Icariin attenuates the calcification of vascular smooth muscle cells through ERα - p38MAPK pathway.

Objective: To investigate the relationship between icariin and the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and the signal pathway involved. Methods: We applied a universally accepted calcification model of VSMCs induced by ß glycerophosphate. Then the VSMCs calcification was observed by treatment with icariin and/or inhibitors of estrogen receptors (ERs) and p38-mitogen-activated protein kinase (MAPK) signaling. Results: Icariin inhibited osteoblastic differentiation and mineralization of VSMCs due to decreased ALP activity and Runx2 expression. Further study demonstrated that icariin exerted this suppression effect through activating p38-MAPK but not extracellular-regulated kinase, JNK or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of icariin on osteoblastic differentiation. Interestingly, treatment of VSMCs with an ER antagonist ICI182780 and a selective ERα receptor antagonist PPT attenuated icariin-mediated inhibition effect of VSMCs calcification, associated with suppression of p38-MAPK phosphorylation. Conclusions: Icariin inhibited the osteoblastic differentiation of VSMCs, and that the inhibitory effects were mediated by p38-MAPK pathways through ERα.

Difficult peroral endoscopic myotomy: definition and management strategies.

Introduction: Peroral endoscopic myotomy (POEM) has been established as an alternative endoscopic method for the treatment of achalasia, and several studies have confirmed its relatively long-term efficacy. Although most of the POEM procedures can be smoothly completed, technical difficulties do arise during the treatment of some patients, which may lead to prolonged procedure duration, increased procedure-related complications, or even aborted POEM.Area covered: In the present review, we provide a comprehensive review of difficult POEM, focusing on its definition, risk factors, and intraoperative management strategies. The present review is expected to provide tips for not so experienced operators who perform POEM.Expert commentary: Submucosal fibrosis and sigmoid-type esophagus are associated with difficult POEM. Sometimes, the following may also be associated with difficult POEM: previous endoscopic or surgical treatments, spastic esophageal disorders (type III achalasia, distal esophageal spasm, and hypercontractile esophagus), achalasia with diverticulum or situs inversus. For operators who begin to perform POEM, I suggest an exclusion of patients with severe submucosal fibrosis or sigmoid-type esophagus, and begin to perform POEM for these patients when they have completed at least 50 cases of 'easy POEM' and the proposed management strategies may be helpful.

LncRNA-ANRIL inhibits cell senescence of vascular smooth muscle cells by regulating miR-181a/Sirt1.

BACKGROUND: Cardiovascular disease is one of the major threats to human life and health, and vascular aging is an important cause of its occurrence. Antisense non-coding RNA in the INK4 locus (ANRIL) is a kind of long non-coding RNA (lncRNA) that plays important roles in cell senescence. However, the role and mechanism of ANRIL in senescence of vascular smooth muscle cells (VSMCs) are unclear. METHODS: Cell viability and cell cycle were evaluated using an MTT assay and flow cytometry analysis, respectively. Senescence-associated (SA)-ß-galactosidase (gal) staining was used to determine cell senescence. Dual luciferase reporter assays were conducted to confirm the binding of ANRIL and miR-181a, as well as miR-181a and Sirt1. The expression of ANRIL, miR-181a, and Sirt1 was determined using qRT-PCR and protein levels of SA-ß-gal and p53-p21 pathway-related proteins were evaluated by Western blotting. RESULTS: ANRIL and Sirt1 were down-regulated, whereas miR-181a was up-regulated in aging VSMCs. In young and aging VSMCs, over-expression of ANRIL could down-regulate miR-181a and up-regulate Sirt1. MTT and SA-ß-gal staining assays showed that over-expression of ANRIL and inhibition of miR-181a promoted cell viability and inhibited VSMC senescence. The dual-luciferase reporter assay determined that miR-181a directly targets ANRIL and the 3'-UTR of Sirt1. Furthermore, over-expression of ANRIL inhibited cell cycle arrest and the p53-p21 pathway. CONCLUSION: ANRIL promotes cell viability and inhibits senescence in VSMCs, possibly by regulating miR-181a/Sirt1, and alleviating cell cycle arrest by inhibiting the p53-p21 pathway. This study provides novel insights for the role of ANRIL in the development of cell senescence.

MicroRNA-588 regulates invasion, migration and epithelial-mesenchymal transition via targeting EIF5A2 pathway in gastric cancer.

BACKGROUND: miRNAs are potential regulators of genes in many cancers. Here, we confirmed that the expression of miR-588 decreased in gastric cancer (GC) tissues and cells. MATERIALS AND METHODS: Sixty-seven GC tissues along with noncancerous tissues adjacent to them were included in the study. Quantitative real-time reverse transcription-PCR study was done to quantify the expression levels of mature miRNA. The expression of proteins was determined by Western blot and transwell chamber assay for invasion and migration studies. Immunohistochemical analysis and luciferase assay were done for evaluating the expression of epithelial-mesenchymal transition (EMT) markers and activity of EIF5A2, respectively. In vivo metastatic assay was done by injecting MGC-803 cells into nude mice. RESULTS: In the 5-year predicted survival study of GC patients included in the study, we found that miR-588 acted as a specific prognostic marker. Overexpression of miR-588 resulted in suppression of cell invasion, migration and progression of EMT, whereas suppression of miR-588 inverted the effects in both in vivo and in vitro experiments. miR-588 retained EIF5A2 by directly binding to the 3'-UTR. EIF5A2 was overexpressed in GC tissue samples, and the expression of miR-588 was inversely correlated to the levels of EIF5A2. The impact of miR-588 on invasion, migration and progression of EMT may be partially due to miR-588-mediated alterations of EiF5A2. CONCLUSION: Overall, the findings of the study suggest that miR-588 acts as a tumor suppressor by regulating the invasion, migration and EMT via EIF5A2 pathway, hence presenting miR-588 as a prognostic marker as well as a therapeutic target for GC.

Construction of a novel vector expressing Survivin-shRNA and fusion suicide gene yCDglyTK and its application in inhibiting proliferation and migration of colon cancer cells.

Despite progress achieved in cancer chemotherapy in recent decades, adverse effects remain a limiting factor for a number of patients with colorectal cancer, suggesting the requirement for novel therapeutic strategies. Gene therapy appears to be a promising strategy for treating cancer. The present study aimed to investigate the anti-tumor effect of a combined gene therapy, using Survivin downregulation by RNAi and a fusion suicide gene yCDglyTK therapy system. A triple-gene vector expressing Survivin-targeted small hairpin RNA (Survivin-shRNA) and fusion suicide gene yCDglyTK was constructed, and administered to HCT116 cells. Survivin expression decreased significantly and yCDglyTK fusion gene expression was confirmed by both reverse transcription-quantitative polymerase chain reaction and western blot analysis. Introduction of Survivin-shRNA into yCDglyTK/prodrug system eradicated colon cancer cells and induced apoptosis more effectively. Furthermore, this therapeutic system is able to inhibit the migration of HCT116 cells. These results indicate that the recombinant plasmid may serve as a novel gene therapy approach to treat colorectal carcinoma.

The Clinical Significance of miR-34a in Pancreatic Ductal Carcinoma and Associated Molecular and Cellular Mechanisms.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) exhibits poor prognosis and resistance to chemotherapy. This study was to identify the biomarkers associated with the progression, poor prognosis and chemoresistance of PDAC. METHODS: miR-34a and miR-150 levels in the plasma and tissues from PDAC patients were measured by real-time PCR. Xenograft PDAC tumor models were established in mice by inoculation of CD133+ stem cells isolated from PDAC tumors. Protein expression was measured by Western blot. RESULTS: The plasma miR-34a and miR-150 levels were significantly lower in PDAC patients than in patients with benign pancreatic lesions and in healthy subjects. The miR-34a and miR-150 levels in the tumor tissues were significantly lower than in pancreatic tissues with benign lesions. The protein levels of CD133, Notch1, Notch2 and Notch4 receptors in PDAC tumor tissues were significantly higher than in pancreatic tissues with benign lesions. miR-34a injection significantly inhibited the tumor growth of PDAC tumors and sensitized the anticancer effects of 5-fluorouracil (5-FU). miR-34a significantly inhibited Notch1, Notch2 and Notch4 expression in xenograft tumor tissues in vivo and BxPC-3 cells in vitro. miR-34a and miR-150 significantly induced apoptosis and inhibited proliferation, invasion and migration in BxPC-3 cells. miR-34a, but not miR-150, significantly sensitized the anticancer effect of 5-FU in BxPC-3 cells in vitro. CONCLUSION: A loss of expression of miR-34a, but not of miR-150, is associated with disease progression and poor prognosis in PDAC patients, and may be involved in the chemoresistance of PDAC cells.

microRNA-214 functions as a tumor suppressor in human colon cancer via the suppression of ADP-ribosylation factor-like protein 2.

microRNAs (miRNAs/miRs) are a conserved class of endogenous, short non-coding RNAs that post-transcriptionally regulate the expression of genes involved in diverse cellular processes. miR-214 has been reported to be associated with several cancers, including human colon cancer. However, the function of miR-214 in colon cancer development is poorly understood. In the current study, miR-214 was demonstrated to be downregulated in colon cancer tissues compared with healthy colon tissues. Functional studies showed that miR-214 overexpression results in the inhibition of cell viability, colony formation and proliferation, and the induction of cell apoptosis. ADP-ribosylation factor-like protein 2 (ARL2) is predicted to be a target candidate of miR-214. A luciferase reporter assay, western blot analysis and quantitative polymerase chain reaction were performed, which revealed that miR-214 negatively regulates ARL2 expression by targeting its 3' untranslated region directly. In conclusion, the results of the present study revealed that miR-214 suppresses colon cancer cell growth via the suppression of ARL2, and indicated that miR-214 may present a significant potential therapeutic target for colon cancer.

Down-regulation of miR-138 promotes colorectal cancer metastasis via directly targeting TWIST2.

BACKGROUND: Colorectal cancer (CRC) is the most common digestive system malignancy. The molecular events involved in the development and progression of CRC remain unclear. Recently, more and more evidences have showed that deregulated miRNAs participate in colorectal carcinogenesis. METHODS: The expression levels of miR-138 were first examined in CRC cell lines and tumor tissues by real-time PCR. The in vitro and in vivo functional effects of miR-138 were examined further. Luciferase reporter assays were conducted to confirm the targeting associations. Kaplan-Meier analysis and log-rank tests were performed to estimate the overall survival and disease free survival rate. RESULTS: miR-138 was found to be down-regulated in human colorectal cancer tissues and cell lines. Ectopic expression of miR-138 resulted in a dramatic inhibition of CRC migration and invasion in vitro and in vivo. Twist basic helix-loop-helix transcription factor 2 gene (TWIST2) was identified as one of the functional target. Restoration of miR-138 resulted in a dramatic reduction of the expression of TWIST2 at both mRNA and protein levels by directly targeting its 3'-untranslated region (3'UTR). Up-regulation of TWIST2 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and is inversely correlated with miR-138 expression. We also identified that down-regulation of miR-138 was associated with lymph node metastasis, distant metastasis, and always predicted poor prognosis. CONCLUSION: These data highlight a pivotal role for miR-138 in the regulation of CRC metastasis by targeting TWIST2, and suggest a potential application of miR-138 in prognosis prediction and CRC treatment.

[Delayed protective effect of morphine preconditioning on rabbit myocardium].

OBJECTIVE: To investigate whether morphine preconditioning has the delayed protective effect on rabbit myocardium. METHODS: Thirty New Zealand white rabbits were randomly divided into a NS group, a Mor-12 group and a Mor-24 group (n=10). In the Mor-12 group and Mor-24 group, morphine (3 mg/kg) was infused into rabbits, while the same volume of normal saline (NS) was given to rabbits in the NS group. Twelve hours after morphine infusion in the Mor-12 group, 24 h after NS or morphine infusion in the NS group and Mor-24 group, rabbits were subjected to 30 min left anterior descending coronary artery occlusions and were reperfused for 120 min. In 8 of the 10 rabbits in each group, arterial blood samples were taken before the ischemia (T1), 30 min after the ischemia (T2) and 120 min after the reperfusion (T3) to determine the concentration of cardiac troponin I (cTnI), and the myocardial infarct area was determined at the end of reperfusion. In the other 2 of the 10 rabbits in each group,the cell ultramicro-structure injury of myocardium was examined by electron microscope at the end of reperfusion. RESULTS: The concentration of cTnI at T2 and T3 in the Mor-24 group was lower than that in the NS group and Mor-12 group.The myocardial infarct size, and cell ultramicrostructure injury of myocardium in the Mor-24 group were decreased compared with the NS group and Mor-12 group. CONCLUSION: Morphine preconditioning has delayed protective effect on rabbit myocardium.

[A comparison of efficacy and safety in the treatment of hyperglycemia with continuous subcutaneous insulin with insulin pump or multiple insulin injections daily in critical elderly patients].

OBJECTIVE: To compare efficacy and safety in the treatment of hyperglycemia with continuous subcutaneous insulin infusion (CSII) or multiple daily insulin injection (MDI) in critical elderly patients. METHODS: Ninety-four elderly patients in critical condition with fasting glucose (10.3+/-2.5) mmol/L were randomly divided into CSII group (46 cases) and MDI group (48 cases). Soluble human insulin was used in both groups, and the treatment lasted for 7 days, and blood glucose level, average insulin dosage, percentage of hypoglycemia during 7 days, blood C-reacting protein (CRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) level and acute physiology and chronic health evaluation II (APACHE II) scores on the 7th day, and mortality during 28 days were observed. RESULTS: Compared with MDI group, blood glucose was better controlled [76.1% (35/46) vs. 33.3% (16/48)], percentage of fair control of blood glucose level was higher [21.7% (10/46) vs. 14.6% (7/48)], percentage of poor control of blood glucose level was lower [2.2% (1/46) vs. 52.1% (25/48)], percentage of hypoglycemia was lower [10.9% (5/46) vs. 22.9% (11/48)], average insulin dosage during 7 days was less [(40.1+/-6.3) U/d vs. (46.2+/-7.1) U/d], serum TNF-alpha level [(11.5+/-2.7) microg/L vs. (19.8+/-4.2) microg/L], IL-6 level [(78.3+/-5.1)microg/L vs.(141.4+/-6.2) microg/L] and CRP level [(53.1+/-3.3) mg/L vs. (72.1+/-4.0) mg/L] on the 7th day was lower, APACHE II score was lower on the 7th day [(6.0+/-1.4) scores vs. (11.6+/-1.0) scores], and 28-day mortality was lower in CSII group [4.3% (2/46) vs. 16.7% (8/48)]. All the above values showed statistically significant difference between two groups (all P < 0.05). CONCLUSION: CSII can better control blood glucose and alleviate inflammatory response and improve prognosis in elderly critically ill patients.