[Expression of anaplastic lymphoma kinase clone 1A4 in pediatric medulloblastoma and its significance].

Objective: To investigate the immunohistochemical staining of anaplastic lymphoma kinase (ALK; clone 1A4) in pediatric medulloblastoma (MB). Methods: Molecular subtyping was performed based on the NanoString and sequencing techniques for 44 pediatric MB cases at Children's Hospital, Zhejiang University School of Medicine from 2014 to 2017. ALK expression was detected with EnVision immunhistochemistry using ALK clone 1A4 on whole section. Statistical analyses were performed to evaluate the correlation of protein expression with molecular subgroups. Results: The age ranged from 0.5 to 13.0 years with an average age of 5.8 years. There were 28 males and 16 females, and 31 classic, 5 desmoplastic nodular, 3 extensive nodular and 5 large cell/anaplastic MBs. Except three cases was unable classified, 41 MBs were classified into the four molecular groups: 5 in WNT group, 12 in SHH group, 9 in Group 3 and 15 in Group 4. Thirteen of 44 MB cases were positive staining for ALK, and the positive rate was 29.5%. Six cases were strong reaction, and 7 cases were weak. The expression of ALK at the protein level was associated with the WNT group (P<0.01). The characteristic perinuclear dot-like staining was only showed in WNT group. Conclusions: The ALK immunhistochemistry using antibody clone 1A4 is a useful marker for the molecular subgroup detection of MB. The strong staining and perinuclear dot-like staining indicate as WNT group.

LncRNA SNHG8 promotes the development and chemo-resistance of pancreatic adenocarcinoma.

OBJECTIVE: The aim of this study was to investigate the role of small nucleolar RNA host gene 8 (SNHG8) in the pathogenesis of pancreatic adenocarcinoma and to explore the possible underlying mechanism. PATIENTS AND METHODS: SNHG8 expression in 40 pairs of pancreatic adenocarcinoma tissues and para-cancerous tissues, as well as 10 normal pancreas tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Survival analysis was conducted to explore the correlation between SNHG8 expression and the prognosis of pancreatic adenocarcinoma patients. After the transfection of SNHG8 siRNA into pancreatic adenocarcinoma cells, the proliferation and cell cycle were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Meanwhile, cell apoptosis was detected by flow cytometry and Western blot. The regulatory effect of SNHG8 on the chemo-sensitivity of pancreatic adenocarcinoma cells was assessed by CCK-8 assay. RESULTS: The expression of SNHG8 in pancreatic adenocarcinoma tissues was significantly higher than that of para-cancerous tissues and normal pancreatic tissues. Pancreatic adenocarcinoma patients with higher expression of SHNG8 presented shorter overall survival than those with lower expression. Meanwhile, SNHG8 expression was correlated with tumor stage and differentiation level, whereas not correlated with age, sex, tumor location and lymph node metastasis of pancreatic adenocarcinoma patients. In vitro results showed that SNHG8 knockdown significantly decreased the proliferative ability, prolonged G0/G1 phase and increased the apoptosis of Hs766T and PANC-1 cells. Western blot results elucidated that SNHG8 knockdown remarkably downregulated the protein expression levels of cleaved caspase-3 and cleaved PARP in Hs766T and PANC-1 cells. In addition, SNHG8 significantly decreased the chemo-sensitivity of pancreatic adenocarcinoma cells. CONCLUSIONS: SNHG8 is highly expressed in pancreatic adenocarcinoma tissues and is negatively correlated with its prognosis. Moreover, SNHG8 promotes cell proliferation and cell cycle, whereas inhibits cell apoptosis and reduces the chemo-sensitivity of pancreatic adenocarcinoma cells.

MiR-377 suppresses cell proliferation and metastasis in gastric cancer via repressing the expression of VEGFA.

OBJECTIVE: In recent years, microRNAs have been identified to participate in tumor genesis and progression of different tumors including gastric cancer. However, the role of miR-377 played in gastric cancer (GC), and its mechanisms have not been demonstrated. PATIENTS AND METHODS: We detected miR-377 expression level in 86 GC and adjacent normal tissue samples by quantitative reverse transcription PCR (qRT-PCR) as well as in GC cell lines. The relationship between miR-377 and clinical pathological features was analyzed. Using miR-377 mimics and inhibitors, we interfered with miR-377 level and employed several functional experiments to study the miR-377 effects on cell proliferation, migration, and invasion. Western blot assay and dual-luciferase assay were used to verify the target of miR-377. RESULTS: miR-377 expressed significantly lower in GC tissues and cell lines compared to normal tissues and GES-1 cells. Overexpression of miR-377 inhibited cell growth, migration and invasion, while downregulation miR-377 obviously promoted cell growth and metastasis. Furthermore, vascular endothelial growth factor A (VEGFA) was confirmed as a direct target of miR-377 and reversed the influence of mir-377 over-expression. CONCLUSIONS: miR-377 expressed lower in GC and suppressed cell proliferation, migration and invasion partly via repressing the VEGFA expression, which could provide a potential target for GC diagnosis and therapy.

Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery.

Current ultrasound (US)-mediated gene delivery methods are inefficient due, in part, to a lack of US optimization. We systematically explored the use of microbubbles (MBs), US parameters and plasmid delivery routes to improve gene transfer into the mouse liver. Co-presentation of plasmid DNA (pDNA), 10% Optison MBs and pulsed 1-MHz US at a peak negative pressure of 4.3 MPa significantly increased luciferase gene expression with pDNA delivered by intrahepatic injection to the left liver lobe. Intraportal injection delivered pDNA and MBs to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. Gene expression was also dependent on acoustic pressure over the range of 0-4.3 MPa, with a peak effect at 3 MPa. An average of 85-fold enhancement in gene delivery was achieved. No enhancement was observed below 0.25 MPa. Increasing pulse length while decreasing pulse repetition frequency and exposure time to maintain a constant total energy during exposure did not further improve transfection efficiency, nor did extend the US exposure pre- or postinjection of pDNA. The results indicate that coupled with MBs, US can more efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation.

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets.

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K(+) channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 microM) and higher (250 microM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca(2+) concentration ([Ca(2+)](i)) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 microM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca(2+)](i), PKC activation elicited glucose-induced insulin secretion at 100 and 250 microM diazoxide, while PKA activation did so only at 100 microM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 microM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression.

Insulin plays a crucial role in the regulation of glucose-homeostasis, and its synthesis is regulated by several stimuli. The transcription of the human insulin gene, enhanced by an elevated intracellular concentration of calcium ions, was completely blocked by Ca2+/calmodulin-dependent protein kinase inhibitor. The activity of the transcription factor activating transcription factor-2 (ATF-2), which binds to the cAMP responsive elements of the human insulin gene, was enhanced by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). Mutagenesis studies showed that Thr69, Thr71, and Thr73 of ATF-2 are all required for activation by CaMKIV. CaMKIV-induced ATF-2 transcriptional activity was not altered by activation of cJun NH2-terminal protein kinase (JNK) or p38 mitogen-activated protein (MAP) kinase. Furthermore, when transfected into rat primary cultured islets, ATF-2 enhanced glucose-induced insulin promoter activity, whereas cAMP response element-binding protein (CREB) repressed it. These results suggest a mechanism in which ATF-2 regulates insulin gene expression in pancreatic beta-cells, with the transcriptional activity of ATF-2 being increased by an elevated concentration of calcium ions.

The MH1 domains of smad2 and smad3 are involved in the regulation of the ALK7 signals.

The biological responses of the transforming growth factor beta (TGF-beta) superfamily are induced by activation of a receptor complex and Smad proteins. We surveyed the TGF-beta superfamily receptors using the degenerate PCR strategy, and found activin receptor-like kinase 7 (ALK7) to be abundantly expressed in fetal rat pancreatic islets. ALK7 is also expressed in adult rat islets and pancreatic beta-cell-derived MIN6 cells. The constitutively active form of ALK7, ALK7(T194D), activated Smad3 and a chimeric Smad protein, Smad3-2, containing the MH1 domain of Smad3 and the MH2 domain of Smad2, and translocated them to nuclei and then induced activation of the human PAI-1 promoter. However, neither Smad2 nor Smad2-3 protein, containing the MH1 domain of Smad2 and the MH2 domain of Smad3 were activated. These results indicate that the ALK7 signal regulates nuclear localization and activation of Smad2 and Smad3, and the MH1 domain of Smad2 has inhibitory effects on the nuclear localization.