CircRNA_0078767 promotes osteosarcoma progression by increasing CDK14 expression through sponging microRNA-330-3p.

Circular RNA (circRNA)-associated competing endogenous RNA (ceRNA) mechanism have emerged as critical mechanism in cancer initiation and progression. However, the roles of the circRNA-microRNA (miRNA)-messenger RNA ceRNA network in osteosarcoma are still not fully characterized. In this study, therefore, circ_0078767-related ceRNA mechanism in osteosarcoma was studied. Bioinformatics tools primarily identified differentially expressed circRNAs and their downstream miRNAs in osteosarcoma, implying the potential interaction between circ_0078767, miR-330-3p, and cyclin-dependent kinase 14 (CDK14) in this malignancy, which were further verified by means of RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays. Aberrant abundance of circ_0078767 was found in both osteosarcoma tissues and cells, relating to dismal prognosis in patients with osteosarcoma. Functionally, circ0078767 strengthened the proliferation, invasiveness, and migration of osteosarcoma cells, which could be neutralized by miR-330-3p. Additionally, miR-330-3p targeted and decreased CDK14 expression whereby motivating the malignant phenotypes of osteosarcoma cells. Through in vivo experiments, we further confirmed that circ_0078767 targeted miR-330-3p to upregulate CDK14, whereby strengthening the in vivo tumorigenic and metastatic ability of osteosarcoma cells. Circ_0078767 promotes the occurrence and development of osteosarcoma by upregulating CDK14 in a miR-330-3p-dependent manner.

Wogonoside alleviates inflammation induced by traumatic spinal cord injury by suppressing NF-κB and NLRP3 inflammasome activation.

Wogonoside possesses anti-oxidative, anti-inflammatory, anti-allergy and anti-tumor properties. The aim of the present study was to evaluate whether wogonoside alleviates spinal cord injury (SCI)-induced inflammation via nuclear factor (NF)-κB and nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation. Sprague-Dawley rats were positioned in the jaws of a calibrated aneurysm clip with a closing pressure of 55 g. The jaws were placed on the dorsal and ventral surfaces of the spinal cord and left in place for 1 min. SCI rats were treated with 12, 25 and 50 mg/kg wogonoside. Following this, the locomotor function was assessed using the Basso Beattie Bresnahan scale. The water content of the spinal cord was measured, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 levels were assessed and western blot analysis was performed to evaluate the expressions of NF-κB and NLRP3. Wogonoside was demonstrated to significantly ameliorate the SCI-induced reduction in Basso Beattie Bresnahan score (P<0.01) and significantly reduce the water content of the spinal cord in rats with SCI-induced inflammation (P<0.01). Results also indicated that treatment with wogonoside significantly reduced the levels of IL-1ß, TNF-α and IL-6 in rats with SCI-induced inflammation (P<0.01), potentially via the phosphorylation of NF-κB inhibitor α. Furthermore, treatment with wogonoside inhibited the expressions of toll-like receptor 4, NLRP3 and caspase-1 protein in SCI model rats (P<0.01). In conclusion, the results of the present study suggest that wogonoside alleviates SCI-induced inflammation by suppressing NF-κB and NLRP3 inflammasome activation.

Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP­2/9 production via the p38 MAPK signaling pathway.

Metastasis is the primary cause of mortality in osteosarcoma. Targeting metastasis is a major strategy in osteosarcoma treatment. As a traditional Chinese medicine, Trillium tschonoskii Maxim has been widely used in the therapy of various diseases, including cancer. However, currently there is no evidence regarding the anti­metastasic effect of Paris saponin VII (PS VII), which is extracted from Trillium tschonoskii Maxim, on osteosarcoma cells and its underling mechanisms. The present study aimed to examine the effect of PS VII on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Migration and invasion of osteosarcoma cells was then detected using scratch wound healing assays and Transwell assays, respectively. Additionally, the expression of matrix metalloproteinase (MMP)­2 and ­9 was determined at the mRNA and protein level following treatment with PS VII. Mitogen­activated protein kinase (MAPK) expression was also detected by western blot analysis. Finally, an inhibitor of p38 MAPK was used to verify the effect of PS VII on the expression of MMP­2 and ­9, as well as the migration and invasion osteosarcoma cells. This demonstrated that the proliferation, migration and invasion of the osteosarcoma cells were suppressed following treatment with PS VII. PS VII downregulated the expression of MMP­2 and ­9 in a dose­ and time­dependent manner. PS VII also exerted its ability to downregulate the phosphorylation of p38 MAPKs. Furthermore, by using a p38 inhibitor, SB203580, the role of PS VII in MMP­2 and ­9 expression and osteosarcoma cell invasion was revealed. Taken together, these results demonstrated that PS VII suppresses the migration and invasion of osteosarcoma cells via the p38 MAPK signaling pathway.

Low dose nicotine attenuates Aß neurotoxicity through activation early growth response gene 1 pathway.

Epidemiological studies indicate that smoking is negatively correlated with the incidence and development of Alzheimer's disease (AD). Nicotine was reported to be the active factor. However, the detailed mechanisms still remain to be fully elucidated. Early growth response gene 1 (EGR-1) plays important roles in several important biological processes such as promoting cell growth, differentiation, anti oxidative stress, and apoptosis, but few in the pathogenesis of AD. In the present study, we show that nicotine can activate the MAPK/ERK/EGR-1 signaling pathway partially through α7 nAChR. In addition, the up-regulation of EGR-1 by nicotine can also increase the phosphorylation of CyclinD1 which contributes to the attenuation of amyloid-ß (Aß(25-35)) -induced neurotoxicity. Although nicotine and Aß(25-35) can activate EGR-1, the expression of EGR-1 is down-regulated following treatment with nicotine and Aß(25-35). This study demonstrates that low dose nicotine attenuates Aß(25-35)-induced neurotoxicity in vitro and in vivo through activating EGR-1 pathway.

Requirement of the phosphatidylinositol 3-kinase/Akt signaling pathway for the effect of nicotine on interleukin-1beta-induced chondrocyte apoptosis in a rat model of osteoarthritis.

Chondrocyte apoptosis is mainly responsible for the progressive degeneration of cartilage in osteoarthritis (OA). Interleukin-1beta (IL-1ß) was widely used as a modulating and chondrocyte apoptosis-inducing agent. Nicotine is able to confer resistance to apoptosis and promote cell survival in some cell lines, but its regulatory mechanism is ambiguous. We aimed to investigate the effect of nicotine on IL-1ß-induced chondrocyte apoptosis and the mechanism underlying how nicotine antagonizes IL-1ß-induced apoptosis of rat chondrocytes. Chondrocytes isolated from newborn rat joints were exposed to IL-1ß. The cell viability was analyzed by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay, and the apoptotic cells were counted with DAPI staining. The levels of Akt, phosphorylated-Akt (p-Akt) and downstream protein targets of Akt were detected by western blotting. The results showed that nicotine neutralized the effect of IL-1ß on chondrocytes by activating PI3K/Akt signaling pathways, including the PI3K/Akt/Bcl-2 pathway, to block IL-1ß-induced cell apoptosis and the PI3K/Akt/p70S6K (p70S6 kinase)/S6 pathway for promoting protein synthesis, modulating its downstream effectors such as TIMP-1 and MMP-13. Activation of the PI3K/Akt pathway is, in part, required for the effect of nicotine on IL-1ß-induced chondrocyte apoptosis in a rat model of osteoarthritis.

Dysfunction of murine dendritic cells induced by incubation with tumor cells.

In vivo studies showed that dendritic cell (DC) dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs (imDCs) are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CD11b, CD11a and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity.

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57x FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells.

HBx-DNA probe preparation and its application in study of hepatocarcinogenesis.

AIM:To study the role of HBV especially HBx Open Reading Frame (ORF) in the development of hepatocellular carcinoma (HCC).METHODS:HBV 3.2kb fragment was retrieved by digesting recombinant plasmid pBR(322)-2HBV with EcoR II, and HBx 0.59kb fragments by digesting HBV-DNA with BamH I and Bgl II. These fragments were labelled with digoxigenin to get HBV-DNA and HBx-DNA probes. HBV-DNA was detected in HCC by dot blot and Southern blot hybridization with HBV-DNA probe, so the positive specimens in which HBV-DNA were integrated were selected. HBx-DNA was subsequently detected in the selected specimens with HBx-DNA probe.RESULTS:HBV-DNA was detected in 75% HCC, among which integrated type, integrated + free type covered 63.6% and 36.4%. There was no free type. HBx-DNA was detected in 90.5% specimens of integrated type.CONCLUSION:Hepatocarcinogenesis was highly related to HBV-DNA integration, and HBV-DNA mainly integrated into chromosome with incomplete virus DNA fragments among which HBx fragment was the predominant one.