👤 Shurong Yang

Micropapillary-predominant adenocarcinoma (MPA) of the lung is associated with extensive lymph node involvement and rapid terminal metastasis. However, this subtype has been recognized for only a few years, and there have been few studies of the molecular mechanisms associated with its highly invasive behaviors. The present study utilized immunohistochemical staining of surgically resected tissue blocks of MPA and lepidic-predominant lung adenocarcinoma to quantify the expression of specific biological markers in the WNT/β-catenin pathway and evaluate their influence on the lymph nodes invasion of these two types of lung adenocarcinomas. Our findings revealed that disruption of the cell membrane cadherin-catenin complex, which weakens the tumor cell adherence of MPA, was caused by the dissociation of β-catenin from the cadherin-catenin complex and the subsequent accumulation of β-catenin in the cytoplasm. This caused abnormal activation of the WNT/β-catenin pathway. We also found that Wnt-1-specific overexpression and Axin1 inhibition in MPA could explain the redistribution and cytoplasmic retention of β-catenin. Collectively, these findings suggest that an abnormality in the WNT/β-catenin pathway could enhance the invasiveness of MPA through the overexpression of Wnt-1 and downregulation of Axin1 molecules. Our data support the need for further research regarding the WNT/β-catenin pathway and the need to develop novel targeted therapies for restoration of tumor cell adherence and improvement of the prognosis of MPA. Show less

Triple-negative breast cancer (TNBC) is one subtype of breast cancer, which is characterized by an aggressive disease. It is commonly accompanied with extremely poor prognosis because of no available molecularly targeted therapy. Thus, understanding the detailed molecular mechanisms of TNBC is urgently needed. The levels of Axis inhibition protein 1 (Axin1), Cyclin D1, c-Myc, and miR-124-3p.1 were measured by quantitative real-time PCR (qRT-PCR). Furthermore, the breast cancer cell proliferation was measured by CCK-8 assay, colony formation assays, and EdU staining. Xenograft model was used to show the tumor genesis of breast cancer cells. The regulatory function of miR-124-3p.1 on Wnt/β-catenin signaling activation through directly targeting Axin1 was proven using qRT-PCR, Western blot analysis, and dual-luciferase reporter assay. To further assess the clinical significance of miR-124-3p.1 in the prognosis of breast cancer patients, we performed Kaplan-Meier survival analysis and log-rank tests. miR-124-3p.1 expression was elevated in advanced TNBC patients, and high miR-124-3p.1 predicts poor overall survival in TNBC patients. Further data showed that miR-124-3p.1 downregulation diminished, while miR-124-3p.1 upregulation increased the growth of TNBC cells in vitro and in vivo. Finally, we proved that miR-124-3p.1 exerted its function via targeting tumor suppressor gene Axin1 and activating the Wnt signaling pathway. In summary, all the results demonstrate that miR-124-3p.1 promotes TNBC cell growth by controlling Axin1, suggesting that targeting miR-124-3p.1 might offer an effective therapeutic strategy for TNBC in the future. Show less

The dysregulation of ubiquitin ligase is the cause of many human diseases. Tripartite motif protein 32 (TRIM32) is an E3 ubiquitin ligase whose role in nucleus pulposus (NP) cell apoptosis is unclear. The expression of TRIM family protein and β-catenin in 40 NP tissue samples was detected by RT-PCR. Interleukin (IL)-1β or tumor necrosis factor (TNF)-α was used to treat rat NP cells. Knockdown and overexpression of Show less

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake. Show less

Lymphoma, a malignant tumor, is mainly characterized by painless lymph node enlargement and hepatosplenomegaly. At present, lymphoma is mainly treated by radiation, chemical drugs, bone marrow transplantation and surgery. However, due to the high degree of heterogeneity, lymphomas are highly different in terms of treatment intensity and prognosis. This study is designed to investigate the function of tripartite motif-containing 11 (TRIM11) in lymphomas. The expression of TRIM11 in lymphoma tissues and multiple lymphoma cell lines was respectively detected by microarray immunohistochemistry, real-time PCR and Western blotting. After TRIM11 knockdown, overexpression, or β-catenin inhibitor XAV939 treatment, proliferation, apoptosis and cell cycle progression, as well as expression of related-genes were detected. Next, Co-Immunoprecipitation (Co-IP) and ubiquitination detection were performed. Elevated expression of tripartite motif-containing 11 (TRIM11) was observed in lymphoma tissues and multiple lymphoma cell lines (Raji, Jurkat, U937 and Hut78). Knockdown of TRIM11 in lymphoma cells significantly suppressed cell proliferation and prevented cell cycle progression from entering S or G2 phase. Concurrently, the expression of β-catenin, Cyclin D1 and c-Myc proteins in TRIM11-silenced lymphoma cells was decreased, while Axin1 was increased. In addition, TRIM11 overexpression had an opposite effect to TRIM11 knockdown, and a β-catenin inhibitor, XAV939, potently attenuated the induction of TRIM11 on lymphoma cells. Co-IP assay showed the interaction of TRIM11 and Axin1, and TRIM11 knockdown inhibited Axin1 ubiquitination degradation. Together all, the results suggested that TRIM11 may be an oncogene in lymphomas, which involving the activation of the β-catenin signaling and the ubiquitination degradation of Axin1. Show less

Hepatocellular carcinoma (HCC) is one of the most prevalent human malignancies worldwide and has high morbidity and mortality. Elucidating the molecular mechanisms underlying HCC recurrence and metastasis is critical to identify new therapeutic targets. This study aimed to determine the roles of aminopeptidase N (APN, also known as CD13) in HCC proliferation and metastasis and its underlying mechanisms. We detected APN expression in clinical samples and HCC cell lines using immunohistochemistry, flow cytometry, real-time PCR, and enzyme activity assays. The effects of APN on HCC metastasis and proliferation were verified in both in vitro and in vivo models. RNA-seq, phosphoproteomic, western blot, point mutation, co-immunoprecipitation, and proximity ligation assays were performed to reveal the potential mechanisms. We found that APN was frequently upregulated in HCC tumor tissues and high-metastatic cell lines. Knockout of APN inhibited HCC cell metastasis and proliferation in vitro and in vivo. Functional studies suggested that a loss of APN impedes the ERK signaling pathway in HCC cells. Mechanistically, we found that APN might mediate the phosphorylation at serine 31 of BCKDK (BCKDK Show less

The animal model of hyperlipidemia in rats was established to investigate the lipid-lowering effect and mechanism of Danhong Injection on hyperlipidemic rats. SD rats were selected as the research object. The rats in normal group were fed with basic diet, and the rats in other groups were fed with high-fat diet to establish hyperlipidemia model. The successfully modeled rats were randomly divided into model group, Danhong Injection low, medium, high dose(1.0, 2.0, 4.0 mL·kg~(-1)) groups, and simvastatin(2.0 mg·kg~(-1)) group. Danhong Injection groups received intraperitoneal administration, and simvastatin group received intragastrical administration, once a day for 4 weeks. At the first, second, third, and fourth weekends after administration, blood was collected from the orbital vein to detect the levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C), and then the atherosclerosis index(AI) was calculated. After 4 weeks of administration, the animals were sacrificed, and their heart, liver, spleen, lung, kidney and adipose tissue were extracted and weighed respectively to calculate the organ index of each group. The expressions of acyl-coaoxidase 1(Acox1), adenosine 5'-monophosphate(AMP)-activated protein kinase alpha(AMPK-α), bile salt export pump(BSEP), peroxisome proliferator-activated receptor gamma(PPAR-γ), catalase(CAT) and superoxide dismutase(SOD) mRNA in liver tissues were detected by fluorescence quantitative PCR; the content of cholesteryl ester transfer protein(CETP) and lecithin cholesterol acyltransferase(LCAT) in serum was detected by ELISA. The results showed that as compared with the normal group, the levels of serum TC, TG and LDL-C in the model group were significantly increased, and the level of HDL-C was significantly decreased, indicating that the hyperlipidemia rat model was successfully constructed. As compared with the model group, Danhong Injection could decrease the contents of TC, TG, LDL-C and increase the content of HDL-C in hyperlipidemia rats; reduce the body weight of hyperlipidemia rats, and reduce the liver weight, liver index, fat weight and fat index; it had no significant effect on the main organ indexes such as heart, spleen, lung and kidney; but it could increase the expressions of Acox1, AMPK-α, BSEP, PPAR-γ, CAT and SOD mRNA in liver tissues of rats; it could also reduce the level of CETP and increase the level of LCAT in serum; and the regulatory effect of Danhong Injection groups all showed a dose-dependent effect. It can be concluded that Danhong Injection can regulate the blood lipid contents, reduce the blood lipid levels and alleviate the accumulation of body fat in rats with hyperlipidemia. The mechanism may be related to inhibiting lipid metabolism disorder and oxidative stress induced by high-fat diet feeding, and improving the imbalance of lipid transport system. Show less

Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes. Show less

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition. Show less

Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10 Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases. Show less

DLG2, also known as postsynaptic density protein-93 (PSD-93) or chapsyn-110, is an excitatory postsynaptic scaffolding protein that interacts with synaptic surface receptors and signaling molecules. A recent study has demonstrated that mutations in the DLG2 promoter region are significantly associated with autism spectrum disorder (ASD). Although DLG2 is well known as a schizophrenia-susceptibility gene, the mechanisms that link DLG2 gene disruption with ASD-like behaviors remain unclear. Mice lacking exon 14 of the Dlg2 gene (Dlg2 Dlg2 These results suggest that homozygous Dlg2 deletion in mice leads to ASD-like behavioral phenotypes, including social deficits and increased repetitive behaviors, as well as reductions in excitatory synaptic input onto dorsolateral spiny projection neurons, implying that the dorsal striatum is one of the brain regions vulnerable to the developmental dysregulation of DLG2. Show less

Postsynaptic density protein-93 (PSD-93) plays an important role in ischemic brain injury through N-methyl-D-aspartate receptor (NMDAR)-triggered neurotoxicity. GTPase-activating protein for Ras (SynGAP) is a GAP specifically expressed in the central nervous system to regulate nerve development and synaptic plasticity. However, the link between PSD-93 and SynGAP and their role in ischemic brain injury remain elusive. Here, we showed that PSD-93 interacted with SynGAP and mediated SynGAP ubiquitination and degradation following ischemic brain injury. Proteasome inhibitor MG-132 could reverse the decrease of SynGAP protein level in wild-type mice following cerebral ischemia reperfusion through inhibiting SynGAP ubiquitination. Furthermore, NMDA receptor inhibitor MK801 could increase SynGAP protein level in wild-type mice following cerebral ischemia reperfusion. However, in PSD-93 knockout mice, MG-132 or NMDAR inhibitor had no significant effect on SynGAP expression. Both MG-132 and PSD-93 knockout reduced infarct volume and improved neurological deficit in mice at different time points after cerebral ischemia reperfusion. Furthermore, we identified that 670-685 amino acid sequence of SynGAP was essential to the binding of SynGAP to PSD-93, and designed a fusion peptide Tat-SynGAP (670-685aa) that could attenuate ischemic brain damage in wild-type mice. In conclusion, we provide the first evidence that PSD-93 directly interacts with SynGAP and mediates its ubiquitination and degradation to aggravate ischemic brain damage. Tat-SynGAP (670-685aa) may be considered as a candidate for treatment of acute ischemic stroke. Show less

A further understanding of the molecular mechanism of hepatocellular carcinoma (HCC) is necessary to predict a patient's prognosis and develop new targeted gene drugs. This study aims to identify essential genes related to HCC. We used the Weighted Gene Co-expression Network Analysis (WGCNA) and differential gene expression analysis to analyze the gene expression profile of GSE45114 in the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas database (TCGA). A total of 37 overlapping genes were extracted from four groups of results. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were performed on the 37 overlapping genes. Then, we used the STRING database to map the protein interaction (PPI) network of 37 overlapping genes. Ten hub genes were screened according to the Maximal Clique Centrality (MCC) score using the Cytohubba plugin of Cytoscape (including FOS, EGR1, EPHA2, DUSP1, IGFBP3, SOCS2, ID1, DUSP6, MT1G, and MT1H). Most hub genes show a significant association with immune infiltration types and tumor stemness of microenvironment in HCC. According to Univariate Cox regression analysis and Kaplan-Meier survival estimation, SOCS2 was positively correlated with overall survival (OS), and IGFBP3 was negatively correlated with OS. Moreover, the expression of IGFBP3 increased with the increase of the clinical stage, while the expression of SOCS2 decreased with the increase of the clinical stage. In conclusion, our findings suggest that SOCS2 and IGFBP3 may play an essential role in the development of HCC and may serve as a potential biomarker for future diagnosis and treatment. Show less

To explore the molecular regulatory mechanisms underlying fibroblast differentiation and dysfunction in the development of adolescent idiopathic scoliosis (AIS) in an effort to identify candidate therapeutic targets for AIS. The GSE110359 dataset, obtained from the bone marrow stromal cells of 12 AIS patients and five healthy controls, was retrieved from the GEO database. The data were preprocessed and differentially expressed genes (DEGs) were identified. KEGG pathway and Gene Ontology (GO)-Biological Process (BP) enrichment analyses were performed to identify the function of the DEGs. A protein-protein interaction (PPI) and a microRNA-transcription factor (TF)-target co-regulatory network were constructed to identify hub genes in the development of AIS. In addition, hub DEGs were evaluated by quantitative PCR (qPCR) and immunohistochemical staining. A total of 188 DEGs including 100 up-regulated and 88 down-regulated genes were obtained. The up-regulated DEGs were related to "p53 signaling pathway", "FoxO signaling pathway", and "cGMP-PKG signaling pathway" terms, while the down-regulated DEGs were significantly enriched in seven terms including "protein processing in endoplasmic reticulum". The key up-regulated genes, PRKG1, CCNG2, and KAT2B, and the key down-regulated genes, MAP2K1 and DUSP6, were identified by the PPI and miRNA-TF-Target regulatory network analyses. mRNA expression patterns for PRKG1, DUSP6, and KAT2B were successfully verified by qPCR. In addition, PRKG1 protein levels were found to be elevated during the immunohistochemical analysis. Increased expression of PRKG1 in AIS patients might be an attractive therapeutic target for AIS. However, further gain or loss-of-function studies should be conducted. Show less

Understanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation RNA Sequencing (TRAPseq) in the Fgf20 lineage. We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number. We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice. Show less

Reduced plasma circular RNA DYM (circDYM) has been detected in patients with major depressive disorder (MDD). Mechanism research has demonstrated that circDYM, acting as a microRNA-9 sponge, suppressed microglial activation by increasing Heat Shock Protein 90 ubiquitination, indicating that circDYM could be a potential biomarker of MDD. Thirty-two normal controls (NCs) and 60 MDD patients were recruited. Enrolled patients were randomly allocated to the real or sham repetitive transcranial magnetic stimulation (rTMS) group, followed by continuous five-day visual cortical rTMS or sham treatment. All participants underwent multidimensional neuropsychological assessments and detection of circDYM levels. Initial scores on all emotional and psychosocial assessments in MDD were significantly different from those of NCs. As compared with the NC group, baseline plasma circDYM levels in MDD patients decreased remarkably (p=0.030) and showed significant positive correlations with the scores of the 24-item Hamilton Depression Rating Scale (r=0.318, p=0.031) and retardation subscale (r=0.323, p=0.029). The increase in circDYM was noteworthy after rTMS (p=0.006), while downregulation with no statistical significance was observed after sham treatment (p=0.170). It was not estimated on the correlation between plasma circDYM levels and long-term efficacy of rTMS. The mechanism of upregulated circDYM expression in response to visual cortical rTMS remained unrevealed, and the sample size was relatively small. This study verified the reduced circDYM levels in MDD patients, and further determined the upregulated circDYM expression after rTMS treatment, revealing the potential of circDYM as a biomarker for MDD diagnosis and antidepressant effect of visual cortical rTMS. Show less

Macrophages differentiated into a classically activated (M1) or alternatively activated phenotype (M2) in infection and tumor, but the precise effects of glycolysis and oxidative phosphorylation (OXPHOS) metabolic pathway remain unclear. Herein, the effects of glycolysis or OXPHOS on macrophage polarizations were investigated using a pharmacological approach in mice. 2-Deoxy-D-glucose (2-DG) treatments, which blocks the key enzyme hexokinase of glycolysis, efficiently inhibits a specific switch to M1 lineage, decreasing the secretion of pro-inflammatory cytokines and expressions of co-stimulatory molecules associated with relieving infectious inflammation Show less

This study aimed to explore more gene markers associated with glioma or its prognosis. The glioma-related RNAseq data from the Gene Expression Omnibus database and The Cancer Genome Atlas dataset in UCSC Xena database were downloaded. There was a total of 971 tumor samples and 102 normal samples in the 2 datasets. The differentially expressed genes (DEGs) data between tumor and normal samples were analyzed, on which were then performed function and pathway enrichment analyses. Pearson correlation coefficient between DEGs was calculated to construct the coexpression network. Finally, prognostic genes were screened. A total of 634 upregulated and 769 downregulated DEGs were identified between tumor and control groups. These DEGs were significantly involved in 15 upregulated pathways, such as p53 signaling pathway, and 16 downregulated pathways, such as neuroactive ligand-receptor interaction, and cell adhesion molecules. In the coexpression network, pseudouridine synthase 7 ( Show less

Hypercholesterolemia- and atherosclerosis-caused vasomotor property dysfunction may be involved in many clinic manifestations of atherosclerosis, including angina, acute myocardial infarction, and sudden cardiac death. However, its underlying mechanism is not clear. The endothelial glycocalyx is a protective surface layer on the endothelial cells, serving as a molecular sieve, cell adhesion modulator, and mechanosensor for blood flow. In the present study, we demonstrated by confocal microscopy in Sprague-Dawley (SD) male rats fed a 12-wk high-cholesterol diet (HC) compared with the normal diet (NC) that the dimension of the endothelial glycocalyx reduced significantly in both the common carotid artery (2.89 ± 0.41 µm and 3.25 ± 0.44 μm, respectively) and the internal sinus region (2.35 ± 0.07 µm and 3.46 ± 0.86 μm, respectively). Furthermore, we showed by real-time PCR that this dimension modification of endothelial glycocalyx may be attributed to a significant downregulation of heparan sulfate proteoglycan (HSPG)-related genes, including syndecan-3, glypican-1, and EXT1, not resulting from an enhanced shedding of sulfated glycosaminoglycans (sGAGs) from the vessel wall to the plasma. Meanwhile, the mean contraction and relaxation forces of the common carotid artery with responses to norepinephrine (NE) and acetylcholine (ACh) decreased ~0.34- and 0.13-fold, respectively, accompanied by a lower level of nitric oxide (NO) release. These findings suggest that the atherogenic high cholesterol diet diminished endothelial glycocalyx and disturbed the local NO release, thus contributing to the impaired vasomotor properties of the vessel. Show less

Chromothripsis is a type of chaotic complex genomic rearrangement caused by a single event of chromosomal shattering and repair processes. Chromothripsis is known to cause rare congenital diseases when it occurs in germline cells, however, current genome analysis technologies have difficulty in detecting and deciphering chromothripsis. It is possible that this type of complex rearrangement may be overlooked in rare-disease patients whose genetic diagnosis is unsolved. We applied long read nanopore sequencing and our recently developed analysis pipeline dnarrange to a patient who has a reciprocal chromosomal translocation t(8;18)(q22;q21) as a result of chromothripsis between the two chromosomes, and fully characterize the complex rearrangements at the translocation site. The patient genome was evidently shattered into 19 fragments, and rejoined into derivative chromosomes in a random order and orientation. The reconstructed patient genome indicates loss of five genomic regions, which all overlap with microarray-detected copy number losses. We found that two disease-related genes RAD21 and EXT1 were lost by chromothripsis. These two genes could fully explain the disease phenotype with facial dysmorphisms and bone abnormality, which is likely a contiguous gene syndrome, Cornelia de Lange syndrome type IV (CdLs-4) and atypical Langer-Giedion syndrome (LGS), also known as trichorhinophalangeal syndrome type II (TRPSII). This provides evidence that our approach based on long read sequencing can fully characterize chromothripsis in a patient's genome, which is important for understanding the phenotype of disease caused by complex genomic rearrangement. Show less

Prostate stem/progenitor cells (PrSCs) are responsible for adult prostate tissue homeostasis and regeneration. However, the related regulatory mechanisms are not completely understood. In this study, we examined the role of heparan sulfate (HS) in PrSC self-renewal and prostate regeneration. Using an in vitro prostate sphere formation assay, we found that deletion of the glycosyltransferase exostosin 1 (Ext1) abolished HS expression in PrSCs and disrupted their ability to self-renew. In associated studies, we observed that HS loss inhibited p63 and CK5 expression, reduced the number of p63+- or CK5+-expressing stem/progenitor cells, elevated CK8+ expression and the number of differentiated CK8+ luminal cells and arrested the spheroid cells in the G1/G0 phase of cell cycle. Mechanistically, HS expressed by PrSCs (in cis) or by neighboring cells (in trans) could maintain sphere formation. Furthermore, HS deficiency upregulated transforming growth factor β (TGFβ) signaling and inhibiting TGFβ signaling partially restored the sphere-formation activity of the HS-deficient PrSCs. In an in vivo prostate regeneration assay, simultaneous loss of HS in both epithelial cell and stromal cell compartments attenuated prostate tissue regeneration, whereas the retention of HS expression in either of the two cellular compartments was sufficient to sustain prostate tissue regeneration. We conclude that HS preserves self-renewal of adult PrSCs by inhibiting TGFβ signaling and functions both in cis and in trans to maintain prostate homeostasis and to support prostate regeneration. Show less

Aim of this study was to identify biomarkers between different grades of bladder cancer (BLCA) and its prognostic value. mRNA expression data from GSE32549 and GSE71576 were extracted for further analysis. Differentially expressed genes (DEGs) were identified using GEO2R web tool. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction (PPI) network were conducted to explore the function and relationship of DEGs. The Cancer Genome Atlas (TCGA) database was used for external validation and Gene set enrichment analysis (GSEA) analysis was used to further identify FADS1 pathways. Bladder cancer cells and patient specimens were used to further demonstrate the function of FADS1. Datasets from GEO identified a panel of DEGs. Functional enrichment analysis highlighted that DEGs were associated with nuclear division, spindle, cell cycle and p53 signaling pathway. External validation from TCGA demonstrated that FADS1 was an independent prognostic marker in BLCA patients. In cell lines and tumor specimen analysis, FADS1 was overexpressed in the tumor specimen, compared with adjacent tissues, and positively correlated with tumor grade of BLCA. Moreover, FADS1 could enhance the proliferation ability and influence cell cycle of bladder cancer cells. FADS1 was an independent prognostic biomarker for BLCA and could confer the bladder cancer cells increased proliferation ability. Show less

Numerous evidences have shown that circular RNAs (circRNAs) play a key role in regulating the pathogenesis of cancer. However, the mechanism of circRNAs in urothelial carcinoma of bladder (UCB) remains largely unclear. In this study, we found circFAM114A2 was significantly downregulated both in UCB tissue specimens and cell lines, and the expression level was highly correlated with pathological TNM stage and grade. Functionally, overexpression of circFAM114A2 dramatically inhibited the migration, invasion and proliferation of UCB cells in vitro, and suppressed tumor growth in vivo. Mechanistically, we confirmed miR-762 was copiously pulled down by circFAM114A2 in 5637 and T24 cells. Fluorescence in situ hybridization (FISH) further indicated the cytoplasmic interactions between circFAM114A2 and miR-762. By using luciferase reporter assay, we found that miR-762 could directly target TP63. Subsequently, we found that circFAM114A2 might increase the expression of ∆NP63 (main isoform of TP63 in UCB) by sponging miR-762. Taken together, our results demonstrated that circFAM114A2 might serve as a competing endogenous RNA (ceRNA) of miR-762 in regulating the expression of ∆NP63, thus suppressed UCB progression through circFAM114A2/miR-762/∆NP63 axis. Show less

The extremely high proliferation rate of tumor cells contributes to pancreatic cancer (PC) progression. Runt-related transcription factor 1(RUNX1), a key factor in hematopoiesis that was correlated with tumor progression. However, the role of RUNX1 in PC proliferation was still unclear. We found that RUNX1 was significantly upregulated in PC tissues and its expression was negatively associated with prognosis of PC patients in a multicenter analysis according to immunohistochemical (IHC). RUNX1 downregulation in PC resulted in a significantly reduced cell proliferation rate, which was consistent with in vivo subcutaneous tumor formation assay results. RNA-seq and ChIP-seq results revealed that a portion of target genes, including HAP1, GPRC5B, PTPN21, VHL and EN2, were regulated by RUNX1, a finding successfully validated by ChIP-qPCR, qRT-PCR and Western blot. Subsequently, IHC and proliferation assays showed these target genes to be dysregulated in PC, affecting tumor growth. Our data suggest that RUNX1 plays an oncogenic role in tumor proliferation and is a potential prognostic biomarker and therapeutic target for PC. Show less

Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process. Show less

Although papillary thyroid carcinoma (PTC) has a favorable prognosis after surgical or medical treatment, its survival rate is still very low. Therefore, finding more reliable therapy methods to limit PTC is a necessity. Compelling evidence has implicated the role of microRNAs (miRNAs or miRs) in PTC. This study aims at investigating the possible effect of microRNA-599 (miR-599) on proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of PTC cells by targeting Hey2 gene. Differentially expressed genes/miRNAs associated with PTC were screened based on microarray analysis. Then, the expression of the candidate gene, as well as, the regulatory miRNA were detected in PTC cells, the related signaling pathway was verified. Afterward, the relationship between the miR and the candidate gene was verified by dual-luciferase reporter gene assay. Subsequently, the effects of overexpressed miR and silenced candidate gene on cell proliferation, cell apoptosis, EMT, migration, and invasion were detected. In PTC tissues and cells, miR-599 was downregulated while Hey2 expressed highly. Hey2 is a target gene of miR-559. In addition, the expression of Bax and E-cadherin was elevated while that of Hey2, Notch1, Delta-like1, Hes1, N1ICD, Jagged1, Snail, Slug, N-cadherin and Vimentin, and Bcl-2 was reduced in cells treated with upregulated miR-599 or downregulated Hey2. Moreover, miR-599 overexpression or Hey2 silencing inhibited cell proliferation, migration, invasion, along with EMT but promoted apoptosis. This study verified that miR-599 promotes apoptosis and represses proliferation, EMT of PTC cells through inactivating the Notch signaling pathway by downregulating Hey2, which has great clinical significance for PTC treatment. Show less

Aging-related cognitive ability impairments are one of the main threats to public health, and impaired hippocampal neurogenesis is a major cause of cognitive decline during aging. However, the regulation of adult neurogenesis in the hippocampus requires further study. Here, we investigated the role of microRNA-153 (miR-153), a highly conserved microRNA in mice and humans, in adult neurogenesis. During the passaging of neural stem cells (NSCs) in vitro, endogenous miR-153 expression was downregulated, with a decrease in neuronal differentiation ability. In addition, miR-153 overexpression increased the neurogenesis of NSCs. Further studies showed that miR-153 regulated neurogenesis by precisely targeting the Notch signaling pathway through inhibition of Jagged1 and Hey2 translation. In vivo analysis demonstrated that miR-153 expression was decreased in the hippocampi of aged mice with impaired cognitive ability, and that miR-153 overexpression in the hippocampus promoted neurogenesis and markedly increased the cognitive abilities of the aged mice. Overall, our findings revealed that miR-153 affected neurogenesis by regulating the Notch signaling pathway and elucidated the function of miR-153 in aging-related, hippocampus-dependent cognitive ability impairments, and neurodegenerative diseases. Show less

The advent of base editors (BEs) holds great potential for correcting pathogenic-related point mutations to treat relevant diseases. However, Cas9 nickase (nCas9)-derived BEs lead to DNA double-strand breaks, which can trigger unwanted DNA damage response (DDR). Here, we show that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. By fusing dCas12a with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A), we further develop the BEACON (base editing induced by human APOBEC3A and Cas12a without DNA break) system to achieve enhanced deamination efficiency and editing specificity. Efficient C-to-T editing is achieved by BEACON in mammalian cells at levels comparable to AncBE4max, with only low levels of DDR and minimal RNA off-target mutations. Importantly, BEACON induces in vivo base editing in mouse embryos, and targeted C-to-T conversions are detected in F0 mice. Show less