Vector Control Group Research Articles

Functions of MiRNA-128 on the regulation of head and neck squamous cell carcinoma growth and apoptosis.

BackgroundIncidence of head and neck squamous cell carcinoma (HNSCC) has continuously increased in past years while its survival rate has not been significantly improved. There is a critical need to better understand the genetic regulation of HNSCC tumorigenesis and progression. In this study, we comprehensively analyzed the function of miRNA-128 (miR-128) in the regulation of HNSCC growth and its putative targets in vitro and in vivo systems.MethodsThe function and targets of miR-128 were investigated in human HNSCC cell lines (JHU-13 and JHU-22), which were stably transfected with the miR-128 gene using a lentiviral delivery system. The expression levels of miR-128 and its targeted proteins were analyzed with qRT-PCR, Western blotting and flow cytometry. The binding capacity of miRNA-128 to its putative targets was determined using a luciferase report assay. MTT, colony formation, and a tumor xenograft model further evaluated the effects of miR-128 on HNSCC growth.ResultsWe generated two miR-128 stably transfected human HNSCC cell lines (JHU-13miR-128 and JHU-22miR-128). Enforced expression of miR-128 was detected in both cultured JHU-13miR-128 and JHU-22miR-128 cell lines, approximately seventeen to twenty folds higher than in vector control cell lines. miRNA-128 was able to bind with the 3′-untranslated regions of BMI-1, BAG-2, BAX, H3f3b, and Paip2 mRNAs, resulting in significant reduction of the targeted protein levels. We found that upregulated miR-128 expression significantly inhibited both JHU-13miR-128 and JHU-22miR-128 cell viability approximately 20 to 40%, and the JHU-22miR-128 tumor xenograft growth compared to the vector control groups.ConclusionsmiR-128 acted as a tumor suppressor inhibiting the HNSCC growth by directly mediating the expression of putative targets. Our results provide a better understanding of miRNA-128 function and its potential targets, which may be valuable for developing novel diagnostic markers and targeted therapy.

Effects of Tissue-Type Plasminogen Site-Specific Transgene in Gelatin-Coated Dacron on the Fibrinolysis Activity of Rabbit Left Atrium.

To investigate the effects of tissue-type plasminogen activator (tPA) gene transfer with left-atrium local positioning on the fibrinolytic activity of rabbit left atrial blood. A total of 48 rabbits were randomly divided into 3 groups (n = 16): gene therapy, vector control, and blank control groups. Each group was divided into 2 subgroups (8 rabbits in each subgroup) according to the sacrifice time on the postoperative 3(rd) and 14(th) days. The tPA mRNA transcriptional level and exogenous tPA protein expression within regional myocardial tissues of the left atrium were detected on the postoperative 3(rd) and 14(th) days. After excluding the animals that died, 6 samples of each subgroup were randomly selected for the statistics (n = 6). The tPA activities in rabbit left atrial blood and peripheral blood were also detected. The tPAmRNA and tPA protein expressions within regional myocardial tissues were detected on the postoperative 3(rd) and 14(th) days. The tPA activity in left atrial blood in the gene therapy group was higher than the tPA activity of other groups (p < 0.02). No significant differences were observed in the tPA activity of peripheral blood among the 3 groups before surgery. A gelatin-coated Dacron piece, which carried the tPA gene, was implanted in the left atrial appendage. The gelatin-coated Dacron piece could express and secrete tPA proteins in the region, thus enhancing the fibrinolytic activity of left atrial blood. Fibrinolytic activity; Gelatin coating; Gene; Left atrium; Tissue-type plasminogen activator.

Role of Smac in apoptosis of lung cancer cells A549 induced by Taxol.

A series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) directly have been discovered and have been shown to promote chemotherapy-induced apoptosis. In this study, we investigate the role of Smac in Taxol-induced apoptosis of lung cancer cell in vitro. PcDNA3.1/Smac recombinants were transfected into the non-small cell lung cancer cell line A549. Smac expression was detected by RT-PCR and Western blot. The invasive ability of cells was examined. Flow cytometry was used to analyze apoptosis of cells induced by Taxol with Annexin V/PI double staining technique. Smac expression was significantly higher in the PcDNA3.1/Smac recombinant group than in the untransfected group at mRNA and protein level (p < 0.05) and lower invasion through a basal membrane was apparent after transfection (p < 0.05). A small number of cells were promoted to apoptosis in the PcDNA3.1/Smac group. There were significant differences compared to the empty vector group and control group. The apoptosis rate was significantly higher in PcDNA3.1/Smac + Taxol group than in other groups (p < 0.05). Transfected Smac can enhance the chemosensitivity of the non-small cell lung cancer cell line A549 to Taxol.

Upregulation of AIOLOS induces apoptosis and enhances etoposide chemosensitivity in Jurkat leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder of immature hematopoietic precursors committed to T-cell lineage. T-ALL accounts for ~15% of pediatric ALL cases and is prone to early relapse. With new and improved treatment protocols, the prognosis of T-ALL has improved particularly in children; however, the outcome of relapsed T-ALL cases remains poor. The AIOLOS gene is necessary to control lymphocyte differentiation and may be a potential target of T-ALL therapy. In the present study, Jurkat cells were divided into three groups: untransfected (UT) control, lentiviral vector control (Lenti-Mock) and AIOLOS-overexpressing (Lenti-AIOLOS) groups. Lenti-AIOLOS Jurkat cells were constructed by lentiviral transduction; cell cycle analysis, apoptosis and cytotoxicity assays were then performed to evaluate the effects of AIOLOS on cell cycle distribution, apoptosis and cell chemosensitivity to etoposide of Jurkat cells in vitro. Moreover, the expression levels of genes associated with apoptosis and cell cycle were investigated by quantitative reverse transcription-polymerase chain reaction. Results showed that the percentage of Jurkat cells in the G0/G1 phase increased from 71.5 (UT) to 85.4% (Lenti-AIOLOS; P<0.05), yet the percentage of cells in the S-phase decreased from 15.1 (UT) to 11.6% (Lenti‑AIOLOS; P<0.05). The percentage of total apoptotic cells was significantly increased in the AIOLOS-transfected Jurkat cells (21.93%) compared with this percentage in the Lenti-Mock (13.35%) or the UT group (13.30%; P<0.05). Consistent with these results, AIOLOS overexpression induced P21 and P27 upregulation and CCND3 and SKP2 downregulation. Furthermore, AIOLOS overexpression synergistically increased the cytotoxic effects of etoposide and downregulated NF-κB expression. Our findings revealed that lentivirus-mediated AIOLOS overexpression in Jurkat cells induced cell apoptosis, arrested the cell cycle at the G0/G1 phase, and synergistically increased the sensitivity of Jurkat cells to etoposide by inhibiting NF-κB activity.

The Microanatomy of the Leukemic Stem Cell Niche in Murine Chronic Myelogenous Leukemia

Objectives and background: Constituents of the bone marrow microenvironment (BMM) influence the proliferation, differentiation and location of hematopoietic stem and progenitor cells (HSPC). Dependent on their maturation stage, different subsets of HSPC are localized at distinct sites in the BMM. This location depends on HSPC-intrinsic, as well as HSPC-extrinsic factors. The BMM protects leukemic stem cells (LSC) from treatment with tyrosine kinase inhibitors or chemotherapy. We, therefore, investigated the microanantomy of the LSC niche hypothesizing that it may differ from the normal HSPC niche.Methods: We used a combination of confocal and 2-photon intravital microscopy (IVM) of the murine calvarium and well-described retroviral models of BCR-ABL1+chronic myelogenous leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL).Results: We show here that BCR-ABL1+Lin–c-Kit+Sca-1+ (LKS) CD150+CD48– (LKS SLAM) cells, which harbor the LSC fraction in the CML model, homed to locations further away from the endosteum than their normal counterparts. Prior in-vitro treatment of BCR-ABL1+ LKS with imatinib mesylate, considered standard of care in CML, reversed this phenotype and the cells were found closer to the endosteum.Native BCR-ABL1, as well as the imatinib-resistant BCR-ABL1 point mutants BCR-ABL1Y253F, BCR-ABL1E255K, BCR-ABL1T315I and BCR-ABL1M351T had similar intrinsic catalytic activity, but the BCR-ABL1Y253F, BCR-ABL1E255K, and BCR-ABL1T315I mutants increased the IL-3-independent proliferative capacity of 32D cells relative to native BCR-ABL1. BCR-ABL1Y253F and BCR-ABL1M351T caused increased transformation of primary BM B-lymphoid progenitors in vitro and led to accelerated induction of B-ALL in mice. In the CML model, BCR-ABL1Y253F and BCR-ABL1T315Iinduced myeloproliferative neoplasia with shortened survival and features of accelerated phase disease compared to native BCR-ABL1, whereas BCR-ABL1T315I LKS cells homed closer to osteoblastic cells than LKS cells expressing native BCR-ABL1.Sequential in vivo tracking of leukemic progenitor growth by IVM showed a similar nadir in the number of cells per leukemic cell 'nest’ 11 days after irradiation and IV transplantation in recipients of DsRed+BCR-ABL1+ or empty vector control-transduced bone marrow. However, between days 18-25 after transplantation there was a significant increase in the number of cells per leukemic cell 'nest’ compared to the empty vector control group. Sequential immunohistochemistry and TUNEL assays of leukemic bone sections in imatinib- or vehicle-treated recipient mice with CML showed that initial BCR-ABL1+ growth tends to occur at locations further away from the endosteum, whereas erythroid islands were found closer to the endosteum and trabeculae. Apoptosis in response to imatinib appeared most prominent in the metaphysis. Lastly, we could demonstrate by IVM in the CML model that treatment of mice with a combination of imatinib plus granulocyte colony-stimulating factor led to 'emptying’ of the LSC niche and superior eradication of BCR-ABL1+ leukemic cells compared to treatment with imatinib alone.Conclusions: In summary, these data suggest that the microanatomy of the LSC niche in CML differs from the normal hematopoietic niche. BCR-ABL1 mutation status may affect the positioning of CML LSC in the microenvironment, and location in the niche may be altered pharmacologically, suggesting that niche location may influence clinical outcome. DisclosuresKrause:Glycomimetics. Inc.: Research Funding.

A Novel Th17-Prone CD146+CCR5+ T-Cell Population As an Early Marker of Intestinal Graft-Versus-Host Disease

GVHD of the gastrointestinal tract (GI) is associated with high mortality. We have identified systemic, skin- and GI-specific plasma biomarkers present at clinical GVHD onset, and more recently biomarkers for treatment responsiveness. However, in order to identify early GI-specific biomarkers prior to GVHD onset, we performed state-of-the-art proteomics on plasma samples taken 14 days prior to clinical manifestation of GI GVHD. We selected candidates that were increased at least 1.5 fold in plasma from GI GVHD patients compared to HSCT patients without GVHD at matched time points. We identified two lead proteins: CD146, a cell adhesion and trafficking molecule expressed on a subset of CD4+ T cells and endothelial cells, and the chemokine (C-C motif) ligand 14 that binds to the chemokine receptor CCR5 on T cells. As these proteins have not been previously identified in proteomics experiments and antibodies for their corresponding receptors on T cells were available, we analyzed their expression profiles on PB cells from 214 HSCT patients (71 GI GVHD, 48 no GVHD, 33 non-GVHD enteritis, 22 skin first GVHD, 40 isolated skin GVHD) at the onset of symptoms. The frequency of CD146+CCR5+ T cells was significantly increased in GI GVHD patients compared to patients without GVHD, non-GVHD enteritis, or with isolated skin GVHD, as well as increased in patients who first experienced skin and then GI GVHD (Fig. 1). When using the median % of CD146+CCR5+ T cells detected in GI GVHD patients to classify patients into low and high-risk groups, patients in the high-risk group had higher 6-month non-relapse mortality (42 vs. 20%, p = 0.02). Importantly, CD146+CCR5+ T cells at onset of clinical symptoms were not correlated with GI histologic severity, suggesting that these cells are not mucosa damage products but rather systemic effectors. Thus, we measured their frequencies in samples taken at a median of 19 days post-transplant and with a median interval of 14 days prior to clinical symptoms and found that CD146+CCR5+ T cells circulate in patients before GI GVHD clinical onset.In GI GVHD patients, these cells expressed a Th1 and Th17 phenotype and expressed high levels of the activation marker ICOS known to be critical for the development of human Th17 cells. To test the hypothesis that CD146+ T cells are Th17-prone, we next investigated whether in vitro polarization of CD4 T cells with defined stimulation conditions will increase the CD146+CCR5+ expression as well as the production of Th1 and Th17 cytokines. Fig. 2A demonstrates that CD4 T cells differentiated with both Th17-inducing cytokines and ICOS co-stimulation had a significantly higher percentage of CD146+CCR5+ T cells and co-expressed more IL-17A+IFNγ+ than T-cells stimulated with Th1-inducing cytokines or by CD28.We then analyzed colonic mucosa biopsies from patients with GI GVHD (N = 18) and non-GVHD enteritis (N = 10) for the expression of CD146 by immunohistochemistry. CD146 expression was detected on CD3 lymphoid cells and was strongly present on the endothelium. The CD146+ vessel count in GI GVHD tissues was significantly higher than in non-GVHD enteritis tissues (p < 0.001).Th17 cells migrated more efficiently through endothelial cell monolayers than their Th1 counterparts (Fig. 2B). Lentivirus-mediated shRNA knock-down of CD146 in either cell type demonstrated that reduced CD146 expression on CD4+ T cells, but not on endothelial cells significantly reduced the T-cell transendothelial migration (Fig. 2C), suggesting that CD146 on T cells is paramount for promoting infiltration of pathogenic T cells into GVHD target organs. As proof of principle for this hypothesis, we tested donor CD146-/- T cells in an allogeneic murine GVHD model and did not find any difference in GVHD severity when compared to wild type CD146+/+ T cells. Finally, we used a xenogeneic GVHD mouse model with injection of human CD4+ T cells lentivirally transduced with CD146 or control shRNA. In comparison to the vector control group, mice transplanted with CD146 shRNA transduced T cells did not lose weight (Fig. 3A), had similar human T cells engraftment (B), had less splenic CD146+CCR5+ T cells (C), and expressed less TBET 53 days after transplant (D).In conclusion, early quantification of a novel CD146+CCR5+ Th17-prone and ICOS-induced population may allow identification of patients at risk for GI GVHD development and subsequent mortality. Targeting CD146 may represent a new avenue to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibitive effect of IL-24 gene on CD133(+) laryngeal cancer cells.

To explore the inhibitive and apoptosis inductive effect of IL-24 genes on CD133(+) laryngeal cancer cells in Hep-2 line. Human peripheral blood monocytes were isolated. The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method. Primers P1 and P2 was designed for the amplification of human IL-24 genes. After confirmation of agarose gel electrophoresis tests, TA was cloned into pMD19-T simple vector. Nhe I and Xho I double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector, and detected by enzyme digestion and gene sequencing methods. Flow cytometry (FCM) was used to isolate CD133(+) cells from Hep-2 cells. CD133(+) cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000. After detection, MTT and FCM were used to observe the effect of IL-24 gene on CD133(+) laryngeal cancer Hep-2 cells. Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133(+) Hep-2 could expressed IL-24 gene in cells stably. MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group (P<0.05); FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group (P<0.05). IL-24 gene expressions can inhibit proliferation of CD133(+) laryngeal cells in Hep-2 line and promote their apoptosis.

Effects of osteopontin on hypercalciuria-induced attachment of calcium oxalate crystals to rat renal epithelial cells

Objective To address the effects of osteopontin (OPN) on hypercalciuria contributing to the kidney stone formation.Methods The normal rat kidney epithelial cell line NRK cells were divided into three groups,which were cultured in the medium containing calcium of 0.1,0.3 and 0.6 g/L,respectively.The cell proliferation,the content of OPN in the cell culture supernatants and the level of OPN gene expression at 3,6 and 24 h were examined.The lentivirus vector targeting OPN was constructed.The NRK cells were infected with the lentivirus and then cultured in the medium containing calcium of 0.6 g/L for 24 h.Eventually,The inhibitory effects of silencing OPN gene on the adhesion of calcium oxalate crystals to NRK cells were investigated.Results High level calcium could significantly suppress the proliferation of NRK cells and stimulate secretion of OPN as compared with the blank control group (P ＜ 0.05).The expression of OPN mRNA and protein was also significantly increased (P ＜ 0.05).Calcium concentrations of the lysate of infected NRK cells in the three groups after calcium chloride stimulation were (19.25 ±3.71),(27.22 ±3.82) and (31.91 ± 1.15) mg/L,respectively.The silencing of OPN gene expression could significantly inhibit the abilities of attachment of calcium oxalate crystals to NRK cells as compared with negative lentivirus vector and blank control group,respectively (P ＜ 0.05).Conclusion High level calcium could induce the adhesion of calcium oxalate crystals to NRK cells probably mediated by OPN. Key words: Hypercalciuria ; Osteopontin; Renal epithelial cell

Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 105 TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted.

Strengthening the Skin with Topical Delivery of Keratinocyte Growth Factor-1 Using a Novel DNA Plasmid

Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein–transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26 ± 2 versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255 ± 36 versus 162 ± 16 µm) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1–treated skin was significantly stronger than control vector–transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.

Experimental Study of Specific Targeted Inhibition from RNAi Silencing CXCR7 to Human Colon Cancer Cell SW620

Object: To investigate the change of CXCR7 protein expression after CXCR7-shRNA transfered into human colon cancer cell SW620 which is lentivirus-mediated. Methods: 1. To design and synthesis three shRNA sequence and one negative control sequence of CXCR7. To synthesis and construct recombinant lentiviral vector by pSilencerTM 4. 1 system and CXCR7. To transfer the vector into HEK293T cell for packaging viral and to detect titer. 2. To transfer the three different recombinant lentiviral vector and one negative control vector into human colon cancer cell SW620. To detect expression and silence efficiency of CXCR7 mRNA by RT-PCR. The group expressing the best silence efficiency of CXCR7-shRNA is selected as expression vector for next experiment. 3. To detect the change of SW620 cell proliferation after CXCR7-shRNA transfection by MTT. 4. To detect the change of SW620 cell invasion and migration after CXCR7-shRNA transfection by cell scratching experiment. 5. To detect the SW620 protein expression after CXCR7-shRNA transfection by Western blot. Results: 1. Packaging the three different recombinant lentiviral vector and one negative control vector is successful by sequencing and titer is 3. 16 × 108TU / ml,4. 27 × 108TU / ml,3. 93 × 108TU / ml,2. 95 × 108TU / ml respectively. 2. Expression of CXCR7 mRNA in three positive group is lower than negative control group after transfection( P 0. 05). The inhibition rate of CXCR7-shRNA-1 to CXCR7 is higher than the another group. 3. The tumor cell proliferation is reduced after CXCR7-shRNA-1 transfection into SW620 cell and there was significant difference comparing to control group( P 0. 05). 4. The migration index of control group and positive group are( 49. 92 ± 6. 41) %,( 29. 13 ± 5. 38) % respectively 24 hours after cell scratching experiment and it has statistical significance( P 0. 05). The migration index of control group and positive group are( 96. 52 ± 7. 44) %,( 72. 03 ± 8. 29) % respectively 48h hours after cell scratching experiment and it has statistical significance( P 0. 05). 5. Expression of CXCR7 is reduced after transfection into SW620 cell comparing to empty virus vector group and control group and it has statistical significance( P 0. 05). Conclusion: Expression of CXCR7 mRNA and CXCR7 protein is down-regulated effectively after CXCR7-shRNA recombinant lentiviral vector transfection into SW620 cell and proliferation and migration of tumor cell is inhibited effectively. It is a good foundation on next study of colon cancer RNAi therapy with lentiviral which target CXCR7 / CXCL12 biological axis. And it is a new direction for colon cancer gene therapy.

Overexpression of CDK5 in Neural Stem Cells Facilitates Maturation of Embryonic Neurocytes Derived from Rats In Vitro

To further understand the effects of cyclin-dependent kinase 5 (CDK5) on the differentiation of neural stem cells, which were cultured and transfected with CDK5-EGFP recombinant overexpression vector (OV-CDK5 Group), successful transfection was confirmed by RT-PCR and Western blot. Our results showed that the CDK5 mRNA expression significantly increased in 6 h after transfection. Increase in the levels of the CDK5 protein expression was observed in 72 h, compared with Empty Vector Control Group (EV-CTL Group) (P < 0.01). Furthermore, in OV-CDK5 Group, the percentage of S-phase cells was significantly higher than in EV-CTL Group (P < 0.01). Differentiated cells were showed with short processes in 24 h and with obviously enlarged cell body, and extended cellular processes in 72 h, in comparison to those in EV-CTL Group (P < 0.01). In 72 h under treatment with 10 μmol/L all-trans retinoic acid (ATRA), in OV-CDK5 Group, processes of the GFP-positive cells were reduced slightly and little GFP-positive debris was found. However, in the EV-CTL Group, processes of the GFP-positive cells were obviously shortened and deformed and much GFP-positive debris were found. Moreover, the percentage of G0/G1-phase cells was lesser, while the percentage of S-phase cells was higher than that in EV-CTL Group (P < 0.01, P < 0.01). In conclusion, our experiment suggested that CDK5 might promote proliferation and differentiation of neural stem cells, lengthen the processes of differentiated neurocytes, and accelerate morphological maturation of such cells. Furthermore, CDK5 might antagonize ATRA-induced inhibition against proliferation and differentiation in differentiated neurocytes.

Effects of glycogen synthase kinase-3β knock-down on growth and angiogenensis of human pancreatic cancer xenografts in nude mice

Objective To study the effects of glycogen synthase kinase-3β (GSK-3β) knock-down on the growth and the angiogenesis of human pancreatic cancer xenografts in nude mice.Methods Panc-1 cells were inoculated subcutaneously in athymic nude mice to establish xenograft models.The mice were divided into negative control group,vector control group and experimental group (n =8 each).After 8 weeks,the mice were killed.Tumor volume and weight were measured in nude mice beating xenografts.The inhibitory rate was calculated according to the weights of xenografts.The expression of proliferating cell nuclear antigen (PCNA) and CD31 proteins was assessed by using immunohistochemitry.Proliferation index (SPF) and microvessel density (MVD) were respectively counted according to PCNA and CD31 staining.The expression level of vascular endothelial growth factor (VEGF) was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.Results As compared with control group,the growth rate of human pancreatic cancer xenografts of nude mice in experimental group was decreased notably (P ＜0.05) and the inhibitory rate was 35.17％.In experimental group,the SPF index [(69.55 ± 2.64) ％] was significantly higher than in the negative control group [(83.26 ±2.83) ％] and the vector control group [(83.65 ± 2.65) ％] (P ＜ 0.05).In experimental group,the MVD index [(17.2 ± 2.9)] was higher than in the negative control group [(27.2 ± 3.1)] and the vector control group [(27.2 ± 3.1)] (P ＜ 0.05).The expression levels of VEGF mRNA and protein [(0.089 38 ±0.008 84 and 0.450 6 ±0.014 6) respectively] were significandy higher in xenografts tissues than in the negative control group (0.139 06 ± 0.003 35 and 0.675 8 ± 0.026 2 respectively) and the vector control group (0.138 89 ± 0.001 75 and 0.664 5 ± 0.010 5 respectively),but there was no significant difference between the negative control group and the vector control group (P ＞ 0.05).Conclusion In vivo GSK-3β knock-down can inhibit the growth and the angiogenesis of human pancreatic cancer xenografts in nude mice,which may be related to the down-regulation of VEGF. Key words: Pancreatic cancer; Glycogen synthase kinase-3β ; Microvessel density ; Vascular endothelial growth factor

Suppression of renal TRPM7 may alleviate kidney injury in the renal transplantation

The aim of this study was to investigate the effect of renal cortex transient receptor potential melastatin 7 (TRPM7) suppression on renal ischemia reperfusion injury induced by transplantation in mice. M7shRNA was used to decrease the expression of TRPM7 in NRK-52e cells. The mice were subjected to renal intra-parenchymal injection with lentivirus containing M7shRNA to produce hypo-expression of TRPM7 in renal cortex. Cell hypoxia mode and syngeneic renal transplantation in vivo were established. Then the effects of M7shRNA were measured by fluorescent probe for reactive oxygen species (ROS), intracellular calcium and magnesium; Western blot was applied for p38-MAPKs and Bax expression in cell studies. In vivo studies, mice were killed 24 h, 48 h, 72 h, 7 days and 21 days, respectively, after transplantation and the kidneys were dissected. Serum creatinine was measured, and the H&E, Masson's trichrome staining, TUNEL, Kim-1 and α-smooth muscle actin were used to evaluate pathological change. In cell model of hypoxia, the level of ROS in NRK-52e-M7shRNA was significantly lower than that in both NRK-52e and NRK-52e control cells, while the activation of p38-MAPKs was limited. In renal transplanted mice, renal function of M7shRNA group was conspicuously better than PBS- and vector-control-treated group. The histological examination showed that renal tubule injury and interstitial fibrosis were lower in M7shRNA-treated group compared with PBS and vector-control group. Suppression of renal cortex TRPM7 could alleviate kidney injury induced by transplantation in mice. The mechanism may involve reducing the early stage of ischemia reperfusion injury by inhibition of intracellular Ca(2+), Mg(2+) and ROS.

Diagnosis of Early Alzheimer's Disease Based on EEG Source Localization and a Standardized Realistic Head Model

In this paper, distributed electroencephalographic (EEG) sources in the brain have been mapped with the objective of early diagnosis of Alzheimer's disease (AD). To this end, records from a montage of a high-density EEG from 17 early AD patients and 17 matched healthy control subjects were considered. Subjects were in eyes-closed, resting-state condition. Cortical EEG sources were modeled by the standardized low-resolution brain electromagnetic tomography (sLORETA) method. Relative logarithmic power spectral density values were obtained in the four conventional frequency bands (alpha, beta, delta, and theta) and 12 cortical regions. Results show that in the left brain hemisphere, the theta band of AD subjects shows an increase in the power, whereas the alpha band shows a decreased activity (P-value <0.05). In the right brain hemisphere of AD subjects, a decreased activity is observed in all frequency bands. It was also noticed that the right temporal region shows a significant difference between the two groups in all frequency bands. Using a support vector machine, control and patient groups are discriminated with an accuracy of 84.4%, sensitivity 75.0%, and specificity of 93.7%.

Effects of KAP-1 in promoting the epithelial-mesenchymal transition of pancreatic cancer cell line Capan-2

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2.Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed.Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LVplenti-GFP-KAP-1),negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10％ fetal calf serum).Capan-2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor),empty vector control group (cells transfected with microRNAs inhibitor),blank control group 2 (cells cultured in 1640 medium plus 10％ fetal calf serum).The lentivirus was identified by double endonuclease restriction and sequencing,and the virus titer was detected.The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells.The expressions of KAP-1,genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction.The protein expressions of KAP-1,EMT proteins in all the groups were detected by Western blot.The measurement data were presented by mean ± standard deviation,and were analyzed using the analysis of variance.Results The lentiviral vector of LV-plentiGFP-KAP-1 was successfully constructed,and the virus titer was 2 × 108 TU/ml.Compared with the control group,the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours.The relative mRNA expressions of KAP-1,N-cadherin,vimentin,E-cadherin,miR-100-5p were 1.77 ± 0.83,2.62 ± 0.71,2.50 ± 0.21,7.20 ± 1.17 and 1.81 ±0.40 in the experimental group,5.03 ±0.29,5.07 ±1.53,3.83 ±0.57,7.83 ±0.78,7.01 ± 0.96 in the negative control group,5.13 ± 1.14,5.81 ± 1.49,4.92 ± 0.90,3.07 ± 0.36,6.87 ± 0.35 in the blank control group 1,with significant difference among the 3 groups (F =5.99,7.62,7.88,6.62,4.64,P ＜0.05).The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 1.56 ± 0.42,4.89 ± 0.61,5.20 ± 0.38,with significant difference among the 3 groups (F =5.14,P ＜ 0.05).The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.10 ± 1.37,3.44 ± 0.94,3.08 ±1.16,with no significant difference among the 3 groups (F =0.49,P ＞ 0.05).The results of western blot showed that the relative protein expressions of KAP-1,N-cadherin,vimentin,E-cadherin were 2.77 ± 1.99,1.31 ±0.38,4.25 ± 0.63,0.62 ± 0.06 in the experimental group,0.83 ± 0.46,0.41 ± 0.37,1.03 ± 0.33,1.17 ± 0.45 in the negative control group,0.71 ± 0.26,0.08 ± 0.04,1.37 ± 0.92,3.04 ± 0.65 in the blank control group 1,with significant difference among the 3 groups (F =5.54,4.68,3.19,8.18,P ＜ 0.05).The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 2.27 ±0.71,0.56 ±0.43,0.61 ±0.39,with significant difference among the 3 groups (F =4.81,P ＜0.05).The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.19 ± 0.55,3.93 ± 0.06,3.61 ± 0.73,with no significant difference among the 3 groups (F =0.04,P ＞ 0.05).Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression. Key words: Pancreatic neoplasms; KAP-1 ; Epithelial-mesenchymal transition ; microRNAs

Effect of angiopoietin-like protein 4 on rat pulmonary microvascular endothelial cells exposed to LPS

Pulmonary microvascular endothelial cells (PMVECs) possess highly proliferative and angiogenic capacities and are localized at the critical interface between the blood and microvessel wall; they are the primary targets of inflammatory cytokines during lung inflammation. Angiopoietin-like protein 4 (Angptl4) is a circulating protein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the effect of Angptl4 on rat PMVECs (RPMVECs) exposed to lipopolysaccharide (LPS). The cell culture was stimulated with LPS. Total Angptl4 cDNA was obtained from Source BioScience. The PCR product was cloned into the pcDNA3.1-eGFP or the pcDNA3.1-eGFP-Angptl4 vector, which were then transfected into the RPMVECs using SuperFect transfection reagent. The Angptl4 mRNA levels, protein levels and cell morphology of the RPMVECs in the experimental groups were detected using real time-PCR, western blot analysis, MTT assay, ELISA and confocal microscopy methods, respectively. The Angptl4 expression vector, pcDNA3.1-eGFP-Angptl4, was successfully constructed. The Angptl4 mRNA level in the LPS-pcDNA3.1-eGFP-transfected group (blank control) was slightly increased and was significantly higher in the experimental group compared with the empty vector and blank control group with significant differences. Pro-apoptotic caspase-8, -9 and Bax protein were inhibited, while p-AKT/AKT and p-MEK1/2 protein expression was also decreased. The rosiglitazone group had significantly decreased levels of the inflammatory cytokine, tumor necrosis factor (TNF)-α (P<0.01). The overexpression of Angptl4 inhibited the LPS-induced increase in the permeability of the RPMVECs, which was associated with the depolymerization of central F-actin in the RPMVECs. In conclusion, our study demonstrates that the overexpression of Angptl4 exerts protective, anti-inflammatory and anti-angiogenic effects. It represents a novel therapeutic target gene for the treatment of acute lung injury induced by LPS.

Inhibition of liposome-mediated recombinant plasmid expressing small interference RNA targeting hypoxiainduced factor-1α on retinal neovascularization in mice

Objective To observe the inhibition of LipofectamineTM2000 (LF2000)-mediated pSUPER recombinant plasmid expressing small interference RNA targeting hypoxia-induced factor (HIF)-1a (pSUPERsiHIF-1α) on retinal neovascularization in mice.Methods pSUPERsiHIF-1α recombinant plasmid was created.Forty-eight (seven-day-old) C57BL/6J mice were randomly divided into a normal group,the control group,empty vector group and gene therapy group with 12 mice in each group.Mice in the normal group were kept in normal room air,while the other three groups retinal neovascularization was induced by hypoxia.The mice in control group were not treated.The mice in the vector group received intravitreous injection of pSUPER and LF2000 (1 μl),and the gene therapy group received pSUPERsiHIF-1α and LF2000 (1 μl) one day before being returned to normal room air.Fluorescent angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections.HIF-lαand vascular endothelial growth factor (VEGF) levels in retinas were measured by immune histochemical staining method and reverse transeriptasepolymerase chain reaction (RT-PCR).Results Fluorescent angiography showed radial branching pattern vessels in the normal group and distorted large vessels,obstructed capillaries,many neovascular tuffs,fluorescence leakage in the peripheral retina in the control group and vector group.The gene therapy group demonstrated a significant reduction in neovascular tufts and fluorescence leakage compared with the control group and the vector group.The number of vascular cell nuclei extending breaking through the internal limiting membrane(ILM) of control group and vector group increased significantly compared with normal group (F=5850.016,P＜0.05),while obviously decreasing in the gene therapy group compared with control group (F=3012.469,P＜0.05).Immunohistochemical staining showed the expression of HIF-1α protein in nucleus and VEGF protein in cytoplasm.The expression of HIF-1α protein in retina was negative,while VEGF protein was weakly positive in normal group.The expression of HIF-1α and VEGF protein were both positive in control group and vector group,while weakly positive in gene therapy group.The results of RT-PCR showed that the expression of HIF-1α mRNA in retina was increased significantly in control group and vector group as compared with normal group (F=3102.326,P＜0.05),while decreasing significantly in gene therapy group as compared with control group (F=3336.425,P＜0.05).Conclusion Retinal neovascularization in the mice is significantly inhibited by intravitreal injection of LF2000-mediated recombinant plasmid pSUPERsiHIF-1α. Key words: Retinal neovascularization/drug therapy; Hypoxia-inducible factor 1,alpha subunit; Liposomes; Gene transfer techniques; Animal experimentation

Antitumor effect of recombinant human interferon-β adenovirus on esophageal squamous cell cancerin vitro

Interferon (IFN)-β has efficient antitumor effect both in vitro and in vivo, but its clinical implication is limited by short half-life and systemic toxicities. Gene therapy could be the choice to avoid the defects. Adenovirus vector containing human IFN-β gene was transfected into esophageal squamous cell carcinoma KYSE150 cells. Expression of human (h)IFN-β was detected by reverse transcription polymerase chain reaction and immunocytochemistry in KYSE150 cells. Cell growth and clonogenic assays, and flow cytometry were used to observe the antiproliferation effect and apoptosis on tumor cells, respectively. Reverse transcription polymerase chain reaction and immunocytochemistry showed obvious hIFN-β expression in KYSE150 cells after transfection and the tumor cell proliferation was obviously inhibited through cell proliferation and clonogenic assays. Flow cytometry analysis showed 27.3% cell apoptosis in adenovirus vector containing human IFN-β gene transfection group compared with 1.12% in empty vector control group. These findings indicate that hIFN-β gene mediated by recombinant adenovirus may have antitumor activity against human esophageal carcinoma cell by inducing apoptosis in vitro.

BTG2 inhibits the proliferation, invasion, and apoptosis of MDA-MB-231 triple-negative breast cancer cells

The purposes of this study were to investigate the effects of B cell translocation gene 2 (BTG2) on the proliferation, apoptosis, and invasion of triple-negative breast cancer and to provide an experimental basis for the future treatment of human triple-negative breast cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the MDA-MB-231 human triple-negative breast cancer cell line using lipofection. Then, relevant changes in the biological characteristics of the BTG2-expressing cell line were analyzed using MTT (tetrazolium blue), flow cytometry, and Transwell invasion chamber assays. Additionally, the effects of BTG2 expression on cyclin D1, caspase 3, and matrix metalloproteinases 1/2 (MMP-1/-2) expression were analyzed. Cell proliferation was significantly lower in the pcDNA3.1-BTG2-transfected group compared to the empty vector and blank control groups (p<0.05). There was no significant difference between the empty vector and blank control groups. FCM results demonstrated that there were significantly more cells in the G1 phase of the cell cycle and fewer S phase cells in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p<0.05). Additionally, the proportion of cells that migrated across the membrane was significantly lower in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p<0.05). Cyclin D1 and MMP-1/-2 expression were significantly lower in MDA-MB-231 cells transfected with pcDNA3.1-BTG2 as compared to the empty vector and blank control groups (p<0.05). Caspase 3 expression was significantly higher in MDA-MB-231 cells from the pcDNA3.1-BTG2 group compared to the empty vector and blank control groups (p<0.05). In conclusion, BTG2 may inhibit MDA-MB-231 proliferation and promote apoptosis. Additionally, BTG2 may also inhibit the invasion of MDA-MB-231 human triple-negative breast cancer cells.