted cell growth and inhibited cell apoptosis, whereas transfection with the anti‑hsa‑miR‑31a‑5p mimic significantly suppressed cell growth and induced cell apoptosis. Taken together, these findings indicated that miR‑31a‑5p is involved in hypertension via the accelerated proliferation of arterial smooth muscle cells and inhibition of apoptosis through targeting TP53.

Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights into future investigations aimed to explore the in-depth mechanisms of miRNAs and other small RNAs including piRNAs in penile carcinogenesis regulation and effective target-specific theragnosis.

It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers.