Hypoxia-inducible lncRNA MIR210HG interacting with OCT1 is involved in glioblastoma multiforme malignancy.

An insufficient oxygen supply within the intratumoral environment, also known as hypoxia, induces glioblastoma multiforme (GBM) invasion, stemness, and temozolomide (TMZ) drug resistance. Long noncoding (lnc)RNAs have been reported to be involved in hypoxia and GBM progression. However, their roles in hypoxic GBM malignancy are still unclear. We investigated the mechanisms of hypoxia-mediated lncRNAs in regulating GBM processes. Using The Cancer Genome Atlas (TCGA) and data mining, hypoxia-correlated lncRNAs were identified. A hypoxia-upregulated lncRNA, MIR210HG, locating in nuclear regions, predicted poor prognoses of patients and modulated hypoxia-promoted glioma stemness, TMZ resistance, and invasion. Depletion of hypoxic MIR210HG suppressed GBM and patient-derived cell growth and increased TMZ sensitivity in vitro and vivo. Using RNA sequencing and gene set enrichment analysis (GSEA), MIR210HG-upregulated genes significantly belonged to the targets of octamer transcription factor 1 (OCT1) transcription factor. The direct interaction between OCT1 and MIR210HG was also validated. Two well-established worse prognostic factors of GBM, insulin-like growth factor-binding protein 2 (IGFBP2) and fibroblast growth factor receptor 1 (FGFR1), were identified as downstream targets of OCT1 through MIR210HG mediation in hypoxia. Consequently, the lncRNA MIR210HG is upregulated by hypoxia and interacts with OCT1 for modulating hypoxic GBM, leading to poor prognoses. These findings might provide a better understanding in functions of hypoxia/MIR210HG signaling for regulating GBM malignancy.

miR-4286 is Involved in Connections Between IGF-1 and TGF-ß Signaling for the Mesenchymal Transition and Invasion by Glioblastomas.

The insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß signal pathways are both recognized as important in regulating cancer prognosis, such as the epithelial-to-mesenchymal transition (EMT) and cell invasion. However, cross-talk between these two signal pathways in glioblastoma multiforme (GBM) is still unclear. In the present study, by analyzing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE) 4412, GBM patients with higher IGF-1 levels exhibited poorer survival. Genes positively correlated with IGF-1 were enriched in EMT and TGF-ß signal pathways. IGF-1 treatment enhanced mesenchymal marker expressions and GBM cell invasion. A significant positive correlation was observed for IGF-1 with TGF-ß1 (TGFB1) or TGF-ß receptor 2 (TGFBR2), both of which participate in TGF-ß signaling and are risk genes in the GBM process. IGF-1 stimulation promoted both TGFB1 and TGFBR2 expressions. LY2157299, a TGF-ß signaling inhibitor, attenuated IGF-1-enhanced GBM cell invasion and mesenchymal transition. By analyzing IGF-1-regulated microRNA (miR) profiles, miR-4286 was found to be significantly downregulated in IGF-1-treated cells and could be targeted to both TGFB1 and TGFBR2. Overexpression of miR-4286 significantly attenuated expressions of the IGF-1-mediated mesenchymal markers, TGFB1 and TGFBR2. Using kinase inhibitors, only U0126 treatment showed an inhibitory effect on IGF-1-reduced miR-4286 and IGF-1-induced TGFB1/TGFBR2 expressions, suggesting that MEK/ERK signaling is involved in the IGF-1/miR-4286/TGF-ß signaling axis. Finally, our results suggested that miR-4286 might act as a tumor suppressive microRNA in inhibiting IGF-1-enhanced GBM cell invasion. In conclusion, IGF-1 is connected to TGF-ß signaling in regulating the mesenchymal transition and cell invasion of GBM through inhibition of miR-4286. Our findings provide new directions and mechanisms for exploring GBM progression.

Glycolysis-associated lncRNAs identify a subgroup of cancer patients with poor prognoses and a high-infiltration immune microenvironment.

BACKGROUND: Long noncoding (lnc)RNAs and glycolysis are both recognized as key regulators of cancers. Some lncRNAs are also reportedly involved in regulating glycolysis metabolism. However, glycolysis-associated lncRNA signatures and their clinical relevance in cancers remain unclear. We investigated the roles of glycolysis-associated lncRNAs in cancers. METHODS: Glycolysis scores and glycolysis-associated lncRNA signatures were established using a single-sample gene set enrichment analysis (GSEA) of The Cancer Genome Atlas pan-cancer data. Consensus clustering assays and genomic classifiers were used to stratify patient subtypes and for validation. Fisher's exact test was performed to investigate genomic mutations and molecular subtypes. A differentially expressed gene analysis, with GSEA, transcription factor (TF) activity scoring, cellular distributions, and immune cell infiltration, was conducted to explore the functions of glycolysis-associated lncRNAs. RESULTS: Glycolysis-associated lncRNA signatures across 33 cancer types were generated and used to stratify patients into distinct clusters. Patients in cluster 3 had high glycolysis scores and poor survival, especially in bladder carcinoma, low-grade gliomas, mesotheliomas, pancreatic adenocarcinomas, and uveal melanomas. The clinical significance of lncRNA-defined groups was validated using external datasets and genomic classifiers. Gene mutations, molecular subtypes associated with poor prognoses, TFs, oncogenic signaling such as the epithelial-to-mesenchymal transition (EMT), and high immune cell infiltration demonstrated significant associations with cluster 3 patients. Furthermore, five lncRNAs, namely MIR4435-2HG, AC078846.1, AL157392.3, AP001273.1, and RAD51-AS1, exhibited significant correlations with glycolysis across the five cancers. Except MIR4435-2HG, the lncRNAs were distributed in nuclei. MIR4435-2HG was connected to glycolysis, EMT, and immune infiltrations in cancers. CONCLUSIONS: We identified a subgroup of cancer patients stratified by glycolysis-associated lncRNAs with poor prognoses, high immune infiltration, and EMT activation, thus providing new directions for cancer therapy.

miR-140 targeting CTSB signaling suppresses the mesenchymal transition and enhances temozolomide cytotoxicity in glioblastoma multiforme.

Temozolomide (TMZ) is a first-line chemotherapeutic agent used against glioblastoma multiforme (GBM), but this disease exhibits recurrence and high lethality. Therefore, it is critical to explore biomarkers which involve in drug resistance and can be represented as different therapeutic effects after a diagnosis. We attempted to investigate the underlying variably expressed genes that contribute to the formation of resistance to TMZ. We analyzed gene and microRNA (miR) data from GBM patients in The Cancer Genome Atlas (TCGA) database to identify genetic factors associated with poor TMZ efficacy. By conducting a gene set enrichment analysis (GSEA), the epithelial-to-mesenchymal transition (EMT) was associated with poor TMZ responses. To identify roles of microRNAs in regulating TMZ resistance, a differential microRNA analysis was performed in TMZ-treated GBM patients. Downregulation of miR-140 was significantly correlated with poor survival. By integrating TCGA transcriptomic data and genomics of drug sensitivity in cancer (GDSC), cathepsin B (CTSB) was inversely associated with miR-140 expression and poor TMZ efficacy. By a pan-cancer analysis, both miR-140 and CTSB were found to be prognostic factors in other cancer types. We also identified that CTSB was a direct target gene of miR-140. Overexpression of miR-140 reduced CTSB levels, enhanced TMZ cytotoxicity, suppressed the mesenchymal transition, and influenced CTSB-regulated tumor sphere formation and stemness marker expression. In contrast, overexpression of CTSB decreased TMZ-induced glioma cell death, promoted the mesenchymal transition, and attenuated miR-140-increased TMZ cytotoxicity. These findings provide novel targets to increase the therapeutic efficacy of TMZ against GBM.

Phthalate plasticizer di(2-ethyl-hexyl) phthalate induces cyclooxygenase-2 expression in gastric adenocarcinoma cells.

The phthalate plasticizer, di(2-ethyl-hexyl) phthalate (DEHP), and its derived metabolites are common anthropogenic environmental toxins, which are known to act as endocrine disruptors. Numerous studies have associated DEHP with disruption of sex hormones, abnormal development of reproductive organs, allergies, and inflammation. Its role in promoting inflammation has been reported by both human epidemiological and animal studies. In stomach tissue, chronic inflammation is known to accompany mucosal damage, and pave the way to gastritis, stomach ulcers, and ultimately gastric cancer. Eastern Asian populations possess the highest gastric cancer incidences in the world. Coincidentally, East Asia is one of the world's major sites for plastics manufacture and export. Thus, possible correlations between DEHP, a common plasticizer, and gastric cancer are of great interest. Our study revealed several critical findings. First, even at very low dosage, mimicking the residual plasticizer exposure, detrimental effects of DEHP on gastric cells can be detected. Second, gastric cells treated with DEHP increased cyclooxygenase-2 (COX-2) in a time-dependent manner. Third, promoter deletion studies revealed a critical role of nuclear factor-kappa B (NF-κB) for COX-2 gene responses. Finally, our results indicated that a low concentration of DEHP is able to trigger COX-2 activation via the extracellular signal-regulated kinase (ERK1/2) and NF-κB signaling pathway. Taken together, we demonstrate that very low doses of DEHP enhance the expression of the prototypical inflammatory gene, COX-2, in gastric cancer cells via ERK1/2 and NF-κB activation. This study provides important insights into the inflammatory process and damages associated with phthalate plasticizers exposure.

Activation of fibroblasts by nicotine promotes the epithelial-mesenchymal transition and motility of breast cancer cells.

The tumor microenvironment plays an important role in tumor initiation and progression. It is well documented that nicotine participates in cigarette smoking-related malignancies. Previous studies focused on the effects of nicotine on tumor cells; however, the role of the microenvironment in nicotine-mediated tumorigenesis is poorly understood. Herein, we investigated the effect and molecular mechanism of nicotine on fibroblasts and its contribution to breast cancer. We found that nicotine induced the epithelial-mesenchymal transition (EMT) of breast cancer cells and promoted activation of fibroblasts. Interestingly, conditioned medium from nicotine-activated fibroblasts (Nic-CM) had a greater impact on promoting the EMT and migratory capability toward cancer cells than did treatment with nicotine alone. Production of connective tissue growth factor (CTGF) and transforming growth factor (TGF)-ß by nicotine-treated fibroblasts was demonstrated to be crucial for promoting the EMT and cancer cell migration, and blocking of CTGF and TGF-ß in Nic-CM-suppressed tumor motility. Moreover, nicotine induced expressions of CTGF, and TGF-ß in fibroblasts as identified through α7 nicotinic acetylcholine receptor (nAChR)-dependent activation of the AKT/TAZ signaling mechanism. Together, our data showed for the first time that activation of fibroblasts is largely responsible for accelerating smoking-mediated breast cancer progression.

miR-4725-3p targeting stromal interacting molecule 1 signaling is involved in xanthohumol inhibition of glioma cell invasion.

Glioblastoma multiforme is the most common brain tumor in adults. Because of its highly invasive nature, it is not easy to treat, resulting in high mortality rates. Stromal interacting molecule 1 (Stim1) plays important roles in regulating store-operated Ca2+ entry, and controls invasion by cancer cells. However, the mechanisms and functions of Stim1 in glioma progression are still unclear. In this study, we investigated the effects of targeting Stim1 expression on glioma cell invasion. By analyzing profiles of glioblastoma multiforme patients from RNA-sequencing data in The Cancer Genome Atlas, higher expression levels of STIM1 were correlated with the poor survival. Furthermore, signaling pathways associated with tumor malignancy, including the epithelial-to-mesenchymal transition (EMT), were activated in patients with high STIM1 expression according to gene set enrichment analyses. Higher Stim1 levels were found in glioma cells compared to human astrocytes, and these higher levels enhanced glioma cell invasion. Xanthohumol (XN), a prenylated flavonoid extracted from the hop plant Humulus lupulus L. (Cannabaceae), significantly reduced cell invasion through inhibiting Stim1 expression. From an micro(mi)RNA array analysis, miR-4725-3p was up-regulated by XN treatment. Over-expression of miR-4725-3p inhibited glioma cell invasion via directly targeting the 3'-untranslated region of STIM1. The extracellular signal-regulated kinase/c-Fos pathway was also validated to participate in XN-up-regulated miR-4725-3p expression according to promoter and chromatin immunoprecipitation assays. These results emphasize that miR-4725-3p-inhibited STIM1 signaling is involved in XN-attenuated glioma cell invasion. These findings may provide insights into novel therapeutic strategies for future glioblastoma therapy and drug development. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

microRNA let-7g suppresses PDGF-induced conversion of vascular smooth muscle cell into the synthetic phenotype.

Platelet-derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let-7g in phenotypic switching. Bioinformatics prediction was used to find let-7g target genes in the PDGF/mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/Krüppel-like factor-4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let-7g transfection were used to confirm let-7g target genes. Two contractile proteins alpha-smooth muscle actin (α-SMA) and calponin were VSMC-specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let-7g gene was injected to apolipoprotein E knockout (apoE-/- ) mice to confirm let-7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF-BB can inhibit α-SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let-7g binding sites, which were confirmed by our reporter assays. Transfection of let-7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let-7g decreased phosphorylated-ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC-specific gene expression by preventing myocardin-serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let-7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let-7g can increase α-SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let-7g gene increased let-7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE-/- mice. We demonstrated that let-7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.

Panobinostat sensitizes KRAS-mutant non-small-cell lung cancer to gefitinib by targeting TAZ.

Mutation of KRAS in non-small-cell lung cancer (NSCLC) shows a poor response to epidermal growth factor receptor (EGFR) inhibitors and chemotherapy. Currently, there are no direct anti-KRAS therapies available. Thus, new strategies have emerged for targeting KRAS downstream signaling. Panobinostat is a clinically available histone deacetylase inhibitor for treating myelomas and also shows potentiality in NSCLC. However, the therapeutic efficacy of panobinostat against gefitinib-resistant NSCLC is unclear. In this study, we demonstrated that panobinostat overcame resistance to gefitinib in KRAS-mutant/EGFR-wild-type NSCLC. Combined panobinostat and gefitinib synergistically reduced tumor growth in vitro and in vivo. Mechanistically, we identified that panobinostat-but not gefitinib-inhibited TAZ transcription, and the combination of panobinostat and gefitinib synergistically downregulated TAZ and TAZ downstream targets, including EGFR and EGFR ligand. Inhibition of TAZ by panobinostat or short hairpin RNA sensitized KRAS-mutant/EGFR-wild-type NSCLC to gefitinib through abrogating AKT/mammalian target of rapamycin (mTOR) signaling. Clinically, TAZ was positively correlated with EGFR signaling, and coexpression of TAZ/EGFR conferred a poorer prognosis in lung cancer patients. Our findings identify that targeting TAZ-mediated compensatory mechanism is a novel therapeutic approach to overcome gefitinib resistance in KRAS-mutant/EGFR-wild-type NSCLC.

Podocalyxin-Like Protein 1 Regulates TAZ Signaling and Stemness Properties in Colon Cancer.

Colon cancer is the third most common cancer in the world and the second most common cause of cancer-related mortality. Molecular biomarkers for colon cancer have undergone vigorous discovery and validation. Recent studies reported that overexpression of podocalyxin-like protein 1 (PODXL) is associated with distant metastasis and poor prognosis across several types of malignancies. Its role and underlying molecular mechanism, however, are not yet fully understood. In the present study, we revealed that the Hippo transducer, the transcriptional coactivator with PDZ-binding motif (TAZ), acts as a downstream mediator of PODXL in colon cancer. Inhibition of PODXL resulted in the suppression of TAZ signaling and the downregulation of Hippo downstream genes. Moreover, PODXL plays a critical role in cancer stemness, invasiveness, and sensitivity to chemotherapies in colon cancer HCT15 cells. Notably, expression of PODXL showed a positive correlation with stem-like and epithelial-mesenchymal transition (EMT) core signatures, and was associated with poor survival outcomes in patients with colon cancer. These findings provide novel insights into the molecular mechanism of PODXL-mediated tumorigenesis in colon cancer.

MicroRNA-302b-inhibited E2F3 transcription factor is related to all trans retinoic acid-induced glioma cell apoptosis.

All-trans retinoic acid (ATRA), a derivative of retinoid, is involved in the onset of differentiation and apoptosis in a wide variety of normal and cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression. Several miRNAs were identified to participate in ATRA-mediated cell differentiation. However, no studies have demonstrated whether miRNA can enhance ATRA cytotoxicity, thereby resulting in cell apoptosis. This study investigated the effects of ATRA-mediated miRNA expression in activating apoptotic pathways in glioblastoma. First, we found that high-dose ATRA treatment significantly reduced cell viability, caspase-dependent apoptosis, endoplasmic reticular (ER) stress activation, and intracellular reactive oxygen species accumulation. From microarray data, miR-302b was analyzed as a putative downstream regulator upon ATRA treatment. Furthermore, we found that ATRA up-regulated miR-302b expression in a dose- and time-dependent manner through retinoic acid receptor α-mediated pathway. Overexpression and knockdown of miR-302b significantly influenced ATRA-mediated cytotoxicity. E2F3, an important transcriptional regulator of glioma proliferation, was validated to be a direct target gene of miR-302b. The miR-302b-reduced E2F3 levels were also identified to be associated with ATRA-mediated glioma cell death. These results emphasize that an ATRA-mediated miR-302b network may provide novel therapeutic strategies for glioblastoma therapy. We propose that high-dose all-trans retinoic acid (ATRA) treatment, a derivative of retinoid, significantly induces glioblastoma cell apoptosis via caspase-dependent apoptosis, endoplasmic reticular (ER) stress, and intracellular reactive oxygen species (ROS) accumulation. The miR-302b overexpression enhanced by ATRA-mediated retinoic acid receptor (RAR)α pathway was also identified. The E2F3 repression, a novel target gene of miR-302b, was involved in ATRA-induced glioblastoma cell cytotoxicity.

The induction of heme oxygenase-1 suppresses heat shock protein 90 and the proliferation of human breast cancer cells through its byproduct carbon monoxide.

Heme oxygenase (HO)-1 is an oxidative stress-response enzyme which catalyzes the degradation of heme into bilirubin, ferric ion, and carbon monoxide (CO). Induction of HO-1 was reported to have antitumor activity; the inhibitory mechanism, however, is still unclear. In the present study, we found that treatment with [Ru(CO)3Cl2]2 (RuCO), a CO-releasing compound, reduced the growth of human MCF7 and MDA-MB-231 breast cancer cells. Analysis of growth-related proteins showed that treatment with RuCO down-regulated cyclinD1, CDK4, and hTERT protein expressions. Interestingly, RuCO treatment resulted in opposite effects on wild-type and mutant p53 proteins. These results were similar to those of cells treated with geldanamycin (a heat shock protein (HSP)90 inhibitor), suggesting that RuCO might affect HSP90 activity. Moreover, RuCO induced mutant p53 protein destabilization accompanied by promotion of ubiquitination and proteasome degradation. The induction of HO-1 by cobalt protoporphyrin IX (CoPP) showed consistent results, while the addition of tin protoporphyrin IX (SnPP), an HO-1 enzymatic inhibitor, diminished the RuCO-mediated effect. RuCO induction of HO-1 expression was reduced by a p38 mitogen-activated protein kinase inhibitor (SB203580). Additionally, treatment with a chemopreventive compound, curcumin, induced HO-1 expression accompanied with reduction of HSP90 client protein expression. The induction of HO-1 by curcumin inhibited 12-O-tetradecanoyl-13-acetate (TPA)-elicited matrix metalloproteinase-9 expression and tumor invasion. In conclusion, we provide novel evidence underlying HO-1's antitumor mechanism. CO, a byproduct of HO-1, suppresses HSP90 protein activity, and the induction of HO-1 may possess potential as a cancer therapeutic.

Negative feedback regulation between microRNA let-7g and the oxLDL receptor LOX-1.

Lectin-like oxidized LDL receptor-1 (LOX-1) is a surface scavenger receptor for oxidized low-density lipoprotein (oxLDL). Several transcription factors have been reported to regulate LOX-1 expression. MicroRNAs are small noncoding RNAs that control gene expression, but there have been no reports of LOX-1 expression being regulated by microRNAs. Because the microRNA let-7g has been predicted to bind to LOX-1 mRNA, we investigated whether let-7g can regulate LOX-1 expression. Our experiments first demonstrated that oxLDL can reduce let-7g expression. We later confirmed that there is a let-7g binding site on the 3'-untranslated region of LOX-1 mRNA. We showed that intracellular Ca(2+)-activated protein kinase C is involved in the oxLDL-LOX-1-let-7g pathway. Bioinformatics predicted that the let-7g promoter has a binding site for the transcriptional repressor OCT-1. We used a promoter assay and chromatin immunoprecipitation to confirm this binding. Consequently, knockdown of OCT-1 was found to increase let-7g expression. Transfection of let-7g inhibited oxLDL-induced LOX-1 and OCT-1 expression, cell proliferation and migration. Mice fed with a high-fat diet showed a decrease in let-7g and an increase in LOX-1 and OCT-1. A study on humans showed the serum levels of let-7g are lower in subjects with hypercholesterolemia compared with normal controls. Our findings identify a negative feedback regulation between let-7g and LOX-1, and indicate that let-7g could be a target to treat cardiovascular disease.

Persistence and dynamic structures of diverse cephalosporinase genes in nontyphoidal Salmonella in cross-sectional surveillance in Taiwan.

OBJECTIVES: Nontyphoidal Salmonella (NTS) is a major foodborne pathogen causing from acute gastroenteritis to bacteraemia, particularly in paediatric and elderly patients. Antimicrobial resistance of NTS, especially resistance to extended-spectrum cephalosporins, has emerged over the past decades. METHODS: Thirteen NTS isolates resistant to ceftriaxone or cefotaxime were collected from a teaching hospital in Taipei, and another three from a tertiary hospital, in New Taipei City, Taiwan, from September 2018 to December 2019. Ten other archived isolates from 2000 to 2017 were also obtained. Complete genomes of the 26 isolates were obtained. Serovars, sequence types, resistomes, genetic relatedness, and sequence comparison of plasmids were analyzed. RESULTS: Serogroups B, C2 and E were significantly associated with ampicillin resistance. Over 90% of these 26 isolates are susceptible to carbapenems and colistin. Genomic epidemiology of these isolates shows that blaCMY-2-harbouring isolates in different serovars were prevalent over two decades, presumably resulting from highly mobile IncI1 plasmid harbouring blaCMY-2. One type of the IncI1 plasmids contained a mobile element, IS26, which might be involved in the acquisition of antimicrobial resistance genes. Two emerging serovars, S. Goldcoast ST358 harbouring blaCTX-M-55 on IncHI2 plasmids and S. Anatum ST64 harbouring blaDHA-1 on IncA/C2 plasmids persisted in Taiwan, possibly through the clonal spread. Integration of complete or partial plasmid sequences into host chromosomes or multiplications of the antimicrobial resistance genes also appears to be mediated by IS26, in the two emerging clones. CONCLUSION: The dynamic movement of cephalosporinase genes mediated by IS26 in NTS is of great concern.

OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: novel mechanisms for cardiovascular diseases.

MicroRNA-29b has been reported to epigenetically regulate proatherogenic genes in response to oxLDL. Since transcription factors and epigenetic regulations are important mechanisms to regulate gene expression, we investigated whether these mechanisms are involved in oxLDL-induced microRNA-29b upregulation. First, we confirmed that microRNA-29b expression was increased in the aorta of mice fed with a high-fat diet, which was consistent with our previous in vitro findings. Next, we found that oxLDL only activated the microRNA-29b-1/microRNA-29a cluster gene on chromosome 7 but not the other distinct microRNA-29b gene located on chromosome 1. Using the promoter reporter assay and chromatin immunoprecipitation, activator protein-1 (AP-1) was shown to bind to the microRNA-29b-1 promoter. We further identified the signaling pathway of LOX-1/Ca(2+)/ROS/ERK/c-Fos was involved in oxLDL-mediated microRNA-29b overexpression after treating with the MAPTAM (Ca(2+) chelator), NAC (ROS scavenger), U0126 (ERK inhibitor) and c-Fos (one of the AP-1 proteins) shRNA, respectively. To investigate epigenetic regulations, we found that microRNA-29b promoter contained no CpG islands for DNA methylation. Therefore we investigated whether histone modifications influence microRNA-29b promoter activity. We showed that down-regulation of HDAC1 and the modifications on histone 3 lysine 4 (H3K4) and H3K9 significantly affected microRNA-29b expression. Furthermore, knockdown of c-Fos expression attenuated the effect of oxLDL-induced histone modifications on the microRNA-29b gene expression. Taken together, our data suggest that both transcription factor activation and histone modifications are important regulatory mechanisms of oxLDL-induced atherogenic process. This article is part of a Special Issue entitled OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: Novel mechanisms for cardiovascular diseases.

MicroRNA-93 inhibits tumor growth and early relapse of human colorectal cancer by affecting genes involved in the cell cycle.

PURPOSE: Colorectal cancer (CRC) is associated with high recurrence and mortality. Because deregulation of microRNAs is associated with CRC development and recurrence, the expression levels of microRNAs can be a simple and reliable biomarker to detect postoperative early relapse, thereby helping physicians to treat high-risk patients more efficiently. EXPERIMENTAL DESIGN: We used microRNA arrays and observed that microRNA-93 had substantially different expression levels in early (recurrence within 12 months after surgery) and non-early relapse CRC patients. The replication study, which included 35 early relapse and 42 non-early relapse subjects, further confirmed overexpression of microRNA-93 in non-early relapse samples. The in vitro and in vivo effects of microRNA-93 were investigated by examining cell proliferation, migration and invasion, as well as cell cycles, target-gene expression and xenograft in null mice. RESULTS: Cellular studies showed that the overexpression of microRNA-93 inhibited colon cancer cell proliferation and migration but not invasion. The cell cycle studies also revealed that microRNA-93 caused an accumulation of the G2 population. However, microRNA-93 could not induce cell apoptosis or necrosis. Functional studies showed that microRNA-93 could suppress CCNB1 protein expression leading to cell cycle arrest in the G2 phase. Moreover, microRNA-93 repressed expression of ERBB2, p21 and VEGF, all of which are involved in cell proliferation. MicroRNA-93 also suppressed tumor growth in null mice. CONCLUSIONS: This study showed that microRNA-93 can inhibit tumorigenesis and reduce the recurrence of CRC; these findings may have potential clinical applications for predicting the recurrence of CRC.

Orai1/CRACM1 overexpression suppresses cell proliferation via attenuation of the store-operated calcium influx-mediated signalling pathway in A549 lung cancer cells.

BACKGROUND: Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known. METHODS: To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay. RESULTS: Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation. CONCLUSIONS: We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca(2+) influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.

Interleukin-28B genetic variants in identification of hepatitis C virus genotype 1 patients responding to 24 weeks peginterferon/ribavirin.

BACKGROUND & AIMS: A substantial proportion of hepatitis C virus genotype 1 (HCV-1) patients achieved a sustained virological response (SVR, HCV RNA seronegative throughout 24 weeks of post-treatment follow-up) after 24 weeks peginterferon/ribavirin therapy. We explored the role of interleukin-28B genotype in identifying patients who responded to the regimen. METHODS: Interleukin-28B rs8099917 genotype was determined in 226 HCV-1 patients with 24 weeks peginterferon/ribavirin. RESULTS: Compared to patients with rs8099917 TG/GG genotype, those with TT genotype had significantly higher rapid virological response (RVR, HCV RNA seronegative at treatment week 4, 54.0% vs. 17.9%, p<0.001) and SVR (64.7% vs. 25.6%, p<0.001) rates, and lower relapse rate (28.0% vs. 54.5%, p=0.01). Logistic regression analysis revealed that the strongest factor predictive of a RVR was the carriage of rs8099917 TT genotype (odds ratio/ 95% confidence intervals [OR/CI]: 6.24/2.34-16.63), followed by lower viral loads (OR/CI: 5.29/2.81-9.93) and age (OR/CI:0.94/0.91-9.97). The most important factor predictive of an SVR was the attainment of a RVR (OR/CI: 22.23/9.22-53.58), followed by the carriage of rs8099917 TT genotype (OR/CI: 3.38/1.18-9.65), lower viral loads (OR/CI: 2.23/1.00-4.93) and ribavirin exposure dose (OR/CI: 1.17/1.06-1.30). The determinant power of rs8099917 genotype on SVR was mainly restricted to non-RVR patients, particularly those with higher baseline viral loads. Combination of the two pretreatment predictors, interleukin-28B genotype and baseline viral loads, could predict treatment efficacy with a positive predictive value of 80% and a negative predictive value of 91%. CONCLUSIONS: Interleukin-28B genotype could help identifying patients who are or are not candidates for an abbreviated regimen before treatment.

Tannic acid suppresses ultraviolet B-induced inflammatory signaling and complement factor B on human retinal pigment epithelial cells.

Ultraviolet B (UVB) radiation may cause the inflammation of retinal pigment epithelium (RPE) cells and play a role in development of age-related macular degeneration (AMD). The activation of the complement factor B (CFB) gene has been shown to be involved in formation of AMD. Here our results revealed that UVB induces IL-6/STAT3 signaling activation and the UVB-induced STAT3 is able to regulate the CFB expression in ARPE-19 cells. Tannic acid (TA) is a kind of water-soluble polyphenol and may have anti-inflammation effects. We also found that TA attenuates the UVB-induced IL-6 protein production, the STAT3 phosphorylation and the CFB expression. Taken together, these findings suggest UVB-induced inflammation of RPE can be mediated through the IL-6/STAT3/CFB pathway, and TA has a protected effect via the inhibition to the inflammatory response.

OxLDL up-regulates microRNA-29b, leading to epigenetic modifications of MMP-2/MMP-9 genes: a novel mechanism for cardiovascular diseases.

MicroRNAs (miRNAs), small noncoding RNAs, can control gene expression by binding to their target genes for degradation and/or translational repression. Epigenetic mechanisms are defined as heritable changes in gene expression that do not involve coding sequence modifications. Both mechanisms play an important role in maintaining physiological functions and are also related to disease development. However, few studies report that miRNA-mediated epigenetic regulations are involved in atherosclerosis. In the present study, oxidized low-density lipoprotein (oxLDL) significantly increased primary human aortic smooth muscle cell (HASMC) migration through MMP-2/MMP-9 up-regulation associated with decreased DNA methylation levels. Either mRNA or protein level of DNA methyltransferase 3b (DNMT3b) showed a dose-dependent down-regulation in oxLDL-mediated HASMCs. Knockdown DNMT3b expression enhanced oxLDL-induced DNA demethylation levels of MMP-2/MMP-9. The expression of miRNA-29b (miR-29b), directly targeting DNMT3b, was up-regulated by oxLDL treatment in a dose-dependent manner. OxLDL-mediated MMP-2/MMP-9 up-regulation, DNMT3b down-regulation, and DNA demethylation were all attenuated after knockdown miR-29b expression by antagomiR-29b. We find that oxLDL can up-regulate miR-29b expression, resulting in DNMT3b down-regulation in HASMCs and epigenetically regulated MMP-2/MMP-9 genes involved in cell migration. These results show that miRNA-mediated epigenetic regulation may be a novel mechanism in atherosclerosis.