LncRNA SNHG7 participates in osteosarcoma progression by down-regulating p53 via binding to DNMT1.

OBJECTIVE: This study aims to explore the role of lncRNA SNHG7 in the development of osteosarcoma, and its underlying mechanism. PATIENTS AND METHODS: The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect SNHG7 expression in tumor tissues and paracancerous tissues harvested from osteosarcoma patients. Meanwhile, the relationship between SNHG7 expression and tumorigenesis was analyzed. The effects of SNHG7 and p53 on cell proliferation, cell cycle and apoptosis were detected by plate cloning and flow cytometry, respectively. The binding relationship between SNHG7 and DNMT1, as well as the regulatory mechanism of DNMT1 on p53, were detected by RIP and ChIP. Western blot was conducted to detect the expression of p53 after the knockdown of SNHG7 in osteosarcoma cells. Rescue experiments were finally conducted to verify whether SNHG7 exerted its biological function by targeting p53. RESULTS: QRT-PCR results demonstrated that the expression of SNHG7 in osteosarcoma tissues was remarkably higher than that in paracancerous tissues. Moreover, SNHG7 expression in osteosarcoma with stage III and IV was higher than those in stage I and II. The inhibition of SNHG7 in osteosarcoma cells U2OS and HOS promoted cell proliferation, arrested cell cycle in the G0/G1 phase and induced apoptosis. RIP and ChIP experiments illustrated that SNHG7 inhibited the expression of p53 by binding to DNMT1. The overexpression of p53 in U2OS cells partially reversed the promoted cell proliferation and apoptosis caused by SNHG7. CONCLUSIONS: Highly expressed SNHG7 can promote the proliferation and inhibit apoptosis of osteosarcoma cells by regulating p53 expression by binding to DNMT1.

[Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

Detection of infectious hepatitis A virus by integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction.

AIMS: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV). METHODS AND RESULTS: The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference. Water samples artificially contaminated with infectious or formalin-inactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively. The tagged RT-PCR had high specificity for HAV negative-strand RNA. By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV. The described method detected infectious HAV at inoculation level of 10(0) TCID50 per flask within 4 days. CONCLUSIONS: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples. This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines. It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water.

Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current.

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes.

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in Xenopus oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.

Insulin modulation of cloned mouse NMDA receptor currents in Xenopus oocytes.

Modulation of recombinant N-methyl-D-aspartate receptor (NMDAR) currents by insulin was studied using the Xenopus oocyte expression system. Insulin (0.8 microM, 10 min) regulated NMDAR currents in a subunit-specific manner. Currents from epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 receptors were variably potentiated, whereas currents from epsilon3/zeta1 receptors were not. Protein tyrosine kinases (PTKs) and protein kinase C were found to be involved in insulin-mediated modulation in an NMDAR subtype-specific way. Pretreatment with a specific PTK inhibitor, lavendustin A, attenuated and blocked the insulin effect on epsilon2/zeta1 and epsilon4/zeta1, respectively. Preincubation with selective protein kinase C inhibitors, staurosporine or calphostin C, depressed the response of epsilon1/zeta1 and epsilon2/zeta1 receptors to insulin. Basal regulation of NMDAR currents by endogenous PTKs and protein tyrosine phosphatases (PTPs) was also investigated. Of the four receptor subtypes, only epsilon1/zeta1 receptor currents were affected by basal PTK inhibition via lavendustin A, whereas PTP inhibition by phenylarsine oxide or orthovanadate enhanced currents from epsilon1/zeta1 and epsilon2/zeta1 receptors. Surprisingly, a stimulatory PTP modulation was observed for epsilon4/zeta1. As NMDAR subunits are differentially expressed in the brain, the observed subtype-specific modulations of NMDAR currents by insulin, PTKs, and PTPs may provide important insights into certain NMDAR-dependent physiological and pathological processes.

Characterization of a glial associated antigen GA-1 by monoclonal antibody.

A glial antigen (GA-1) was identified by monoclonal antibodies (MAb) raised against C6 rat glioma cells. MAb-7D3 (IgG2a kappa) revealed GA-1 as a single protein band with a Rf value of 0.09 by the use of basic-PAGE Western blot. SDS-PAGE Western blot and radioimmunoprecipitation (RIP) further resolved GA-1 into two subunits with a molecular weight of 200 and 78 kDa respectively. Subcellular localization by immunocytochemical staining revealed its cytosolic presence with a punctate pattern perinuclearly. Significant expression of GA-1 may be detected in 4 glioma or glial cell lines derived from rat brain. However, no expression may be detected in the rest of the 18 mammalian cell lines or primary neural cell cultures examined. All of the above data thereby suggest that GA-1 may be glial specific whereas the epitope of GA-1 defined by MAb-7D3 is species (rat) specific.