Neuron-derived exosomes mediate sevoflurane-induced neurotoxicity in neonatal mice via transferring lncRNA Gas5 and promoting M1 polarization of microglia.

Sevoflurane exposure during rapid brain development induces neuronal apoptosis and causes memory and cognitive deficits in neonatal mice. Exosomes that transfer genetic materials including long non-coding RNAs (lncRNAs) between cells play a critical role in intercellular communication. However, the lncRNAs found in exosomes derived from neurons treated with sevoflurane and their potential role in promoting neurotoxicity remain unknown. In this study, we investigated the role of cross-talk of newborn mouse neurons with microglial cells in sevoflurane-induced neurotoxicity. Mouse hippocampal neuronal HT22 cells were exposed to sevoflurane, and then co-cultured with BV2 microglial cells. We showed that sevoflurane treatment markedly increased the expression of the lncRNA growth arrest-specific 5 (Gas5) in neuron-derived extracellular vesicles, which inhibited neuronal proliferation and induced neuronal apoptosis by promoting M1 polarization of microglia and the release of inflammatory cytokines. We further revealed that the exosomal lncRNA Gas5 significantly upregulated Foxo3 as a competitive endogenous RNA of miR-212-3p in BV2 cells, and activated the NF-κB pathway to promote M1 microglial polarization and the secretion of inflammatory cytokines, thereby exacerbating neuronal damage. In neonatal mice, intracranial injection of the exosomes derived from sevoflurane-treated neurons into the bilateral hippocampi significantly increased the proportion of M1 microglia, inhibited neuronal proliferation and promoted apoptosis, ultimately leading to neurotoxicity. Similar results were observed in vitro in BV2 cells treated with the CM from HT22 cells after sevoflurane exposure. We conclude that sevoflurane induces the transfer of lncRNA Gas5-containing exosomes from neurons, which in turn regulates the M1 polarization of microglia and contributes to neurotoxicity. Thus, modulating the expression of lncRNA Gas5 or the secretion of exosomes could be a strategy for addressing sevoflurane-induced neurotoxicity.

Association of Epstein Barr virus A73 gene polymorphism with nasopharyngeal carcinoma.

OBJECTIVE: To study the association between Epstein Barr (EB) virus A73 gene polymorphism and nasopharyngeal carcinoma (NPC) in a Chinese Han population. METHODS: A case-control study was designed, including 510 nasopharyngeal cancer patients and 520 healthy controls, A157154C genotypes of the A73 gene in EB virus were detected, genotype and allele frequency distribution between the two groups were compared. RESULTS: The C allele frequency in the NPC group was significantly higher than that in the control group (68.4% vs. 61.2%; p<0.001). The CC genotype frequency in the NPC group was significantly higher than that in the control group, the difference was significant (47.4% vs. 41.2%; p<0.001). The CC genotype frequency in male patients was significantly higher than that in female patients in the NPC group, the difference was significant (50.3% vs. 34.7%; p<0.001). CONCLUSION: A157154C polymorphism of the A73 gene in EB virus was associated with NPC susceptibility.

[Effects of parecoxib sodium preemptive analgesia on perioperative cytokine responses and stress responses in patients undergoing ophthalmology surgery].

OBJECTIVE: To investigate the effects of parecoxib sodium preemptive analgesia on perioperative cytokine responses and stress responses and postoperative analgesia in patients undergoing ophthalmology surgery. METHODS: One hundred ASAI-II patients undergoing ophthalmology surgery were randomly divided into 2 groups of fifty: the parecoxib group received parecoxib 40 mg by muscle injection, the control group received an equal volume of 0.9% normal saline two ml. Venous blood samples were obtained at 10 minutes before parecoxib or 0.9% normal saline was injected (T1), 30 minutes (T2) and 60 minutes (T3) after surgery was initiated, the moment when surgery was finished (T4), 6 hours (T5), 12 hours (T6) and 24 hours (T7) after surgery was finished for determination of the plasma levels of IL-6, IL-8, IL-1RA, TNF and IL-1ß. At the same time, the plasma levels of epinephrine, norepinephrine, cortisol and adrenocorticotropic hormone at T1-T6 were measured. VAS scores with patients were recorded at 1, 2, 6, 12, 24 h after surgery. RESULT: The change of the plasma levels of IL-6, IL-8, IL-1RA, TNF, IL-1ß in the two groups:in the parecoxib group the plasma levels of TNF and IL-1ß did not decreased significantly as compared with the control group (P > 0.05). The plasma levels of IL-6, IL-8 at T4-T6 decreased significantly as compared with the control group (P < 0.01). The plasma levels of IL-1RA at T3-T6 decreased significantly as compared with the control group (P < 0.01). The change of the plasma levels of epinephrine, norepinephrine, cortisol and adrenocorticotropic hormone in the two groups:in the parecoxib group the plasma levels of epinephrine, norepinephrine at T2, T3 decreased significantly as compared with the control group (P < 0.01). In the parecoxib group the plasma levels of cortisol at T2-T4 decreased significantly as compared with the control group (P < 0.01). In the parecoxib group the plasma levels of adrenocorticotropic hormone at T2-T5 decreased significantly as compared with the control group (P < 0.01). Postoperation VAS scores:VAS scores of the parecoxib group were less than that of the control group (P < 0.05). CONCLUSION: Preoperatively-administered parecoxib during ophthalmology surgery can produce better analgesia effect, reduce the production of cytokines, decrease central nervous system sensitization so as to improve the quality of postoperative pain relief. At the same time, it can reduce perioperative stress hormone release so as to have a positive effect on post operative recovery.

[Effects of hyperbaric oxygen on the acute lung injury induced by oleic acid in rats].

OBJECTIVE: To explore the effect of hyperbaric oxygen (HBO) on acute lung injury (ALI) induced by oleic acid (OA) in rats. METHODS: Eighty healthy Sprague-Dawley (SD) rats were randomly divided into four groups. In OA group (n=30), ALI was produced by injection of OA 0.15 ml/kg through tail vein. Ten rats were randomly selected and sacrificed after injection of OA at the time of 4 hours, 3 days, and 7 days, respectively. In OA plus HBO group (n=20), rats received HBO intervention in a special box with oxygen of 2.5 atm (1 atm=101.325 kPa) for 90 minutes. Ten rats were randomly respectively sacrificed at 3 days and 7 days. In simple HBO group, 20 rats were sacrificed at 3 days and 7 days of HBO intervention, respectively. Other 10 rats were assigned as control group. Blood, lung specimens were collected after sacrifice. Serum contents of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and IL-6 were measured. Gross changes and pathological findings of the left lung were recorded. The wet to the dry weigh (W/D) of the right lung was determined. RESULTS: Partial pressure of oxygen in arterial blood (PaO2, mm Hg, 1 mm Hg=0.133 kPa) fell from 107.70+/-5.37 to 57.40+/-2.63 in OA group. Congestion, bleeding and edema could be seen grossly. They could also be found under microscope with disappearance of normal structure, and accumulation of fluid in interstitium with inflammatory cell infiltration and hyaline membrane formation were also found. Lung W/D ratio was increased as compared with the control group (6.94+/-0.44 vs. 4.59+/-0.44, P<0.05). A marked increase was found in serum TNF-alpha, IL-1 beta, IL-6 levels [TNF-alpha (microg/L): 18.52+/-1.20 vs. 5.27+/-0.61, IL-1 beta (microg/L): 13.73+/-1.37 vs. 6.13+/-1.51, IL-6 (microg/L): 14.51+/-1.21 vs. 11.14+/-0.89]. After HBO therapy for 3 days and 7 days, PaO2 (mm Hg, 3 days: 79.20+/-1.68 vs. 59.00+/-2.70, 7 days: 94.30+/-3.77 vs. 74.00+/-3.85) and lung W/D (3 day: 7.43+/-0.73 vs. 9.82+/-0.99, 7 days : 6.75+/-1.14 vs. 8.77+/-1.60) of HBO group were ameliorated to varying degrees compared with OA model group (P<0.05 or P<0.01). HBO therapy for 3 days could lower levels of IL-1 beta (microg/L) in the serum (6.46+/-1.99 vs. 9.09+/-1.09, P<0.05). CONCLUSION: It is suggested that HBO treatment for ALI in rats had effects of improving arterial blood gases and the lung water transport, and inhibiting inflammatory mediators production.

Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells.

BACKGROUND: Eukaryotic initiation factor eIF4E, an important regulator of translation, plays a crucial role in the malignant transformation, progression and chemoresistance of many human solid tumors. The overexpression of this gene has been found in a variety of human malignancies including breast carcinoma. In the present study, we attempted to explore the possibility of eIF4E as a therapeutic target for the treatment of human breast carcinoma using breast carcinoma cell line (MCF-7). MATERIALS AND METHODS: The survivin promoter-driven eIF4E-shRNA vector was constructed on the basis of pSUPER.retro vector. Then, we established stably transfected MCF-7 and MCF210 (normal human mammary epithelial cell) cells expressing eIF4E-shRNAs or control-shRNAs. Firstly, the changes of eIF4E expression were detected by RT-PCR and Western blot assays. Next, the optimal shRNA vector (eIF4E-shRNA2) was selected to knock down eIF4E expression and investigate the effect of eIF4E-shRNA on eIF4E-regulated gene expression and cell proliferation both in vitro and in vivo. Followingly, the changes of cell cycle and apoptosis in the stably transfectants (MCF-7) were detected by flow cytometry and TUNEL methods, while we also explored possible apoptosis pathways. Finally, we investigated the effect of shRNA targeting eIF4E on the chemosensitivity of breast carcinoma cells to cisplatin in vitro and in vivo. RESULTS: Two survivin promoter-driven eIF4E-shRNA vectors were successfully constructed. eIF4E-shRNA2 but not eIF4E-shRNA1 efficiently downregulated the levels of eIF4E expression in the stably transfected MCF-7-s2 cells but not in the stably transfected MCF210-s2 cells, while MCF-7-s2 showed obvious proliferation suppression but MCF210-s2 did not. The downregulation of eIF4E expression significantly reduced the levels of VEGF, FGF-2 and cyclinD1 expression, suppressed cell growth, induced cell cycle arrest in G(0)/G(1) phase and subsequent apoptosis by activating caspase 3 in MCF-7 cells. The results of FCM and TUNEL staining assays indicated that the classic apoptosis characters of the MCF-7 cells stably expressing eIF4E-shRNA2 manifested an apoptosis rate of 18.3%, significantly higher than those in the control groups (P < 0.05). Moreover, we found that downregulation of c-IAP1, c-IAP2 and c-Myc but not Bcl-2 family proteins were involved in the apoptosis induced by eIF4E-shRNA2. In tumorigenicity assay, xenograft tumors developed from MCF-7-s2 cells in mice showed a significant slowdown in the growth speed and formation rate compared with control groups. Furthermore, we also testified that eIF4E-shRNA could synergistically enhance the cytotoxicity effects of cisplatin to MCF-7 cells both in vitro and in vivo. CONCLUSION: Survivin promoter-driven RNA interference system could efficiently and specifically downregulate eIF4E expression in human breast carcinoma cells but not in normal human mammary epithelial cell cells. Thus, eIF4E might play an important role in chemosensitivity to cisplatin, and survivin promoter-driven RNAi targeting eIF4E can be used as adjuvant therapy for human breast carcinomas with tumor specificity and high efficacy.

Knockdown of RCK/p54 expression by RNAi inhibits proliferation of human colorectal cancer cells in vitro and in vivo.

Colorectal cancer is the third most common cancer in both men and women around the world. Although much progress of the mechanism of colorectal carcinogenesis has been made, the studies centering on the mechanisms of tumorigenesis are much needed to be further exploited. The overexpression of RCK/p54 gene, a member of the DEAD box protein/RNA helicase family, has been found in this malignancy. Roles of RCK in the development of colon cancer, however, are unknown. In this report, we explored whether RCK/p54 plays a role in maintaining the malignant phenotype and functions in the canonical Wnt signaling pathway of colorectal cancer cells harboring an APC mutation. The ectopic overexpression of RCK/p54 gene in colorectal cancer cells by transfection with RCK/p54 cDNA could lead to a significant increase of Tcf transcriptional activity and expression levels of Wnt target genes. By RNAi assay, we also observed that the Tcf transcriptional activity in LoVo-shRNA cells was significantly decreased by approximately 61.3%, while the mRNA and protein expression levels of Wnt target genes were also obviously decreased. Furthermore, the anti-tumour effects and its possible mechanisms of actions in LoVo cells elicited by a decrease in the level of RCK/p54 by RNAi were examined. Results showed that RCK/p54 downregulation could significantly reduce the viability of LoVo cells, increased cell number of S phase, led to cell apoptosis induction, and inhibited tumor growth in nude mice. Taken together, RCK/ p54 might be a determinant of colorectal cancer proliferation by activating the canonical Wnt pathway and RCK/p54-shRNA might be a potential strategy for colorectal cancer gene therapy.

Hypoxia-inducible enhancer/alpha-fetoprotein promoter-driven RNA interference targeting STK15 suppresses proliferation and induces apoptosis in human hepatocellular carcinoma cells.

STK15 (Aurora A/BTAK) is an oncogenic serine/threonine kinase that plays a role in centrosome separation and in the formation of the mitotic bipolar spindle. It is highly expressed and constitutively activated in various human tumors including hepatocellular carcinoma (HCC). To investigate its possibility as a molecular target for future therapies directed against hepatocellular carcinoma, we constructed a tissue-specific RNA interference (RNAi) system mediated by hypoxia-inducible (HI) enhancer/alpha-fetoprotein (AFP) promoter and employed it to downregulate exogenous reporters (LUC and EGFP) and endogenous STK15 gene expression and analyzed the phenotypical changes in HCC cells. Results showed that the expression of exogenous reporters (LUC and EGFP) was specifically downregulated in hepatoma cells but not in non-hepatoma cells. Moreover, the specific downregulation of STK15 expression in hepatocellular carcinoma cells (HepG2) significantly inhibited in vitro cellular proliferation and in vivo tumorigenicity. Furthermore, we also found that the downregulation of STK15 expression led to cell arrest in the G(2)/M phase and finally apoptosis induction of HepG2 cells. Thus, the HI enhancer/AFP promoter-mediated RNAi targeting STK15 may be a potential therapeutic strategy for the treatment of hepatocellular carcinoma with tumor specificity and high efficacy.

Inducing apoptosis and enhancing chemosensitivity to gemcitabine via RNA interference targeting Mcl-1 gene in pancreatic carcinoma cell.

PURPOSE: Resistance to chemotherapy is a major cause of treatment failure and poor prognosis in pancreatic carcinoma. Myeloid cell leukemia-1 (Mcl-1) is highly up-regulated in pancreatic carcinoma and is associated with the anti-apoptosis and the resistance to chemotherapy drugs. Suppression of Mcl-1 would be an approach to induce apoptosis and enhance the chemosensitivity. METHODS: In this study, three pancreatic cancer cell lines (PANC-1, BxPC-3 and SW1900) stably expressing shRNAs targeting Mcl-1 gene were established and gene expression inhibition was assessed by Real-Time QPCR and Western blotting. The effects of Mcl-1 downregulation mediated by RNAi were explored in vitro and in vivo. RESULTS: We showed that the specific downregulation of Mcl-1 strikingly inhibited cell growth, colony formation, cell cycle arrest and induced apoptosis in pancreatic cancer cells in vitro, and markedly decreased the tumorigenicity in a mouse xenograft model. Moreover, knockdown of Mcl-1 significantly increased the chemosensitivity to Gemcitabine in pancreatic carcinoma cells. CONCLUSIONS: Our data suggests that the specific downregulation of Mcl-1 by RNAi is a promising approach to induce apoptosis and enhance the chemosensitivity for pancreatic carcinoma gene therapy.

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer.

BACKGROUND: The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-gamma-seminoprotein E(4)B(7) murine mAb by guided selection. METHODS: Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E(4)B(7) mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified gamma-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for gamma-seminoprotein were selected and further identified. RESULTS: First, using the E(4)B(7) Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against gamma-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E(4)B(7) mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-gamma-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. CONCLUSIONS: Through guided-selection, we successfully produced a human anti-gamma-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.

[Inhibition of survivin gene expression in PC-3 cells by RNAi].

OBJECTIVE: To construct eukaryotic expression vectors by using the pSilencer3. 1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells. METHODS: Three target gene segments were synthesized and cloned into the pSilencer3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors: pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT-PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method. RESULTS: Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemical staining indicated that pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down. CONCLUSION: The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors (pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3) in the prostate cancer cell line PC-3. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration.

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.