Relationship between cytokine mRNA expression and organ damage following cecal ligation and puncture

AIM: To investigate the role of cytokine gene expression inorgan damage at different tissue sites during sepsis.METHODS: Male NIH mice were subjected to cecal ligationand puncture (CLP) or sham operetion ( Sham ). Pro-inflammatory cytokine (TNFα, IL-1β and IL-6) and anti-inflammatory cytokine (IL-4) gene expression in the liverand lung tissue were assessed by RT-PCR. The permeabilityof microvascular and water content in the lungs and liverwere also examined.RESULTS: Significant increase in TNFα, IL-1β and IL-6 geneexpression was observed at 3 and 12 h after CLP both in theliver and lungs ( P＜ 0.01). The level of IL-4 gene expressionwas not changed after CLP in the lungs, but increased at 12h after CLP ( P ＜ 0.01) in the liver tissue. Both the liver andlungs showed a significant increase in microcirculatorypermeability at 12 h after CLP (P＜ 0.01), and the increasein the lungs was higher than that in the liver. The watermaes fractions in the liver ( P＜ 0.05) and lungs ( P＜ 0.01)were increased after CLP, and the increase in the lungshappened earlier and more severely than that in the liver.CONCLUSION: The inflammatory response in the liver andlungs wes different during sepsis. At the early stage ofsepsis, pro-inflammatory reaction dominates both in theliver and lungs. But at the later stage of sepsis, induction ofcompensatory anti-inflammatory response was seen in theliver but not in the lungs. This difference in situ activity maycontribute to the different vulnerability of organ damageduring sepsis. The strategy of systemic administration ofanti-inflammatory drugs to sepsis should be reconsidered.

PrP 106-126 Altered PrP mRNA Gene Expression in Mouse Microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases.The pathogenic agent is an abnormal prion protein aggregate.Microglial activation in the centre nervous system is a characteristic feature of prion disease.In this study,we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR.PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126,with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly.Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126,and indicate that microglial cells might play a critical role in prion pathogenesis.

RAT GDNF GENE TRANSFECTION AND EXPRESSION OF ITS mRNA AND PROTEIN IN SCHWANN CELLS

Objective To investigate the possibility of the transfection of glial-cell line derived neurotro-phic factor (GDNF) gene into Schwann cells (SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs. Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay (ELISA). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold) ,and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.

Enhanced proliferation,invasion,and epithelial-mesenchymal transition of nicotine-promoted gastric cancer by periostin

AIM:To investigate the contribution of periostin in nicotine-promoted gastric cancer cell proliferation,survival,invasion,drug resistance,and epithelial-mesenchymal transition(EMT).METHODS:Gastric cancer cells were treated with nicotine and periostin protein expression was determined by immunoblotting.Periostin mRNA in gastric cancer cells was silenced using small interfering RNA(siRNA) techniques and periostin gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction.Gastric cancer cells transfected with control or periostin siRNA plasmid were compared in terms of cell proliferation using the methylthiazolyldiphenyl-tetrazolium bromide assay.Cell apoptosis was compared using annexin V-fluoresceine isothiocyanate and propidium iodine double staining.Tumor invasion was determined using the Boyden chamber invasion assay,and the EMT marker Snail expression was evaluated by immunoblotting.RESULTS:Nicotine upregulated periostin in gastric cancer cells through a COX-2 dependent pathway,which was blocked by the COX-2-specific inhibitor NS398.Periostin mRNA expression was decreased by ～87.2% by siRNA in gastric cancer cells,and stable periostinsilenced cells were obtained by G418 screening.Periostin-silenced gastric cancer cells exhibited reduced cell proliferation,elevated sensitivity to chemotherapy with 5-fluorouracil,and decreased cell invasion and Snail expression(P < 0.05).CONCLUSION:Periostin is a nicotine target gene in gastric cancer and plays a role in gastric cancer cell growth,invasion,drug resistance,and EMT facilitated by nicotine.

Inhibition of SIRT1 Increases EZH2 Protein Level and Enhances the Repression of EZH2 on Target Gene Expression

Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter region of EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases. Conclusions Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expres-sion.

Gradient decrease in telomerase and its RNA in progressive passage of human endometrial stromal stem cells

The purpose of this study was to explore the change of telomerase in passage from human endometrial stromal stem cells isolated from human endometrium.Telomerase activity of cultured endometrial stromal cells was assessed at mRNA and protein levels using RT-PCR and immunohistochemistry technique.Telomerase mRNA and protein levels were higher at early passages,and then had a gradually decreased trend of immunoreactive intensity and gradually weakened positive cells with progressive passage in endometrial stromal stem cells.These results suggest that telomerase is contributed to the insenecence of endometrial stem cell.

All-trans Retinoic Acid Induced the Differentiation of Human Glioma Cells

OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

Objective： To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. Methods： T24-3 ceil subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 10^7 hu-PBL and subcutaneously injected with 3 × 10^6 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3^＋, CD4^＋, CD8^＋ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhlL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. Results： All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group （P 〈 0.01）. Conclusion： DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.

mRNA stability in the nucleus

Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases （RNases）. The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase Ⅱ in eukaryotes.

Expression and cellular localization of interleukin 8 mRNA and protein in the area of xenogenic bone implant

Objective: To observe the expression and cellular localization of interleukin 8 (IL 8) mRNA and protein in the area of xenogenic bone implant. Methods: The bovine cancellous bone granules were implanted into the thigh muscles of mice. The samples were taken 4, 7, 14 and 21 days after implantation. IL 8mRNA and protein in the site of implant were assayed by in situ hybridization and immunocytochemical techniques. Results: The expression of IL 8mRNA and protein were observed in all specimens 4, 7, 14 and 21 days after implantation. IL 8mRNA was expressed mainly by the neutrophils, monocytes, macrophages and fibroblasts at 7th day post implantation. Some mesenchymal cells, multinucleated giant cells, vascular endothelial cells and smooth muscle cells also expressed IL 8mRNA in the area of xenogenic bone implant at 14th and 21st days. Immunocytochemical studies demonstrated the same results as that of in situ hybridization. Conclusions: Many different kinds of cells express IL 8mRNA and secret IL 8 in the area of xenogenic bone implant, suggesting that IL 8 may play an important role in local immunity of xenogenic bone graft.

The non-canonical NF-κB pathway promotes NPC2 expression and regulates intracellular cholesterol trafficking

Niemann-Pick type C2(NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of lowdensity lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2(NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor(LTβR) activation or Baff receptor(BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system.