Identification of a dysregulated CircRNA-associated gene signature for predicting prognosis, immune landscape, and drug candidates in bladder cancer.

Increasing evidences have demonstrated that circular RNA (circRNAs) plays a an essential regulatory role in initiation, progression and immunotherapy resistance of various cancers. However, circRNAs have rarely been studied in bladder cancer (BCa). The purpose of this research is to explore new circRNAs and their potential mechanisms in BCa. A novel ceRNA-regulated network, including 87 differentially expressed circRNAs (DE-circRNAs), 126 DE-miRNAs, and 217 DE-mRNAs was constructed to better understanding the biological processes using Cytoscape 3.7.1 based on our previously high-throughput circRNA sequencing and five GEO datasets. Subsequently, five randomly selected circRNAs (upregulated circ_0001681; downregulated circ_0000643, circ_0001798, circ_0006117 and circ_0067900) in 20 pairs of BCa and paracancerous tissues were confirmed using qRT-PCR. Functional analysis results determined that 772 GO functions and 32 KEGG pathways were enriched in the ceRNA network. Ten genes (PFKFB4, EDNRA, GSN, GAS1, PAPPA, DTL, TGFBI, PRSS8, RGS1 and TCF4) were selected for signature construction among the ceRNA network. The Human Protein Atlas (HPA) expression of these genes were consistent with the above sequencing data. Notably, the model was validated in multiple external datasets (GSE13507, GSE31684, GSE48075, IMvigor210 and GSE32894). The immune-infiltration was evaluated by 7 published algorithms (i.e., TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL and EPIC). Next, Correlations between riskscore or risk groups and clinicopathological data, overall survival, recognized immunoregulatory cells or common chemotherapeutic agents of BCa patients were performed using wilcox rank test, chi-square test, cox regression and spearman's correlation analysis; and, these results are significant. According to R package "GSVA" and "clusterProfiler", the most significantly enriched HALLMARK and KEGG pathway was separately the 'Epithelial Mesenchymal Transition' and 'Ecm Receptor Interaction' in the high- vs. low-risk group. Additionally, the functional experiments in vitro also revealed that the overexpression of has_circ_0067900 significantly impaired the proliferation, migration, and invasion capacities of BCa cells. Collectively, the results of the current study provide a novel landscape of circRNA-associated ceRNA-regulated network in BCa. The ceRNA-associated gene model which was constructed presented a high predictive performance for the prognosis, immunotherapeutic responsiveness, and chemotherapeutic sensitivity of BCa. And, has_circ_0067900 was originally proposed as tumor suppressor for patients with BCa.

miR-146a contributes to atherosclerotic plaque stability by regulating the expression of TRAF6 and IRAK-1.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease. The vulnerable plaque of atherosclerotic can lead to the development of many diseases including acute coronary syndrome and coronary heart disease. It is well known that miR-146a is the key brake miRNA of the inflammatory signal transduction pathway. However, the effect of miR-146a on the stability of atherosclerotic plaque remains to be elucidated. METHODS AND RESULTS: We constructed animal models of atherosclerosis and foam cell models, and overexpressed and knocked-down miR-146a in models. After staining with Hematoxylin-Eosin (HE), Oil Red O, immunocytochemistry (IHC) and Sirius Red, we used the proportion of (Lipids area + Macrophage area) and (SMCs area + collagen area) to evaluate atherosclerotic plaque stability. TUNEL and flow cytometry were performed to detect the apoptosis level of macrophages. Levels of inflammatory factors were detected via ELISA assay. The results showed that miR-146a, IRAK1 and TRAF6 were abnormally expressed in plaques of atherosclerotic animals. Overexpression of miR-146a contributed to the stability of plaques that inhibited plaque formation, macrophage apoptosis and levels of pro-inflammatory factors. The Dual-luciferase reporter gene assay, IF and FISH were used to verify the regulatory mechanism of miR-146a on IRAK1 and TRAF6. We found that IRAK1 and TRAF6 promoted lipid uptake, apoptosis, and release of pro-inflammatory factors of RAW264.7 macrophages, whereas miR-146a restored RAW264.7 macrophages phenotype by inhibiting IRAK1 and TRAF6 expression. CONCLUSIONS: We display for the first time that miR-146a inhibits the formation of foam cells, RAW264.7 macrophage apoptosis and pro-inflammatory reaction through negative regulation of IRAK1 and TRAF6 expression, thereby enhancing the stability of atherosclerotic plaques.

miR-155 inhibits oxidized low-density lipoprotein-induced apoptosis in different cell models by targeting the p85α/AKT pathway.

The apoptosis of vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and macrophages directly causes the instability or rupture of atherosclerotic plaques. Accumulating evidence suggests that oxidized low-density lipoprotein (OxLDL) could induce apoptosis via endogenous or exogenous pathways. Interestingly, it has been reported that microRNA155 (miR-155) plays a pivotal role in the regulation of apoptosis. Here, we hypothesized that overexpression of miR-155 could inhibit OxLDL-induced apoptosis by targeting the p85α/AKT pathway. In this study, we established models of OxLDL-induced apoptosis in mouse VECs, VSMCs, and macrophages. Furthermore, we explored the effects of miR-155 expression on the apoptosis of different cells, and ultimately revealed whether miR-155 regulated apoptosis by targeting the p85α/AKT pathway. The results demonstrated that miR-155 inhibited p85α expression and attenuated VEC, VSMC, and macrophage apoptosis, at least in part by suppressing the expression of p85α-activated AKT to inhibit apoptosis. Our findings collectively suggested that miR-155 attenuated OxLDL-mediated apoptosis in different cells by targeting p85α, supporting its possible therapeutic role in atherosclerosis.

Inflammation-Related MicroRNAs Are Associated with Plaque Stability Calculated by IVUS in Coronary Heart Disease Patients.

OBJECTIVES: This study aimed to investigate the association between inflammation-related microRNAs (miR-21, 146a, 155) and the plaque stability in coronary artery disease patients. METHODS: The expression of miR-21, 146a, and 155 was measured by real-time PCR in 310 consecutive patients. The level of hs-CRP, IL-6, and IL-8 was measured by ELISA. The plaque stability of coronary stenotic lesions was evaluated with intravascular ultrasound (IVUS). RESULTS: (1) The levels of hs-CRP, IL-6, and IL-8 were significantly increased in the UAP and AMI groups compared with the CPS group (P < 0.01). (2) The expression of miR-21 and miR-146a in peripheral blood mononuclear cells (PBMCs) and plasma was significantly higher in CAD patients compared with non-CAD patients, whereas the miR-155 expression in PBMCs and plasma was significantly lower in patients with CAD. (3) The miR-21 expression in PBMCs was higher in UAP and AMI groups compared with CPS group. The miR-146a expression in PBMCs was higher in SAP, UAP, and AMI groups than in CPS group. Although the level of miR-155 in PBMCs was lower in SAP, UAP, and AMI groups than in CPS group. The expression patterns of miR-21, miR-146a, and miR-155 in plasma were consistent with those of PBMCs. (4) The expressions of miR-21 and miR-146a in PBMCs and plasma were significantly higher in the vulnerable plaque group than those in stable plaque group. While miR-155 in PBMCs and plasma was significantly lower in vulnerable plaque group compared with stable plaque group. (5) The levels of miR-21 and miR-146a in PBMCs and plasma were significantly higher in soft plaque group than in fibrous plaque group and calcified plaque group. However, miR-155 in PBMCs and plasma was significantly lower in soft plaque group. CONCLUSIONS: The expression of miR-21 and miR-146a are associated with the plaque stability in coronary stenotic lesions, whereas miR-155 expression is inversely associated with the plaque stability.

Twist induces epithelial-mesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network.

Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-ß, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity Pathways Analysis showed that integrin ß1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells.

LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway.

Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self-renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial-mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non-coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT-associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist-positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs-associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh-GLI1 pathway associated lncRNA-Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere-formation efficiency (MFE) and the self-renewal capacity in vitro, and oncogenicity in vivo in Twist-positive breast cancer cells are elevated. lncRNA-Hh silence in Twist-positive breast cells attenuates the activated Shh-GLI1 signaling and decreases the CSC-associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist-induced EMT cells to gain the CSC-like stemness properties.

[A negative correlation between Drosha expression and gastric adenocarcinoma malignancy and invasion].

OBJECTIVE: To investigate the role of Drosha expression in the progression of gastric adenocarcinoma and its impact on the invasive ability of SGC-7901 human gastric cancer cells. METHODS: Drosha expression was detected in 889 gastric carcinoma samples on tissue microarrays by immunohistochemistry staining and quantified by Image-Pro Plus software. Statistical analysis was used to evaluate the correlations between Drosha expression and the clinicopathological characteristics of the 889 tumor cases or the outcomes of 309 gastric adenocarcinoma patients. Drosha was knocked down in SGC-7901 cells by small interfering RNA (siRNA), and cell invasive ability was assessed by Transwell(TM) assay. RESULTS: Drosha expression was the highest in the well differentiated gastric adenocarcinoma (median absorbance, 0.4195), and the lowest in the poorly differentiated samples. Drosha expression was significantly related to Laren classification, tumor size, tumor invasion depth, lymph node metastasis, tumor pathological grade and stage. Patients in Drosha positive group had a higher survival rate than those in Drosha negative group. Silencing Drosha in SGC-7901 cells resulted in an enhanced cell invasion. CONCLUSION: Drosha expression was reduced gradually with the degrading histological differentiation of gastric adenocarcinoma, and the knock-down of Drosha expression could promote gastric adenocarcinoma cell invasion.

Twist promotes reprogramming of glucose metabolism in breast cancer cells through PI3K/AKT and p53 signaling pathways.

Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated ß1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the ß1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the ß1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR.

Oxidized ATM promotes abnormal proliferation of breast CAFs through maintaining intracellular redox homeostasis and activating the PI3K-AKT, MEK-ERK, and Wnt-ß-catenin signaling pathways.

Abnormal proliferation is one characteristic of cancer-associated fibroblasts (CAFs), which play a key role in tumorigenesis and tumor progression. Oxidative stress (OS) is the root cause of CAFs abnormal proliferation. ATM (ataxia-telangiectasia mutated protein kinase), an important redox sensor, is involved in DNA damage response and cellular homeostasis. Whether and how oxidized ATM regulating CAFs proliferation remains unclear. In this study, we show that there is a high level of oxidized ATM in breast CAFs in the absence of double-strand breaks (DSBs) and that oxidized ATM plays a critical role in CAFs proliferation. The effect of oxidized ATM on CAFs proliferation is mediated by its regulation of cellular redox balance and the activity of the ERK, PI3K-AKT, and Wnt signaling pathways. Treating cells with antioxidant N-acetyl-cysteine (NAC) partially rescues the proliferation defect of the breast CAFs caused by ATM deficiency. Administrating cells with individual or a combination of specific inhibitors of the ERK, PI3K-AKT, and Wnt signaling pathways mimics the effect of ATM deficiency on breast CAF proliferation. This is mainly ascribed to the ß-catenin suppression and down-regulation of c-Myc, thus further leading to the decreased cyclinD1, cyclinE, and E2F1 expression and the enhanced p21(Cip1) level. Our results reveal an important role of oxidized ATM in the regulation of the abnormal proliferation of breast CAFs. Oxidized ATM could serve as a potential target for treating breast cancer.

[Twist gene promotes the formation and migration of mammospheres of BT-549 breast cancer cells].

OBJECTIVE: To investigate the effect of Twist gene deficiency on the enrichment and migration of breast cancer stem-like cells (CSCs). METHODS: Twist gene in the BT-549 cell line was knocked down by shRNA interference technology, and the cell line BT-549-shVec was used as the negative control group. Interference efficiency was determined by real-time quantitative RT-PCR (qRT-PCR) and Western blotting. Mammospheres with the characteristics of stem-like cells were collected by long-term serum-free suspension cultivation. The migration ability of mammospheres was analyzed by Transwell(TM) assay. RESULTS: The BT-549-shTwist breast cancer cells were successfully established by shRNA interference technology. The enrichment and migration abilities of CSCs from the BT-549-shTwist cell line decreased significantly. CONCLUSION: Twist gene promotes the enrichment and migration of CSCs from the BT-549 cell line.

Complete nucleotide sequence of a norovirus GII.4 genotype: evidence for the spread of the newly emerged pandemic Sydney 2012 strain to China.

A newly emerged pandemic Sydney GII.4-like norovirus (NoV) (Jingzhou GII.4) was isolated in Jingzhou, China in April, 2013, demonstrating the rapid spread of the variant to China. The complete nucleotide sequence was compared with the prototype Sydney 2012 variant and its VP1 gene with that of Huzhou strain (isolated in January 2013 in Huzhou, China). The result demonstrates that the new variant has evolved rapidly, including mutations in the hypervariable P2 domain of the major capsid protein VP1. Our study also shows that the new Jingzhou GII.4 variant co-circulated with GII.3 and GI.2 at the same time, supporting further monitoring of the evolution of the new NoV variant in China.

[Construction of retroviral vector carrying Twist gene and its induction of epithelial-mesenchymal transition in human mammary epithelial cells].

OBJECTIVE: To construct a retroviral vector carrying Twist gene and investigate its effect on human mammary MCF10A epithelial cells. METHODS: Myc-Twist was digested from pcDNA3/myc-Twist and subcloned into the retroviral vector pBABE-puro to construct a recombinant plasmid (pBABE-myc-Twist). The inserted Twist gene was confirmed by restriction enzyme digestion and DNA sequencing. The plasmid pBABE-myc-Twist and the packaging plasmid pAmpho were co-transfected into HEK293T cells for packaging of retrovirus. Meanwhile, the control plasmid pBABE-puro and the packaging plasmid were co-transfected into the other HEK293T cells as a control group. Human mammary MCF10A epithelial cells were infected with the retroviruses carrying Twist gene or the controls, and selected by puromycin. The expression of Twist in the MCF10A-Twist and MCF10A-Vector cells was determined by RT-PCR and Western blotting. The expressions of epithelial-mesenchymal transition (EMT) marker proteins induced by Twist in MCF10A cells were detected using immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were analyzed by Transwell(R); assay. RESULTS: The myc-tagged Twist gene was correctly inserted into the retroviral expression vector as a recombinant plasmid (pBABE-myc-Twist) as identified by restriction analysis and DNA sequencing. The Twist gene was efficiently delivered into human mammary MCF10A epithelial cells by the retrovirus, resulting in the stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT-PCR and Western blotting, respectively. The expression of the epithelial biomarker E-cadherin was downregulated whereas, the mesenchymal marker vimentin upregulated in MCF10A-Twist cells as shown by immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were enhanced notably in MCF10A-Twist cells as compared with MCF10A-Vector control cells (P<0.01). CONCLUSION: Twist induces EMT of MCF10A cells and plays an important role in the promotion of cell migration and invasion.

[Bronchial artery embolization with polyvinyl alcohol foam and gelatinum sponge for serious hemoptysis].

OBJECTIVE: To evaluate the effect of bronchial artery embolization (BAE) with polyvinyl alcohol (PVA) foam and gelatinum sponge (GS) for serious hemoptysis. METHOD: Of 46 patients with severe hemoptysis, BAE with only GS was performed in 21 cases and with both PVA and GS in 25 cases. RESULT: The total efficacy rate of BAE was 91.3%; and comparable between PVA+GS and GS groups (92.0%; vs 90.5%;, P>0.05). The total recurrence rate after BAE was 26.2%;, but lower in PVA+GS group than in GS group (11.3%; vs 42.1%;, P<0.05). No serious complications occurred in these patients after BAE. CONCLUSION: BAE is effective and safe for management of serious hemoptysis, and treatment with PVA+GS may effectively lower the recurrence rate.