Up-regulation of long non-coding RNA SNHG1 contributes to proliferation and metastasis in laryngeal squamous cell carcinoma.

OBJECTIVE: To investigate the expression of human long non-coding ribonucleic acid (RNA) small nucleolar RNA host gene 1 (SNHG1) in laryngeal carcinoma tissues, and to study the effect of SNHG1 on biological functions of laryngeal carcinoma HEp-2 cells. PATIENTS AND METHODS: The expression levels of SNHG1 in 20 pairs of laryngeal carcinoma tissues and para-carcinoma tissues were detected via Real-time fluorescence quantitative polymerase chain reaction (PCR). Laryngeal carcinoma cells were transfected with small interfering (si)-SNHG1 transiently using the RNA interference technique. The effects of si-SNHG1 on proliferation, apoptosis, invasion, and migration of laryngeal carcinoma HEp-2 cells were detected via cell counting kit-8 (CCK-8), colony formation assay, flow cytometry, and wound healing and Transwell assay, respectively. RESULTS: Results of PCR showed that the expression of SNHG1 in carcinoma tissues was increased compared with that in para-carcinoma tissues. Results of CCK-8 and colony formation assay revealed that SNHG1 knockdown could significantly inhibit the proliferation of laryngeal carcinoma HEp-2 cells. Flow cytometry showed that transfection with si-SNHG1 could promote the apoptosis of HEp-2 cells. Moreover, results of wound healing and Transwell assay showed that SNHG1 knockdown could inhibit invasion and migration of HEp-2 cells through inhibiting the epithelial-mesenchymal transition (EMT) process and expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. CONCLUSIONS: The expression of SNHG1 in laryngeal carcinoma tissues is significantly higher than that in para-carcinoma tissue. Patients with high expression of SNHG1 have a poor prognosis. SNHG1 knockdown in HEp-2 cells can inhibit cell proliferation, invasion, and metastasis, and can promote apoptosis.

Isolation and characterization of microsatellite markers for Eucommia ulmoides (Eucommiaceae), an endangered tree, using next-generation sequencing.

Eucommia ulmoides Oliver, a single extant species of Eucommiaceae, is an endemic dioecious tree in China. The natural resources of E. ulmoides have rapidly declined in recent years because of the over-collection of its cortex. To design a suitable protection strategy, it is necessary to develop a set of molecular markers to investigate genetic diversity and population structure of E. ulmoides. Pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform was used to isolate simple sequence repeats (SSRs) for E. ulmoides. A total of 1568 SSRs that contained enough flanking sequences for primer pair design were identified from 45,236 raw sequence reads. One hundred SSRs were randomly selected to design primer pairs and polymerase chain reaction was performed. Among these 100 tested primer pairs, 16 were polymorphic across 18 individuals from three E. ulmoides populations. The number of alleles ranged from 3 to 8, with an average of 5.1. The expected heterozygosity ranged from 0.110 to 0.830, with an average of 0.648, and the observed heterozygosity ranged from 0.111 to 0.833, with an average of 0.524. The inbreeding coefficient ranged from -0.349 to 0.547. This set of microsatellite markers could be valuable for landscape genetic structure assessment and molecular marker-assisted breeding in E. ulmoides.

Highly polysialylated neural cell adhesion molecule (PSA-NCAM) positive cells are increased and change localization in rat hippocampus by exposure to repeated kindled seizures.

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is involved in migration of neural stem cells as well as in neural plasticity. Immunoreactive PSA-NCAM expression was examined in rats with repeated exposure to amygdaloid kindled generalized seizures (GS). The number of PSA-NCAM positive cells in the bilateral dentate gyrus (DG) increased significantly from GS. Although the total number of positive cells was not significantly different between animals with 3 times GS (3 GS) and 30 times GS (30 GS), in the latter group a greater number of positive cells was observed in the outer granule cell layer (GCL) and a marked extension of immunopositive dendrites to the molecular layer. These observations indicate that increased migration of newly generated cells as well as plastic changes of preexisting neural cells occur in response to recurrent GS. This may contribute to an abnormal reconstruction of the synaptic network in the hippocampus and, thus, epileptogenicity from kindling.

Enhanced phosphorylation of PTEN in rat brain after transient middle cerebral artery occlusion.

A phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene that suppresses cell growth, inhibits cell migration, and induces apoptosis. Phosphorylated form of PTEN (p-PTEN) is a key survival factor relating PI3K-Akt pathway and their downstream effectors. A spatiotemporal profiles of PTEN and p-PTEN expression were immunohistochemically examined after 90 min of transient middle cerebral artery occlusion in rats. In the ischemic core, PTEN progressively decreased by 3 days, whereas a rapid but transient increase of p-PTEN was found with a peak at 1 h after the reperfusion. In contrast, in the ischemic penumbra, PTEN showed a minor change and a gradual but sustained p-PTEN expression was observed in the ischemic penumbra with a peak at 12 h. In addition, the balance of population among strongly, moderately, and weakly stained cells was different between the ischemic core and penumbra at their peak time points. These results suggest an important role of p-PTEN for cell survival after ischemia as an upstream regulator for PI3K-Akt.

Enhanced expression of phospho-Akt by electro-acupuncture in normal rat brain.

Electro-acupuncture (EA) is an effective curative method for various diseases in oriental medicine. To investigate a detailed molecular mechanism of EA stimulation, an induction of phospho-Akt (p-Akt) was examined in normal adult rat brain after 60 min of EA with acupoints of Baihui (D20) and Renzhong (D26). In the sham control brain, strong neuronal p-Akt expression was found in ventral posterolateral thalamic nucleus (VPL) and medial habenular nuclei (MHb), but moderate to weak in cortex, caudate, CA1 sector and dentate gyrus of hippocampus, and ventral posteromedial thalamic nucleus. EA stimulation generally enhanced and sustained p-Akt expression for at least 24 h especially in the regions listed above, except VPL and MHb where no apparent change was found. Western blot analysis of p-Akt confirmed the enhanced signal intensity after EA at 8 and 24 h. These results suggest that the EA on D20 and D26 acupoints activates the survival Akt signal pathway, which may be maintaining the neural functions such as cell survival and memory formation in normal brain.

Therapeutic time window of adenovirus-mediated GDNF gene transfer after transient middle cerebral artery occlusion in rat.

The time dependent influence of adenovirus-mediated glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with Ad-GDNF significantly reduced the infarct volume when immediately administered after the reperfusion, but became insignificant when administered at 1 h after the reperfusion as were the cases treated with vehicle- and adenoviral vector containing the E. coli lacZ gene (Ad-LacZ)-treated groups. The protective effect of GDNF was related to the significant reduction of the number of TUNEL positive cells as well as immunohistochemical positive cells for active caspase-3 but not -9. These results showed that exogenous GDNF gene transfer successfully reduced the infarct size in a time-dependant manner by suppressing active caspase-3 but not active caspase-9. However, the therapeutic time window was shorter than the effect of GDNF protein itself previously reported.

Induction of PML immunoreactivity in rat brain neurons after transient middle cerebral artery occlusion.

Promyelocytic leukemia (PML) protein is involved in apoptotic death of cultured neuronal cells, but its role in ischemic brain damage remains uncertain. In this study, we investigated change of immunoreactivity for PML protein in rat brain after transient middle cerebral artery occlusion, and compared the results with that of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). Western blotting analysis revealed that PML immunoreactivity was only scant in the sham-control brain, but it increased at 1 h and 1 day after reperfusion, and decreased in density thereafter. Immunohistochemical analysis revealed that nuclei of neurons were most densely stained. TUNEL positive cells appeared at 1 day and peaked at 3 days of reperfusion, indicating that PML protein induction preceded DNA fragmentation in neurons. The present results suggest that PML protein may be one of the key molecules in ischemic neuronal cell death.

Different expression of glycogen synthase kinase-3beta between young and old rat brains after transient middle cerebral artery occlusion.

Ischemia is a common stress to human brain and is difficult to cure in older individuals. To examine the differences of the response to cerebral ischemia between young and old rat brains, distributions of glycogen synthase kinase-3beta (GSK3beta) and tau proteins were analyzed after 90 min of transient middle cerebral artery occlusion (MCAO) in young (10-11 weeks) and old (15 months) rats by immunohistochemical analyses. At 4 h of reperfusion, strong cytoplasmic and nuclear immunoreactivity for GSK3beta was induced in neurons of lamina I, II, V and VI of the cerebral cortex and dorsal caudate in young brains, while the induction was not observed in lamina I and II of old cerebral cortex. The staining in lamina V and VI and dorsal caudate then gradually decreased until seven days of reperfusion in both animal groups. The staining of tau protein and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) did not show any positive signals in the control brain, but showed positive signals after ischemia with a peak at 24 h and 3 days, respectively. No significant difference was observed in the temporal and spatial patterns of tau and TUNEL stainings between these two groups. These data suggest that GSK3beta may have a role in ischemic neuronal cell death, and that the different spatial expression of GSK3beta between young and old rat brains may partly explain the vulnerability of older neurons after ischemia.

Attenuation of oxidative DNA damage with a novel antioxidant EPC-K1 in rat brain neuronal cells after transient middle cerebral artery occlusion.

EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt, is a novel antioxidant. In this study, we investigated a reduction of oxidative neuronal cell damage with EPC-K1 by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat brain with 60 min transient middle cerebral artery occlusion, in association with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and staining for total and active caspase-3. Treatment with EPC-K1 (20 mg kg(-1) i.v.) significantly reduced infarct size (p < 0.05) at 24 h of reperfusion. There were no positive cells for 8-OHdG and TUNEL in sham-operated brain, but numerous cells became positive for 8-OHdG, TUNEL and caspase-3 in the brains with ischemia. The number was markedly reduced in the EPC-K1 treated group. These reductions were particularly evident in the border zone of the infarct area, but the degree of reduction was less in caspase-3 staining than in 8-OHdG and TUNEL stainings. These results indicate EPC-K1 attenuates oxidative neuronal cell damage and prevents neuronal cell death.

Expression of polysialylated neural cell adhesion molecule in rat brain after transient middle cerebral artery occlusion.

The highly polysialylated form of neural cell adhesion molecule (PSA-NCAM) is important for neurite outgrowth. With this molecule as a marker of plastic change in neurons, we investigated its temporal expression in rat brain after transient middle cerebral artery (MCA) occlusion. In sham-control brain, only subependymal neurons showed a positive immunoreactivity for PSA-NCAM. After 90 min of transient MCA occlusion, neurons in the piriform cortex began to be positively stained at 1 h, while neurons in the cortex and caudate of the MCA territory became positive after 8 h. The stainings persisted for 1 and 3 days after reperfusion. The present results indicate that neurons in the cerebral cortex and caudate have the capability of plastic change in the adult brain, and that those in the piriform cortex rapidly undergo plastic change probably in response to transneuronal injury.

Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation.

We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia.

Time dependent amelioration against ischemic brain damage by glial cell line-derived neurotrophic factor after transient middle cerebral artery occlusion in rat.

Time dependent influence of glial cell line-derived neurotrophic factor (GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with GDNF significantly reduced the infarct volume stained with 2,3,5-triphenyltetrazolium chloride (TTC) when GDNF was topically applied at 0 and 1 h of reperfusion, but became insignificant at 3 h as compared to vehicle group. The protective effect of GDNF was closely related to the significant reduction of the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) positive cells as well as immunofluorescently positive cells for active forms of caspases, especially active caspase-3 but not -9. Thus, the present study showed that topical application of GDNF significantly reduced infarct size in a time-dependent manner, while the therapeutic time window was shorter than other chemical compounds such as an NMDA receptor antagonist (MK-801) and a free radical scavenger (alpha-phenyl-tert-butyl-nitrone, PBN). The effect of GDNF was stronger in suppressing active caspase-3 than active caspase-9.

Reduction of ischemic damage by application of insulin-like growth factor-1 in rat brain after transient ischemia.

In order to investigate a possible effect of insulin-like growth factor-1 (IGF-1) on ischemic brain injury, IGF-1 was applied topically on the brain surface of reperfused rat brain after 60 min of transient middle cerebral artery occlusion. In contrast to the cases treated with vehicle, the infarct volume was greatly reduced at 24 h of reperfusion by the treatment with IGF-1. Immunohistochemical analysis in the middle cerebral artery territory showed that Caspase-3 staining was markedly reduced in the cases with IGF-1 treatment, but 72-kDa heat shock protein staining remained almost unchanged. The present results suggest that treatment with IGF-1 exerts a significant effect on ameliorating brain injury after transient focal brain ischemia. Moreover, this effect is greatly associated with the reduction of Caspase-3 staining, but is only minimally associated with a decreasd stress response at the cellular level.

Phosphorylation of retinoblastoma protein in rat brain after transient middle cerebral artery occlusion.

Although mature neurones do not replicate genomic DNA, some cell cycle-related kinases are aberrantly activated in neurones after ischaemia. As hyper-phosphorylation of retinoblastoma (Rb) protein is the common pathway in mitotic signal cascade, this study investigated the phosphorylation state of the Rb protein as well as its mRNA level in rat brain after transient middle cerebral artery (MCA) occlusion. Immunohisto-chemical analysis revealed that neurones in the sham-operated brain expressed Rb protein without the hyperphosphorylated form. Immunoreactivity for the hyperphosphorylated form of Rb protein progressively increased from 1 h to 3 days after ischaemia in neurones in the MCA territory. Western blot analysis demonstrated a similar change. However, reverse transcription-polymerase chain reaction study revealed that Rb showed no definite change at the mRNA level. These results suggest that Rb protein is progressively hyper-phosphorylated in the brain after ischaemia, which may activate apoptotic mechanisms in neuronal cells of the brain after ischaemia.

Protective effect of ginkgo extract on rat brain with transient middle cerebral artery occlusion.

It has been empirically known that Ginkgo extract is useful for reducing many symptoms associated with cerebral blood flow (CBF) insufficiency, but its mechanisms have been uncertain. In the present study, therefore, we gave Ginkgo extract to rats with per os digestion, and investigated its effect on CBF and ischemic brain damage with middle cerebral artery occlusion (MCAO). The treatment with Ginkgo extract (10 mg 100 g-1 rat) increased CBF in the normal condition, but the degree of increase in CBF was lesser during and after MCAO. TTC staining showed that infarct volume was reduced with Ginkgo treatment. TUNEL and HSP72 immunostaining confirmed the protective effect of Ginkgo treatment reducing numbers of TUNEL and HSP72 positive cells. Immunohistochemical analysis showed that caspase-3 expression was less abundant in Ginkgo treated rats. The present results suggest that Ginkgo extract contains a substance which increases normal CBF and reduces ischemic brain damage.

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats.

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 1. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats.

c-Jun N-terminal kinase (JNK) and JNK interacting protein response in rat brain after transient middle cerebral artery occlusion.

c-Jun response is involved in the development of ischemic brain injury, which is activated by c-Jun N-terminal kinase-1 (JNK-1). The activity of JNK-1 is strictly regulated, and only the phosphorylated form of JNK (phospho-JNK) which is translocated to the nucleus has an ability to activate c-Jun response. There is a protein which inhibits JNK-1 activation, and known as JNK interacting protein-1 (JIP-1). In this study, we investigated change in JNK-1, phospho-JNK, and JIP-1 immunoreactivity in rat brain after transient middle cerebral artery (MCA) occlusion. Immunoreactive JNK-1 was scant in the sham-control brain, but it was induced at 1 h after reperfusion, which was slightly increased at 3 h of reperfusion. By contrast, phospho-JNK remained negative till 3 h. At 8 h, JNK-1 and phospho-JNK became distinctly positive, and nuclei as well as cytoplasm were stained. Thereafter, immunoreactivity for JNK-1 and phospho-JNK became furthermore dense, and most neurons revealed positively stained nuclei. Immunoreactivity for JIP-1 remained negative till 8 h of reperfusion, but at 24 and 72 h, cytoplasm of cortical neurons at the MCA boundary area was positively stained. This JIP-1 induction got behind the JNK-1 activation, and therefore, may be a vain effort for neurons to survive. Inhibition of JNK-1 activation might become an innovative means of therapy for stroke treatment in the future.