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Background: Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis (SAP)-related acute lung injury (ALI). Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI. However, the precise mechanism associated with p38 is unclear, particularly in pulmonary microvascular endothelial cell (PMVEC) injury., Aim: To determine its role in the tumor necrosis factor-alpha (TNF-α)-induced inflammation and apoptosis of PMVECs in vitro . We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI., Methods: In vitro , PMVEC were transfected with mitogen-activated protein kinase kinase 6 (Glu), which constitutively activates p38, and then stimulated with TNF-α. Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels, respectively. In vivo , SAP-ALI was induced by 5% sodium taurocholate and three different doses of SB203580 (2.5, 5.0 or 10.0 mg/kg) were intraperitoneally injected prior to SAP induction. SAP-ALI was assessed by performing pulmonary histopathology assays, measuring myeloperoxidase activity, conducting arterial blood gas analyses and measuring TNF-α, interleukin (IL)-1β and IL-6 levels. Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration, Evans blue extravasation and ultrastructural changes in PMVECs. The apoptotic death of pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2, Bax, Bim and cle-caspase3 levels. The proteins levels of P-p38, NFκB, IκB, P-signal transducer and activator of transcription-3, nuclear factor erythroid 2-related factor 2, HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580., Results: In vitro , mitogen-activated protein kinase (Glu) transfection resulted in higher apoptotic rates and cytokine (IL-1β and IL-6) levels in TNF-α-treated PMVECs. In vivo , SB2035080 attenuated lung histopathological injury, decreased inflammatory activity (TNF-α, IL-1β, IL-6 and myeloperoxidase) and preserved pulmonary function. Furthermore, SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration, Evans blue accumulation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers, apoptosis-related proteins (cle-caspase3, Bim and Bax) and endothelial microstructure. Moreover, SB203580 significantly reduced the pulmonary P-p38, NFκB, P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels., Conclusion: p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflict of interest., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objective: To assess the safety and feasibility of the application of the laparoscopic modality in the perioperative treatment of central liver tumors. Methods: Collecting all the clinical information of a total of 40 patients with central liver tumors who received laparoscopic resection treatment carried out at Department of Hepatological Surgery of People's Hospital of Hunan Provincial from January 2016 to December 2018 to take a retrospective review. There were 19 males and 21 females.The age was (59.5±14.5) years (range: 15 to 71 years) . There were 26 cases of primary hepatic carcinoma (24 cases of hepatocellular carcinoma, 2 cases of cholangiocellular carcinoma) , 8 cases of hepatic cavernous hemangioma, 1 case of metastatic hepatic carcinoma, 5 cases of hepatocellular adenoma. The maximum diameter of tumors were (6.2±2.9) cm (range: 2 to 13 cm) . The patient's information about hepatectomy methods, blocking mode and time of blood flow, operation time, intraoperative blood loss, intraoperative blood transfusion rate, post-operative hospitalization time, perioperative reoperation and postoperative complications were collected. Results: A total of 40 patients all were treated with laparoscopic surgery. The surgical procedure was as follows: 2 patients received the right hepatic lobectomy (Ⅴ, Ⅵ, Ⅶ and Ⅷ segments) , 2 patients received the left hepatic lobectomy (Ⅱ, III and Ⅳ segments) , 13 patients received mesohepatectomy (Ⅳ, Ⅰ and Ⅷ segments) , 2 patients received left hepatic trisegmentectomy (Ⅱ, Ⅲ, Ⅳ and Ⅷ segments) , 2 patients received right hepatic trisegmentectomy (Ⅳ, Ⅴ, Ⅵ, Ⅶ and Ⅷ segments) , 7 patients received Ⅷ segmentectomy, 1 patient received Ⅳ segmentectomy, 3 patients received Ⅴ and Ⅷ segmentectomy, 5 patients received hepatic caudate lobe resection (Ⅰ, Ⅸ segments) , and 3 patients received local tumors resection.Pathological results: there were 26 cases of primary hepatic carcinoma (24 cases of hepatocellular carcinoma, 2 cases of cholangiocellular carcinoma) , 8 cases of hepatic cavernous hemangioma, 1 case of metastatic hepatic carcinoma, 5 cases of hepatocellular adenoma; the pathological reports of all malignant tumor cases all showed negative incisal edge. The operative time was (333±30) minutes (range: 280 to 380 minutes) ; the intraoperative hepatic portal occlusion period was (58±13) minutes (range: 30 to 90 minutes) ; the intraoperative hemorrhage was (173±129) ml (range: 20 to 600 ml) ; the intraoperative blood transfusion rate was 2.5% (1/40) ; the postoperative incidence of bile leakage was 2.5% (1/40) , the hospital discharge of 1 patient with bile leakage was approved after conservative treatments like T pipe decompression and adequate drainage; there was 1 case of abdominal infection and 1 case of pulmonary infection, both of which were discharged from the hospital with conservative treatments; there were no other serious postoperative complications. The postoperative hospital stay was (10.7±2.7) days (range: 6 to 16 days) ; there were no perioperative mortality and reoperation cases. Conclusion: In the centers with abundant laparoscopic hepatectomy experiences, the laparoscopic resection is proved to be safe and feasible in the perioperative treatments of central liver tumors by the highly selective cases, the adequate preoperative assessment and reasonable surgical techniques and approach.

Objective: To investigate the protective effect of (2R, 3R)-dihydroquercetin 7-O-β-D-glucopyranose (C1) extracted from Coreopsis tinctoria Nutt. in a mouse model of alcoholic acute pancreatitis (FAEE-AP) induced byfatty acid ethyl ester (FAEE)., Methods: The 30 healthy SPF mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group, 6 in each group. Alcoholic pancreatitis was induced by ethanol and palmitoleic acid administration (1.75 g/kg ethanol, 200 mg/kg palmitoleic acid, 2 times peritoneal injections). The three treatment groups were given C1 (0 h, 4 h, 8 h) at the dose of 12.5, 25 and 50 mg/kg, respectively. After 24 h of molding, the serum amylase, lipase and IL-6 levels were detected. The trypsin level in pancreatic tissue and myeloperoxidase (MPO) level in pancreatic and lung tissue were detected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of pancreatic tissue and immunohistochemical (IHC) staining was used to detect the expression of nuclear factor-erythroid 2 related factor 2 (Nrf2) in pancreatic tissue., Results: The pancreatic histopathological scores, serum amylase and lipase activity, trypsin level in pancreatic tissue, serum IL-6 level, MPO level of pancreas and lung were significantly higher in the model group than in the control group ( P < 0.01). Compared with the model group, the pancreatic histopathologies of the low dose group was significantly improved ( P < 0.05), as well as the serum amylase and lipase activity, trypsin level of pancreas, serum IL-6 level, the pancreas andthe lung's MPO level decreased significantly ( P < 0.05), and up-regulate that expression of Nrf2 in pancreatic tissue., Conclusion: 12.5 mg/kg of (2R, 3R) -dihydroquercetin 7-O-β-D-glucopyranose (C1) improved the expression of Nrf2, reduced the expression of inflammatory factor IL-6, and protected acute pancreatitis caused by FAEE., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

As the key producers of reactive oxygen species (ROS), NADPH oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), play crucial roles in various biological processes in plants with considerable evolutionary selection and functional diversity in the entire terrestrial plant kingdom. However, only limited resources are available on the phylogenesis and functions of this gene family in wheat. Here, a total of 46 NOX family genes were identified in the wheat genome, and these NOXs could be classified into three subgroups: typical TaNOXs, TaNOX-likes, and ferric reduction oxidases (TaFROs). Phylogenetic analysis indicated that the typical TaNOXs might originate from TaFROs during evolution, and the TaFROs located on Chr 2 might be the most ancient forms of TaNOXs. TaNOXs are highly expressed in wheat with distinct tissue or organ-specificity and stress-inducible diversity. A large-scale expression and/or coexpression analysis demonstrated that TaNOXs can be divided into four functional groups with different expression patterns under a broad range of environmental stresses. Different TaNOXs are coexpressed with different sets of other genes, which widely participate in several important intracellular processes such as cell wall biosynthesis, defence response, and signal transduction, suggesting their vital but diversity of roles in plant growth regulation and stress responses of wheat.

This study seeks to compare the impact of selective partial portal vein ligation (PPVL) or the combination of simultaneous hepatic artery ligation (PPVAL) with in situ splitting (ISS) on liver regeneration and injury. Rats were randomized into three groups; namely: selective PVL, PPVL + ISS and PPVAL + ISS. The changes in hepatic hemodynamics, liver regeneration and hepatocytic injury were examined. Blood flow to the left portal branch and the microcirculation of the left median lobe after PPVL or PPVAL was significantly reduced. Liver regeneration of PPVAL + ISS group was more pronounced than that in the PPVL + ISS and PVL groups at 48 and 72 hours as well as 7 d postoperatively. The serum biochemical markers and histopathological examination demonstrated reduced levels of liver injury in the PPVL + ISS group. Injury to hepatocytes was more pronounced with PPVAL + ISS than PVL. HGF, TNF-α and IL-6 expression in the regenerated lobes in both PPVAL + ISS and PPVL + ISS groups increased significantly when compared to the PVL group. We demonstrated that both PPVL + ISS and PPVAL + ISS were effective and feasible means of inducing remnant liver hypertrophy and could serve as a rapid clinical application for qualified patients.

Transforming growth factor-β (TGF-β) has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) during cancer progression, mainly by activating a set of pleiotropic transcription factors including SNAI2/Slug. However, the involvement of long non-coding RNAs (lncRNAs) in TGF-β-induced Slug activation and EMT remains largely unknown. Methods: In this study, we used microarray analysis to compare lncRNA expression profiles between TGF-β treated and untreated breast cancer cells. Then, the clinical significance of lncRNAs in breast cancer was investigated by qPCR and Kaplan-Meier survival analysis. The molecular mechanisms and EMT-promoting effects in vitro were analyzed by confocal laser microscopy, Western blotting, chromosome conformation capture (3C), chromatin isolation by RNA purification (ChIRP), ChIP, luciferase reporter assay and transwell migration assay. Lastly, the pro-metastatic effects in vivo were evaluated by bioluminescent imaging and hematoxylin and eosin (H&E) staining. Results: We observed that TGF-β induced genome-wide changes in lncRNA levels in breast cancer cells, among which AC026904.1 and UCA1 were highly expressed in metastatic breast cancer and closely associated with poor prognosis. Mechanistic study revealed that AC026904.1 and UCA1 were upregulated by non-canonical and canonical TGF-β pathways, respectively. Further analysis showed that AC026904.1 functions as an enhancer RNA in the nucleus, whereas UCA1 exerts a competitive endogenous RNA (ceRNA) activity in the cytoplasm. In addition, the biological functions of these two lncRNAs converged on the activation and maintenance of Slug, constituting a one-two punch in promoting EMT and tumor metastasis. Conclusion: These findings uncover for the first time that AC026904.1 and UCA1 could cooperatively upregulate Slug expression at both transcriptional and post-transcriptional levels, exerting critical roles in TGF-β-induced EMT. The present work provides new evidence that lncRNAs function as key regulators of EMT and hold great promise to be used as novel biomarkers and therapeutic targets for metastatic breast cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

This study reports the complications occurred during the management of open fractures of the lower limbs, resulting in secondary amputations, observed in clinicians' practice in recent years as well as different risk factors and possible deficiencies in management at the origin of these complications. We conducted a retrospective study over a period of 06 years (January 2006 - January 2012). It included patients with open fracture of the lower limb initially treated in our institution and whose complications resulted in amputation. All patient undergoing emergency amputation after examination at the Emergency Department were excluded. All patient treated in another hospital before being referred to us were excluded by the study, even if secondary amputation had been performed in our institution. We collected data by analyzing the records of patients (clinical and complementary examinations, surgical reports). We evaluated our management of open fractures of the lower limb according to the guidelines and recommendations found in the literature. These complications were observed in 9 out of 306 open fractures of the lower limb treated in the same period (January 2006 - January 2012), reflecting a rate of 2.9%. The average age was 42.6(26-57) years, all patients were male. We recorded 1 case of fracture of the femur, 7 cases of fracture of 2 leg bones and 1 case of foot crushing. These were open fractures including 1 case of type 1 fracture, 3 cases of type II fracture and 5 cases of type III fracture, according to Gustilo and Anderson classification. We performed 5 amputations of the thigh and 4 amputation of the upper third of the leg. Various complications motivated these amputations, including bone infections or soft-tissue gangrenes of ischemic or infectious origin. The patients had no morbidities such as diabete and were not chronic smokers. No patient died. Open fractures deserve special attention of the surgeon on immediade complications both from a diagnostic and therapeutic point of view in order to reduce the amputation rates after primary surgery giving patients the impression that they had an incorrect procedure performed on them. Particular attention should be paid to the degree of initial contamination and to the presence of a virulent germ at the site of trauma which may motivate particular attitudes during primary management.

Tuberculous cold abscesses are a rare and unusual form of extrapulmonary tuberculosis. We here report the case of a 27-year old patient with a 5-month history of left dorsolumbar swelling presenting with dorsolumbar cold abscess revealing Pott's disease without neurological complications. Examination of pus sample collected at the time the abscess was incised and drained helped to confirm the diagnosis of cold abscesses of TB origin. The patient underwent 12 months of anti-tuberculosis treatment which lead to a faster recovery; the sequelae was slight gibbosity.

Amputation of a limb is a serious consequence of snakebite poisoning. It is caused by the toxicity of the venom and often the use of a tourniquet in some patients, which can lead to limb ischemia. Management of the victim aims to ensure survival and preserve the function of the bitten limb. Antivenom immunotherapy is the only specific treatment for ophidian envenomation. It is indicated in cases of general symptoms and signs related to the bite, but also if local damage could lead to the loss of limb function. The authors report on a case of snakebite that led to amputation at the thigh., (Copyright © 2017. Published by Elsevier SAS.)

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75 % oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92 % reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02 % reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production.

Aim: To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV), especially the involvement of bone marrow-derived cells (BMCs) and underlying mechanisms., Methods: BMCs from firefly luciferase (Fluc)/green fluorescent protein (GFP) double transgenic mice were transplanted into C57BL/6J wide-type mice. The recipient mice were injected intraperitoneally with streptozotocin (STZ) daily for 5 consecutive days to induce diabetes mellitus (DM), followed by CNV laser photocoagulation. The BMCs recruitment in CNV exposed to hyperglycemia was firstly examined in Fluc/GFP chimeric mice by in vivo optical bioluminescence imaging (BLI) and in vitro Fluc assays. The CNV severity was evaluated by H&E staining and choroidal flatmount. The expression of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1) was detected by Western Blot., Results: BLI showed that the BMCs exerted dynamic effects in CNV model in Fluc/GFP chimeric mice exposed to hyperglycemia. The signal intensity of transplanted Fluc(+)GFP(+) BMCs in the DM chimeric mice was significantly higher than that in the control chimeric mice with CNV induction at days 5, 7, 14 and 21 (121861.67±9948.81 vs 144998.33±13787.13 photons/second/cm(2)/sr for control and DM mice, P 5d<0.05; 178791.67±30350.8 vs 240166.67±22605.3, P 7d<0.05; 124176.67±16253.52 vs 196376.67±18556.79, P 14d<0.05; 97951.60±10343.09 vs 119510.00±14383.76, P 21d<0.05), which was consistent with in vitro Fluc assay at day 7 [relative light units of Fluc (RLU1)], 215.00±52.05 vs 707.33±88.65, P<0.05; RLU1/ relative light units of renilla luciferase (RLU2), 0.90±0.17 vs 1.83±0.17, P<0.05]. The CNVs in the DM mice were wider than those in the control group at days 5, 7, 14 and 21 (147.83±17.36 vs 220.33±20.17 µm, P 5d<0.05; 212.17±24.63 vs 326.83±19.49, P 7d<0.05; 163.17±18.24 vs 265.17±20.55, P 14d<0.05; 132.00±10.88 vs 205.33±12.98, P 21d<0.05). The average area of CNV in the DM group was larger at 7d (20688.67±3644.96 vs 32218.00±4132.69 µm(2), P<0.05). The expression of VEGF and SDF-1 was enhanced in the DM mice., Conclusion: Hyperglycemia promots the vasculogenesis of CNV, especially the contribution of BMCs, which might be triggered by VEGF and SDF-1 production.

Hepatic ischaemia/reperfusion (I/R) injury is of primary concern during liver surgery. We propose a new approach for preserving low liver blood perfusion during hepatectomy either by occlusion of the portal vein (OPV) while preserving hepatic artery flow or occlusion of the hepatic artery while limiting portal vein (LPV) flow to reduce I/R injury. The effects of this approach on liver I/R injury were investigated. Rats were randomly assigned into 4 groups: sham operation, occlusion of the portal triad (OPT), OPV and LPV. The 7-day survival rate was significantly improved in the OPV and LPV groups compared with the OPT group. Microcirculatory liver blood flow recovered rapidly after reperfusion in the OPV and LPV groups but decreased further in the OPT group. The OPV and LPV groups also showed much lower ALT and AST levels, Suzuki scores, inflammatory gene expression levels, and parenchymal necrosis compared with the OPT group. An imbalance between the expression of vasoconstriction and vasodilation genes was observed in the OPT group but not in the OPV or LPV group. Therefore, preserving low liver blood perfusion by either the OPV or LPV methods during liver surgery is very effective for preventing hepatic microcirculatory dysfunction and hepatocyte injury.

Excessive N-methyl-D-aspartate receptor (NMDAR) activation and the resulting activation of neuronal nitric oxide synthase (nNOS) cause neuronal injury. Homer1b/c facilitates NMDAR-PSD95-nNOS complex interactions, and Homer1a is a negative competitor of Homer1b/c. We report that Homer1a was both upregulated by and protected against NMDA-induced neuronal injury in vitro and in vivo. The neuroprotective activity of Homer1a was associated with NMDA-induced Ca2+ influx, oxidative stress and the resultant downstream signaling activation. Additionally, we found that Homer1a functionally regulated NMDAR channel properties in neurons, but did not regulate recombinant NR1/NR2B receptors in HEK293 cells. Furthermore, we found that Homer1a detached the physical links among NR2B, PSD95 and nNOS and reduced the membrane distribution of NMDAR. NMDA-induced neuronal injury was more severe in Homer1a homozygous knockout mice (KO, Homer1a-/-) when compared with NMDA-induced neuronal injury in wild-type mice (WT, Homer1a+/+). Additionally, Homer1a overexpression in the cortex of Homer1a-/- mice alleviated NMDA-induced neuronal injury. These findings suggest that Homer1a may be a key neuroprotective endogenous molecule that protects against NMDA-induced neuronal injury by disassembling NR2B-PSD95-nNOS complexes and reducing the membrane distribution of NMDARs.

Background: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes., Methodology/principal Findings: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes., Conclusions: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

Unlabelled: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01)., Conclusions: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC., (© 2014 by the American Association for the Study of Liver Diseases.)

NDRG2 mRNA and protein levels can be upregulated in a p53-dependent manner. NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth, regardless of whether p53 is mutated. However, the complicated mechanism by which NDRG2 suppresses tumor cell growth and enhances apoptosis mediated by p53 is not fully understood. Here, we demonstrated that Ad-NDRG2 enhanced the apoptosis of HepG2 cells (wild-type p53). Additionally, Ad-NDRG2 combined with rAd-p53 enhanced the apoptosis of Huh7 cells (mutant p53) after chemotherapy, and the expression of the ERCC6 gene (Cockayne syndrome group B protein gene) was suppressed in this process. Ad-NDRG2 combined with rAd-p53 induced the apoptosis of tumor cells (HepG2 and Huh7 cells); however, apoptosis was attenuated after transfection with ERCC6. Our results indicate that Ad-NDRG2 enhances the p53-mediated apoptosis of hepatocarcinoma cells (HepG2 and Huh7) by attenuating the nucleotide excision repair capacity (i.e., by downregulating ERCC6), and ERCC6 is a NDRG2-inducible target gene that is involved in the p53-mediated apoptosis pathway.

The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.

Objective: To investigate the relationship between mild consciousness and mental disorders after arthroplasty and incomplete cerebral fat embolism., Methods: A retrospective analysis of 12 patients with incomplete cerebral fat embolism after arthroplasty was performed from June 2004 to December 2011. There were 5 males and 7 females,ranging in age from 36 to 82 years old,averaged 56.8 years old. Four patients had femoral neck fractures; 3 patients had avascular necrosis of the femoral head; 3 patients had rheumatoid arthritis; 1 patient had ankylosed hip and 1 patient had knee osteoarthritis. The patients had consciousness and mental disorders after arthroplasty (femoral head replacement in 3 cases, total hip replacement in 7 cases, and knee joint surface replacement in 2 cases), changes of vital sign and abnormal brain MRI examination., Results: Twelve patients had mild consciousness and mental disorders,and the NIHSS score was 1.92+/-3.78,which was correlated with incomplete cerebral fat embolism after arthroplasty. The patients recovered conscious within 24 to 72 hours after treatment with expansion of blood volume,dehydrating agent and neuroprotective drugs,improving respiratory and circulatory function, hormone protection and antibiotic application. The patients were followed up with a mean period of 18 months (ranging from 10 to 36 months). The patients had neurological function recovering to normal without sequelae, and the NIHSS score decreased to 0., Conclusion: Incomplete cerebral fat embolism after arthroplasty is the main reason causing mild awareness and mental disorders, which is often to be misdiagnosed or ignored because of not typical clinical manifestations.

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of PD. Previous studies have revealed that Astragaloside IV (AS-IV) can reduce inflammation and oxidation, making it a potential therapeutic agent for neurodegenerative disease. In this study, we investigated whether AS-IV protect against 1-methyl-4-phenylpyridnium ion (MPP(+))-induced dopaminergic neurotoxicity in SH-SY5Y cells and determined the mechanism of AS-IV neuroprotection. We found that pretreatment with AS-IV significantly reversed the loss of cell viability, nuclear condensation, the generation of intracellular reactive oxygen species (ROS), and the increase in Bax/Bcl-2 ratio and the activity of caspase-3 induced by MPP(+). Our study suggests that the neuroprotective effect of AS-IV is related to mechanisms including ROS production and the inhibition of Bax-mediated pathway. The present study supports the notion that AS-IV may be a promising neuroprotective agent for the treatment of neurodegenerative disorders such as PD.

Objective: To study the characteristics of genotype spectrum and hematologic parameters in children with HbH disease in the North Guangxi region., Methods: HbH disease was identified by clinical manifestations, routine blood tests and hemoglobin electrophoresis in 166 children who came form the North Guangxi region. Genotypes were determined by Multi-PCR combined with PCR reverse dot blot. DNA sequencing was used when the genotype could not be identified by regular methods., Results: Of the 166 children with HbH disease, 8 genotypes were identified: --SEA/-α3.7 (82 cases), --SEA/-α4.2 (40 cases), --SEA/αCSα (38 cases), --SEA/αQSα (1 case), --SEA/αWSα (1 case), --SEA/αCD43/44 (-C) α (1 case), --SEA/-α3.7 plus CD17 (A→T) (1 case) and --SEA/-α4.2 plus CD41-42(-TTCT) (1 case). One case was confirmed as the heterozygote of --SEA and an unknown mutation. In the 134 cases with complete medical data, 2 had normal hemoglobin levels, 36 manifested mild anemia, 90 manifested moderate anemia, and 6 (genotype: --SEA/αCSα) showed severe anemia because of the coexistence of infection. Children with the genotype of --SEA/-α3.7 (69 cases), --SEA/-α4.2 (31 cases) and --SEA/αCSα (34 cases) had hemoglobin levels of 62-120, 69-127 and 34-110 g/L respectively. The hemoglobin level in the --SEA/αCSα group was significantly lower than in the deletional HbH disease group (genotypes: --SEA/-α3.7 and --SEA/-α4.2 ) (P<0.05). In contrast, MCV levels in the --SEA/αCSα group were significantly higher than in the deletional HbH disease group (P<0.05)., Conclusions: The genotype spectrum of HbH disease is diverse in the North Guangxi region. Deletional genotype is prevalent. The disease is heterogeneous. The children with --SEA/αCSα HbH disease have severer anemia and higher MCV levels than those with deletional HbH disease.

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our results reveal a potential strategy for the treatment and prevention of diseases involving CNV.

Background & Aims: Human epidermal growth factor receptor 2 (HER2) (neu/ERBB2) is overexpressed on many types of cancer cells, including gastric cancer cells; HER2 overexpression has been associated with metastasis and poor prognosis. We investigated the mechanisms by which HER2 regulates cell migration and invasion., Methods: HER2 expression or activity was reduced in gastric cancer cell lines using small interfering RNAs or the monoclonal antibody, trastuzumab. We identified proteins that interact with HER2 or microRNAs (miRNAs) involved in HER2 signaling. We used various software programs to identify miRNAs that regulate factors in the HER2 signaling pathway. We analyzed expression patterns of these miRNAs in gastric cancer cell lines and tumor samples from patients., Results: We found that CD44 binds directly to HER2, which up-regulates the expression of metastasis-associated protein-1, induces deacetylation of histone H3 lysine 9, and suppresses transcription of microRNA139 (miR-139) to inhibit expression of its target gene, C-X-C chemokine receptor type 4 (CXCR4). Knockdown of HER2 and CD44 reduced invasive activity of cultured gastric cancer cells and suppressed tumor growth in nude mice. Lymph node metastasis was associated with high levels of HER2, CD44, and CXCR4, and reduced levels of miR-139 in human metastatic gastric tumors. Cultures of different types of metastatic cancer cells with histone deacetylase inhibitors and/or DNA methyltransferase resulted in up-regulation of miR-139., Conclusions: HER2 interaction with CD44 up-regulates CXCR4 by inhibiting expression of miR-139, at the epigenetic level, in gastric cancer cells. These findings indicate how HER2 signaling might promote gastric tumor progression and metastasis., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

N-myc downstream regulated gene 2 (NDRG2) is involved in invasion and metastasis of cancer, furthermore it is frequently down-regulated in prostate cancer. Herein we evaluated the effect of NDRG2 overexpression on invasiveness and bone destruction in prostate cancer. The human prostate cancer cell line PC-3 and DU145 were infected with Ad-NDRG2 or Ad-LacZ. Overexpression of NDRG2 not only inhibited the growth of the cells, but also suppressed invasiveness of the cells in an in vitro assay. PC-3 cells infected with Ad-NDRG2 or Ad-LacZ were injected into the tibias of nude mice. Four weeks later, we found the mice injected with PC-3 cells overexpressing NDRG2 had smaller tumors and less bone destruction. These results demonstrate that NDRG2 overexpression can inhibit tumor growth and invasion, furthermore, it can decrease bone destruction caused by prostate cancer bone metastasis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age-related macular degeneration (AMD). Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) plays a critical role in homing of bone marrow-derived cells (BMCs) to choroidal neovascularization (CNV). In this study, we investigated the contribution of hypoxia specific HIF-1α-induced SDF-1 expression in retinal pigment epithelium (RPE) cells and the potential role of SDF-1 in CNV formation., Materials and Methods: Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells of gfp(+/+) transgenic mice to sublethally irradiated C57BL/6J mice. CNV was induced by laser photocoagulation. Ocular tissue was processed for immunofluorescence to detect HIF-1α and SDF-1 expression, and cell surface markers such as CXCR4, CD34 and CD31 and so on during CNV formation. In vitro, adult human RPE (hRPE) cells were cultured under conditions of chemical hypoxia using CoCl2 administration. And RNAi technique was used to knock down HIF-1α gene to observe the expression of HIF-1α and SDF-1 in hRPE cells., Results: BMCs trafficked around laser lesion adjacent to RPE layer 4 h after laser photocoagulation, where SDF-1 expression was relatively higher. With increasing expression of SDF-1, more BMCs were infiltrated into laser lesion to participate in CNV, and both reached peak at 3 d (p < 0.05). About 81% BMCs involved in CNV were CXCR4+. Many of them acquired the surface marker of endothelial precursor cells (CD34+) and endothelial cells (CD31+). The constituent ratio of CD34+ and CD31+ BMCs increased with SDF-1 expression. In vitro, we proved that hypoxia specific-HIF-1α influenced SDF-1 expression in hRPE cells., Conclusions: These findings suggested that hypoxia-induced SDF-1 expression in RPE might be a critical initiator for recruitment of BMCs in CNV. SDF-1 might be another important factor in BMCs' differentiation into endothelial cells to participate in the CNV.

Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis.

Objective: To investigate the DNA methylation status of the promoter region within the coding gene hsa-miR-124a in human gastric cancer tissue, and examine its association with the expression of Rb and CDK6 protein and clinicopathological factors., Methods: Methylation-specific PCR (MS-PCR) was used to detect the DNA methylation status of hsa-miR-124a in gastric cancer tissues and adjacent normal tissues from 30 patients. The expression of Rb and CDK6 protein within these tissues was examined using immunohistochemistry., Results: The overall methylation rate of gastric cancer tissues was 73.3%, which was significantly higher than that in the adjacent tissues(10.0%, P<0.05). The overall positive rates of Rb and CDK6 protein in gastric cancer tissues were 86.7% and 80.0%, respectively. DNA methylation of hsa-miR-124a was positively correlated with the expression of Rb and CDK6 proteins. Significant associations were found between hypermethylation of hsa-miR-124a and tumor size, differentiation, lymphatic metastasis, and invasion depth(P<0.01)., Conclusions: Hypermethylation of hsa-miR-124a is present in gastric cancer, and is associated with increased expression of CDK6 and Rb protein. These may be related to the proliferation and development of malignancy in the stomach.

Background: Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer's disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract., Methodology/principal Findings: Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H(2)O(2) to simulate senescence. After being exposed to 50 µM H(2)O(2) for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50-200 µM H(2)O(2) for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses., Conclusions/significance: NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.

MicroRNAs, a large family of small regulatory RNAs, are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner, thereby controlling diverse aspects of cell function, including immune reaction. In this study, we screened and identified a group of differentially expressed miRNAs in naive and activated CD4(+) T cells. Among the miRNAs studied, miR-181c was proven to have the potential to regulate CD4(+) T cell activation. miR-181c was downregulated in the process of CD4(+) T cell activation, and transfection of miR-181c mimics partially repressed the activation of both Jurkat cells and human peripheral blood mononuclear cells (PBMC) CD4(+) T cells. We further showed that miR-181c can bind to the IL-2 3' UTR and repress its expression by inhibiting translation. Moreover, miR-181c mimics reduced activated CD4(+) T cell proliferation. Taken together, our results show that miR-181c serves as a negative regulator that modulates the activation of CD4(+) T cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described., Methods: The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot., Results: The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell., Conclusions: We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.

HER2 overexpression is associated with metastasis-the main cause of death in individuals with gastric cancer. In this study, we demonstrated that vector-based shRNA significantly knocked down the expression of HER2 and considerably inhibited both the migration and invasion of gastric cancer cells. HER2 knockdown resulted in the downregulation of the expression of MMP-1, while HER2 overexpression improved the transcription of MMP-1 through the activation of an MMP-1 promoter. The promoter region of MMP-1 between -2500 and -2000 bp was found to be crucial for the upregulation of HER2-mediated transcription. Furthermore, a truncated promoter (-70 to+63) did not display any transcriptional activity. Cell invasion activity was almost completely inhibited when MMP-1 was knocked down. Conversely, the overexpression of MMP-1 partly rescued the invasion ability of cell strains with knocked-down HER2. These findings help further understanding of the molecular mechanisms through which HER2 promotes malignancy, and suggest that targeting both HER2 and MMP-1 may be required to effectively block HER2 signaling in gastric cancer therapy., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: The HER2 antigen is a recognized target on breast cancer cells for immunotherapy. To overcome the immunogenicity and systemic toxicity of traditional immunotoxins, a novel human immunoproapoptotic molecule was developed and its antitumor activity was investigated., Experimental Design: Recombinant e23sFv-TD-tBID, consisting of a single-chain anti-HER2 antibody fragment linked to a human active truncated Bid by a 10-amino acid residue furin cleavage sequence, was bacterially expressed. Purified e23sFv-TD-tBID was tested for binding, internalization, and cytotoxic activity in cell and for tumor localization and antitumor activity in athymic nude mice bearing established human tumor xenografts., Results: e23sFv-TD-tBID selectively binds to HER2-positive cells and induces apoptotic cell death in vitro and in vivo. An investigation of its mechanism of action has revealed that e23sFv-TD-tBID was internalized on binding to the surface of HER2-positive tumor cells, proteolytically cleaved and transported directly to cytosol. The antitumor activity of e23sFv-TD-tBID was shown in a dose-dependent manner when injected i.p. into immunodeficient mice bearing human breast carcinomas. Moreover, this immunoproapoptotic protein, either given as a single dose or in combination with chemotherapy agents, significantly inhibited tumor growth without any observed toxic side effects on mice. Magnetic resonance imaging further showed the specific targeting and good penetration of tumors by e23sFv-TD-tBID in vivo. The therapeutic value of e23sFv-TD-tBID to human was shown by its cytotoxic effects on primary patient-derived breast tumor cells but not on endothelial cells., Conclusion: These data suggest that recombinant e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and warrant further testing for clinical applications.

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X-XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells.

Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch's membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1alpha) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1alpha and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV.

Objective: To determine the expression of new candidate tumor suppressor gene N-Myc downstream-regulated gene 2(Ndrg2) in colorectal cancer with different differentiation, and analyze its clinical significance., Methods: Specimens of 50 colorectal cancer patients with different differentiation were collected. Immunohistochemistry and Western blot were used to examine the expression of Ndrg2. Colorectal cancer tissue array in large scale was applied to analyze the expression of Ndrg2 and the statistics analysis was performed referring to the patients information of the array., Results: Among 50 cases, Ndrg2 expression level of colorectal cancer was significantly lower in 32 cases as compared to adjacent and normal tissue of the same individual, while Ndrg2 expression of adjacent tissue was significantly lower than that of normal tissue. Ndrg2 protein levels increased from poor-differentiated to well-differentiated carcinomas(P=0.005)., Conclusions: The expression of Ndrg2 in different differentiated colorectal cancer tissues show a significant distinction. Ndrg2 may be involved in the regulation of differentiation in colorectal cancer.

Tumor cells have developed immune evasion mechanisms such as considerably heterogenous FasL expression on their surface via which they could induce apoptosis of tumor-specific cytotoxic T lymphocytes (CTLs) in the immune system. Meanwhile, the competition of normal immune cells with tumor cells results in relative growth factors shortage for growth and proliferation of nontumor cells, which improves a susceptibility to early apoptosis of CTL. In an attempt to develop strategies for prolonging the survival of adoptively transferred T cells in a hostile pro-apoptotic tumor microenvironment, we used synthetic siRNA and vector-based shRNA to suppress the expression of Bid in human uterocervical carcinoma HeLa cells, followed by the further achievement of Bid gene silencing in human primary cells-CD8(+) lymphocytes via retrovirus-delivered siRNAs. Our results indicated that Bid knockdown HeLa cells are partially resistant to Fas antibody- or serum deprivation-induced apoptosis. Additionally, the blockade of Bid expression in CD8(+) lymphocytes resulted in a less susceptiveness to Fas antibody-induced apoptosis and a survival advantage following recombinant human interleukin-2 (rhIL-2) withdrawal or under lower rhIL-2 concentrations compared with control lymphocytes. These data suggest that knockdown of Bid might serve as an approach to enhancing the survival and tumoricidal activity of T lymphocytes in adoptive immunotherapy.

Despite the widespread application of RNA interference (RNAi) as a research tool for diverse purposes, the key step of strand selection of siRNAs during the formation of RNA-induced silencing complex (RISC) remains poorly understood. Here, using siRNAs targeted to the complementary region of Survivin and the effector protease receptor 1 (EPR-1), we show that both strands of the siRNA duplex can find their target mRNA and are equally eligible for assembly into Argonaute 2 (Ago2) of RISC in HEK293 cells. Transfection of the synthetic siRNA duplexes with different thermodynamic profiles or short hairpin RNA (shRNA) vectors that generate double-stranded RNAs (dsRNAs), permitting processing specifically from either the 5' or 3' end of the incipient siRNA, results in the degradation of the respective target mRNAs of either strand of the siRNA duplex with comparable efficiencies. Thus, while most RNAi reactions may follow the thermodynamic asymmetry rule in strand selection, our study suggests an exceptional mode for certain siRNAs in which both strands of the duplex are competent in sponsoring RNAi, and implies additional factors that might dictate the RNAi targets.

The second domain of Pseudomonas exotoxin A (PEAII, residues 253-364) has been shown to facilitate translocation of extracellular and vesicular contents into the cytoplasm, and can transport heterologous molecules into target cells. Because full length PEAII may elicit a host immune response, we tried to identify the minimal PEAII translocation motif and use this fragment in combination with an antibody and constitutively active granzyme B (ImmunoGrB) to kill HER2-positive tumor cells. We constructed four ImmunoGrB fusion proteins containing different PEAII deletions and tested their abilities to kill HER2-positive cells. Our data showed that while a complete deletion of PEAII in ImmunoGrB resulted in an inability to kill cancer cells, ImmunoGrBs containing either PEAII (253-358aa) or PEAII (275-358aa) could efficiently kill HER2-positive SK-BR-3 cells. Most interestingly, the construct which contains only a furin cleavage site, named PEAII (275-280aa), could still induce SK-BR-3 apoptosis, although less efficiently. Moreover, delivery of the recombinant proteins by intramuscular plasmid injection led to an apparent tumor regression and prolonged animal survival in a nude mouse xenograft SK-BR-3 tumor model, indicating in vivo antitumor activity of the different PEAII containing ImmunoGrBs. Our results may help in understanding PEAII translocation and may lead to the development of useful tools or alternative therapy.

BH3-interacting domain death agonist (BID) is a crucial element in death signaling pathways and is recognized as an intracellular link connecting the intrinsic mitochondrial apoptotic and extrinsic death receptor-mediated apoptotic pathways. Herein, we describe experiments conducted with a fusion protein, which was generated by fusing a human epidermal growth factor receptor-2 (HER2)-specific single-chain antibody with domain II of Pseudomonas exotoxin A and the truncated active BID (tBID). These experiments extend our previous work on several other immuno-proapoptotic proteins. Specifically, by excluding cells with undetectable HER2, we showed that the secreted immuno-tBID molecule selectively recognized and killed HER2-overexpressing tumor cells in vitro by attacking their mitochondria and inducing their apoptotic death. This apoptosis could only be inhibited partially by caspase pan-inhibitor zVAD and mitochondrial protector TAT-BH4. Subsequently, we transferred the immuno-tbid gene into BALB/c athymic mice bearing HER2-positive tumors together with other immuno-proapoptotic proteins using i.m. injections of liposome-encapsulated vectors. The expression of the immuno-tbid gene suppressed tumor growth and prolonged animal survival significantly. We also shortened the translocation domain of Pseudomonas exotoxin A II to only 10-amino acid sequence, which were crucial for furin cleavage. The new recombinant molecule retained the translocation efficiency and the ability of specific killing HER2-positive tumor cells. Our data showed that, compared with the toxins employed before, the chimeric immuno-tBID molecule can not only specifically recognize HER2-positive tumor cells but also certainly induce apoptosis even in the presence of zVAD and TAT-BH4, thereby suggesting an alternative approach to treating HER2/neu-positive tumors.

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.

In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre-LoxP-mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal-binding protein Jkappa (RBP-J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age-related macular degeneration, RBP-J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP-J could modulate bone marrow-derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP-J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP-J-mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.