NamiRNA-enhancer network of miR-492 activates the NR2C1-TGF-ß/Smad3 pathway to promote epithelial-mesenchymal transition of pancreatic cancer.

Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-ß/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-ß, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-ß/Smad3 pathway.

Identification of prognostic immune-related lncRNAs in pancreatic cancer.

Long noncoding RNAs (lncRNAs) play a critical role in the immune regulation and tumor microenvironment of pancreatic cancer (PaCa). To construct a novel immune-related prognostic risk model for PaCa and evaluate the prognostic prediction of lncRNAs, essential immune-related lncRNAs (IRlncRNAs) were identified by Pearson correlation analysis of differentially expressed immune-related genes (IRGs) and IRlncRNAs in PaCa from The Cancer Genome Atlas (TCGA) and GTEx databases. Least absolute shrinkage and selection operator (LASSO) regression was also applied to construct a prognostic risk model of IRlncRNAs, and gene set enrichment analysis (GSEA) was further applied for functional annotation for these IRlncRNAs. A total of 148 IRlncRNAs were identified in PaCa to construct a prognostic risk model. Among them, lncRNA LINC02325, FNDC1-AS1, and ZEB2-AS1 were significantly upregulated in 69 pairs of PaCa tissues by qRT-PCR. ROC analyses showed that LINC02325 (AUC = 0.80), FNDC1-AS1 (AUC = 0.76), and ZEB2-AS1 (AUC = 0.75) had a good predictive effect on 5-year survival prognosis. We demonstrated that high expression levels of ZEB2-AS1 and LINC02325 were not only positively associated with tumor size and CA199, but elevated levels of ZEB2-AS1 and FNDC1-AS1 were also positively correlated with tumor stage. GSEA further revealed that immune-related pathways were mainly enriched in the high-risk groups. Several immune-related algorithms demonstrated that four IRlncRNAs were related to immune infiltration, immune checkpoints, and immune-related functions. Thus, the prognostic risk model based on IRlncRNAs in Paca indicates that the four IRlncRNA signatures may serve as predictors of survival and potential predictive biomarkers of the pancreatic tumor immune response.

Comprehensive RNA-seq reveals molecular changes in kidney malignancy among people living with HIV.

To heighten the awareness of kidney malignancy in patients with HIV infection to facilitate the early diagnosis of kidney cancer, the differentially expressed mRNAs were analyzed in this malignant tumor using RNA sequencing. We identified 2,962 protein-coding transcripts in HIV-associated kidney cancer. KISS1R, CAIX, and NPTX2 mRNA expression levels were specifically increased in HIV-associated kidney cancer while UMOD and TMEM213 mRNA were decreased in most cases based on real-time PCR analyses. These findings were similar to those noted for the general population with renal cell carcinoma. Immunohistochemical staining analysis also showed that a total of 18 malignant kidney cases among the people living with HIV (PLWH) exhibited positive staining for KISS1R and CAIX. Pathway analysis of the differentially expressed mRNAs in HIV-associated kidney cancer revealed that several key pathways were involved, including vascular endothelial growth factor-activated receptor activity, IgG binding, and lipopolysaccharide receptor activity. Altogether, our findings reveal the identified molecular changes in kidney malignancy, which may offer a helpful explanation for cancer progression and open up new therapeutic avenues that may decrease mortality after a cancer diagnosis among PLWH.

LncRNAs as epigenetic regulators of epithelial to mesenchymal transition in pancreatic cancer.

Pancreatic cancer is the leading cause of cancer-related mortality because of tumor metastasis. Activation of the epithelial-to-mesenchymal transition (EMT) pathway has been confirmed to be an important driver of pancreatic cancer progression from initiation to metastasis. Long noncoding RNAs (lncRNAs) have been reported to exert essential physiological functions in pancreatic cancer progression by regulating the EMT program. In this review, we have summarized the role of EMT-related lncRNAs in human pancreatic cancer and the potential molecular mechanisms by which lncRNAs can be vital epigenetic regulators of epithelial to mesenchymal transition. Specifically, EMT-activating transcription factors (EMT-TFs) regulate EMT via TGF-ß/Smad, Wnt/ß-catenin, and JAK/STAT pathways. In addition, the interaction between lncRNAs and HIF-1α and m6A RNA methylation also have an impact on tumor metastasis and EMT in pancreatic cancer. This review will provide insights into lncRNAs as promising biomarkers for tumor metastasis and potential therapeutic strategies for pancreatic cancer.

Circular RNAs as novel potential biomarkers for pancreatic cancer.

Pancreatic cancer (PaCa) is the fourth leading cause of cancer-related deaths in the United States, and the vast majority of these malignancies are pancreatic ductal adenocarcinomas (PDAC), but there is still a lack of early detection biomarkers for PaCa. Unlike linear RNAs, circRNAs form covalently closed continuous loops and can act as mammalian gene regulators. They may be diagnostic or predictive biomarkers for some tumors, also be novel potential therapeutic targets in different diseases. This review focuses on (1) the biogenesis of circRNAs, RNA binding proteins (RBPs) and complementary sequences of circRNAs; (2) the characteristics of circRNAs which allow them to interact with miRNAs; (3) the roles of circRNAs playing in the regulation of gene expression, cell behavior and cancer, and their potential role as novel biomarkers and therapeutic targets in pancreatic cancer.

Apple polyphenol-rich drinks dose-dependently decrease early-phase postprandial glucose concentrations following a high-carbohydrate meal: a randomized controlled trial in healthy adults and in vitro studies.

BACKGROUND: Previous research demonstrated that a high dose of phlorizin-rich apple extract (AE) can markedly inhibit early-phase postprandial glycemia, but efficacy of lower doses of the AE is unclear. OBJECTIVE: To determine whether lower AE doses reduce early-phase postprandial glycemia in healthy adults and investigate mechanisms. DESIGN: In a randomized, controlled, double-blinded, cross-over acute trial, drinks containing 1.8 g (HIGH), 1.35 g (MED), 0.9 g (LOW), or 0 g (CON) of a phlorizin-rich AE were consumed before 75 g starch/sucrose meal. Postprandial blood glucose, insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP) and polyphenol metabolites concentrations were measured 0-240 min, acetaminophen concentrations to assess gastric emptying rate, and 24 h urinary glucose excretion. Effects of AE on intestinal glucose transport were investigated in Caco-2/TC7 cells. RESULTS: AE significantly reduced plasma glucose iAUC 0-30 min at all doses: mean differences (95% CI) relative to CON were -15.6 (-23.3, -7.9), -11.3 (-19.6, -3.0) and -8.99 (-17.3, -0.7) mmol/L per minute for HIGH, MEDIUM and LOW respectively, delayed Tmax (HIGH, MEDIUM and LOW 45 min vs. CON 30 min), but did not lower Cmax. Similar dose-dependent treatment effects were observed for insulin, C-peptide, and GIP. Gastric emptying rates and urinary glucose excretion did not differ. Serum phloretin, quercetin and epicatechin metabolites were detected postprandially. A HIGH physiological AE dose equivalent decreased total glucose uptake by 48% in Caco-2/TC7 cells. CONCLUSIONS: Phlorizin-rich AE, even at a low dose, can slightly delay early-phase glycemia without affecting peak and total glycemic response.

Inhibition of the facilitative sugar transporters (GLUTs) by tea extracts and catechins.

Tea polyphenolics have been suggested to possess blood glucose lowering properties by inhibiting sugar transporters in the small intestine and improving insulin sensitivity. In this report, we studied the effects of teas and tea catechins on the small intestinal sugar transporters, SGLT1 and GLUTs (GLUT1, 2 and 5). Green tea extract (GT), oolong tea extract (OT), and black tea extract (BT) inhibited glucose uptake into the intestinal Caco-2 cells with GT being the most potent inhibitor (IC50 : 0.077 mg/mL), followed by OT (IC50 : 0.136 mg/mL) and BT (IC50 : 0.56 mg/mL). GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (Ki ) = 0.0317 and 0.0571 mg/mL, respectively, whereas BT was pure non-competitive, Ki  = 0.36 mg/mL. Oocytes injected to express small intestinal GLUTs were inhibited by teas, but SGLT1 was not. Furthermore, catechins present in teas were the predominant inhibitor of glucose uptake into Caco-2 cells, and gallated catechins the most potent: CG > ECG > EGCG ≥ GCG when compared to the non-gallated catechins (C, EC, GC, and EGC). In Caco-2 cells, individual tea catechins reduced the SGLT1 gene, but not protein expression levels. In contrast, GLUT2 gene and protein expression levels were reduced after 2 hours exposure to catechins but increased after 24 hours. These in vitro studies suggest teas containing catechins may be useful dietary supplements capable of blunting postprandial glycaemia in humans, including those with or at risk to Type 2 diabetes mellitus.

Hepatomas are exquisitely sensitive to pharmacologic ascorbate (P-AscH-).

Rationale: Ascorbate is an essential micronutrient known for redox functions at normal physiologic concentrations. In recent decades, pharmacological ascorbate has been found to selectively kill tumour cells. However, the dosing frequency of pharmacologic ascorbate in humans has not yet been defined. Methods: We determined that among five hepatic cell lines, Huh-7 cells were the most sensitive to ascorbate. The effects of high-dose ascorbate on hepatoma were therefore assessed using Huh-7 cells and xenograft tumour mouse model. Results: In Huh-7 cells, ascorbate induced a significant increase in the percentage of cells in the G0/G1 phase, apoptosis and intracellular levels of ROS. High doses of ascorbate (4.0 pmol cell-1), but not low doses of ascorbate (1.0 pmol cell-1), also served as a pro-drug that killed hepatoma cells by altering mitochondrial respiration. Furthermore, in a Huh-7 cell xenograft tumour mouse model, intraperitoneal injection of ascorbate (4.0 g/kg/3 days) but not a lower dose of ascorbate (2.0 g/kg/3 days) significantly inhibited tumour growth. Gene array analysis of HCC tumour tissue from xenograft mice given IP ascorbate (4.0 g/kg/3 days) identified changes in the transcript levels of 192 genes/ncRNAs involved in insulin receptor signalling, metabolism and mitochondrial respiration. Consistent with the array data, gene expression levels of AGER, DGKK, ASB2, TCP10L2, Lnc-ALCAM-3, and Lnc-TGFBR2-1 were increased 2.05-11.35 fold in HCC tumour tissue samples from mice treated with high-dose ascorbate, and IHC staining analysis also verified that AGER/RAGE and DGKK proteins were up-regulated, which implied that AGER/RAGE and DGKK activation might be related to oxidative stress, leading to hepatoma cell death. Conclusions: Our studies identified multiple mechanisms are responsible for the anti-tumour activity of ascorbate and suggest high doses of ascorbate with less frequency will act as a novel therapeutic agent for liver cancer in vivo.

Sugar sensor genes in the murine gastrointestinal tract display a cephalocaudal axis of expression and a diurnal rhythm.

Distributed along the length of the gastrointestinal (GI) tract are nutrient sensing cells that release numerous signaling peptides influencing GI function, nutrient homeostasis and energy balance. Recent studies have shown a diurnal rhythm in GI nutrient sensing, but the mechanisms responsible for rhythmicity are poorly understood. In this report we studied murine GI sugar sensor gene and protein expression levels in the morning (7 AM) and evening (7 PM). Sweet taste receptor ( tas1r2/tas1r3/gnat3/gnat1) sugar transporter ( slc5a1, slc2a2, slc2a5) and putative sugar sensor ( slc5a4a and slc5a4b) gene expression levels were highest in tongue and proximal and distal small intestine, respectively. Clock gene ( cry2/arntl) activity was detected in all regions studied. Slc5a4a and slc5a4b gene expression showed clear diurnal rhythmicity in the small intestine and stomach, respectively, although no rhythmicity was detected in SGLT3 protein expression. Tas1r2, tas1r3, gnat1, and gcg displayed a limited rhythm in gene expression in proximal small intestine. Microarray analysis revealed a diurnal rhythm in gut peptide gene expression in tongue (7 AM vs. 7 PM) and in silico promoter analysis indicated intestinal sugar sensors and transporters possessed the canonical E box elements necessary for clock gene control over gene transcription. In this report we present evidence of a diurnal rhythm in genes that are responsible for intestinal nutrient sensing that is most likely controlled by clock gene activity. Disturbances in clock gene/nutrient sensing interactions may be important in the development of diet-related diseases, such as obesity and diabetes.

Apple and blackcurrant polyphenol-rich drinks decrease postprandial glucose, insulin and incretin response to a high-carbohydrate meal in healthy men and women.

Postprandial glycemic responses to meals are inhibited by polyphenol-rich plant foods. Combinations of polyphenols may be particularly effective through complementary mechanisms. A randomized, controlled, double-blinded cross-over trial was conducted in healthy volunteers to test the hypothesis that apple and blackcurrant polyphenol-rich drinks would reduce postprandial blood glucose concentrations. Secondary outcomes included insulin and glucose-dependent insulinotropic polypeptide (GIP) secretion. Twenty men (mean age 26 y, SD 8) and 5 postmenopausal women (mean age 57 y, SD 3) consumed a placebo drink (CON) and 2 polyphenol-rich drinks containing fruit extracts: either 1200 mg apple polyphenols (AE), or 600 mg apple polyphenols+600 mg blackcurrant anthocyanins (AE+BE), in random order with a starch and sucrose meal. Incremental areas under the curve (iAUC) for plasma glucose concentrations were lower following AE+BE over 0-30 and 0-120 min compared with CON; mean differences (95% CI) -32 mmol/L·min (-41, -22, P<.0005) and -52 mmol/L min (-94, -9, P<.05), respectively. AE significantly reduced iAUC 0-30 min (mean difference -26 mmol/L min, -35, -18, P<.0005) compared with CON, but the difference over 120 min was not significant. Postprandial insulin, C-peptide and GIP concentrations were significantly reduced relative to CON. A dose response inhibition of glucose transport was demonstrated in Caco-2 cells, including total and GLUT-mediated transport, and SGLT1-mediated glucose transport was strongly inhibited at all doses in Xenopus oocytes, following 10 min incubation with 0.125-4 mg apple polyphenols/ml. In conclusion, ingestion of apple and blackcurrant polyphenols decreased postprandial glycemia, which may be partly related to inhibition of intestinal glucose transport.

Inhibition of the intestinal glucose transporter GLUT2 by flavonoids.

We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal luminal compounds that are inefficiently absorbed and naturally present in foods. Because of their abundance in fruits and vegetables, flavonoids were selected as model compounds. Robust inhibition of glucose and fructose transport by GLUT2 expressed in Xenopus laevis oocytes was produced by the flavonols myricetin, fisetin, the widely consumed flavonoid quercetin, and its glucoside precursor isoquercitrin [corrected]. IC50s for quercetin, myricetin, and isoquercitirin [corrected]were approximately 200- to 1000-fold less than glucose or fructose concentrations, and noncompetitive inhibition was observed. The two other major intestinal sugar transporters, GLUT5 and SGLT1, were unaffected by flavonoids. Sugar transport by GLUT2 overexpressed in pituitary cells and naturally present in Caco-2E intestinal cells was similarly inhibited by quercetin. GLUT2 was detected on the apical side of Caco-2E cells, indicating that GLUT2 was in the correct orientation to be inhibited by luminal compounds. Quercetin itself was not transported by the three major intestinal glucose transporters. Because the flavonoid quercetin, a food component with an excellent pharmacology safety profile, might act as a potent luminal inhibitor of sugar absorption independent of its own transport, flavonols show promise as new pharmacologic agents in the obesity epidemic.

Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues.

Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H(2)O(2), and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H(2)O(2) may be beneficial.

Estrogen stimulates microglia and brain recovery from hypoxia-ischemia in normoglycemic but not diabetic female mice.

Diabetic hyperglycemia increases ischemic brain damage in experimental animals and humans. The mechanisms are unclear but may involve enhanced apoptosis in penumbral regions. Estrogen is an established neuroprotectant in experimental stroke. Our previous study demonstrated that female diabetic db/db mice suffered less damage following cerebral hypoxia-ischemia (H/I) than male db/db mice. Here we investigated the effects of diabetes and estrogen apoptotic gene expression following H/I. Female db/db and nondiabetic (+/?) mice were ovariectomized (OVX) and treated with estrogen or vehicle prior to H/I; brains were analyzed for damage and bcl-2 family gene expression. OVX increased ischemic damage in +/? mice; estrogen reduced tissue injury and enhanced antiapoptotic gene expression (bcl-2 and bfl-1). db/db mice demonstrated more damage, without increased bcl-2 mRNA; bfl-1 expression appeared at 48 hours of recovery associated with infarction. To our knowledge, this is the first description of bfl-1 in the brain with localization to microglia and macrophages. Early induction of bfl-1 expression in +/? mouse brain was associated with microglia; delayed bfl-1 expression in diabetic brain was in macrophages bordering the infarct. Furthermore, estrogen replacement stimulated early postischemic expression of bcl-2 and bfl-1 and reduced damage in normoglycemic animals but failed to protect the diabetic brain.

Vitamin C as an antioxidant: evaluation of its role in disease prevention.

Vitamin C in humans must be ingested for survival. Vitamin C is an electron donor, and this property accounts for all its known functions. As an electron donor, vitamin C is a potent water-soluble antioxidant in humans. Antioxidant effects of vitamin C have been demonstrated in many experiments in vitro. Human diseases such as atherosclerosis and cancer might occur in part from oxidant damage to tissues. Oxidation of lipids, proteins and DNA results in specific oxidation products that can be measured in the laboratory. While these biomarkers of oxidation have been measured in humans, such assays have not yet been validated or standardized, and the relationship of oxidant markers to human disease conditions is not clear. Epidemiological studies show that diets high in fruits and vegetables are associated with lower risk of cardiovascular disease, stroke and cancer, and with increased longevity. Whether these protective effects are directly attributable to vitamin C is not known. Intervention studies with vitamin C have shown no change in markers of oxidation or clinical benefit. Dose concentration studies of vitamin C in healthy people showed a sigmoidal relationship between oral dose and plasma and tissue vitamin C concentrations. Hence, optimal dosing is critical to intervention studies using vitamin C. Ideally, future studies of antioxidant actions of vitamin C should target selected patient groups. These groups should be known to have increased oxidative damage as assessed by a reliable biomarker or should have high morbidity and mortality due to diseases thought to be caused or exacerbated by oxidant damage.