Effects of interfering plasmid on the pathogenicity of gastric cancer by cell proliferation and apoptosis testing

Background To study the effects of recombinant interfering plasmids (pCDNA3.1-miR340) on the pathogenicity of gastric cancer by assessing cell proliferation and apoptosis. Methods Microarrays were used to analyse the function of pCDNA3.1-miR340. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate miRNA-340 expression in different patients, while the pCDNA3.1-miR340 plasmid was constructed for use in wound-healing and migration assays. Expression of the miRNA-340 target, RhoA, was assessed by Western blotting. Results miRNA-340 expression was significantly higher in patients with gastric cancer. Treatment with pCDNA3.1-miR340 significantly slowed tumour growth compared to treatment with empty plasmid. Additionally, when miRNA-340 expression was reduced, apoptosis decreased significantly, while RhoA protein expression increased 1.90-2.02-fold in SGC-7901 cells. Conclusions pCDNA3.1-miR340 had a positive therapeutic effect on the pathogenicity of gastric cancer.

Exogenous Tissue Inhibitor of Metalloproteinase-2 Affects Matrix Metalloproteinase-2 Expression in Conjunctival Filtering Blebs and Bleb Scarring in Rats

Objective. To examine the effect of tissue inhibitor of metalloproteinase-2 (TIMP-2) on conjunctival filtering bleb scarring. Methods. A model of conjunctival filtering bleb was established whereby rats were injected with saline, blank adenoviral vector, or adenoviral vector carrying TIMP-2 into the bleb. Filtration bleb formation and matrix metalloproteinase-2 (MMP-2) expression were examined. Results. All operated eyes formed obvious elevated blebs on day 1. In the normal saline group, empty plasmid group, and gene transfection group maintenance time of filtrating blebs was 5–14, 5–14, and 6–16 days, and average survival time was 8.24, 8.16, and 9.44 days, respectively. MMP-2 expression increased slightly in the gene transfection group at 3 and 5 days after surgery, reached a peak after 14 days, and then gradually decreased. MMP-2 expression was weakly positive in the normal conjunctival epithelium, but was hardly detected in the lamina propria. Seven days after surgery, the epithelium and lamina propria of the conjunctival filtering bleb exhibited strong positive expression in the empty plasmid group but only weak expression in the adenovirus group. Conclusion. Exogenous TIMP-2 interfered with local MMP-2 expression, delaying peak expression of MMP-2 and slowing the scarring of filtering blebs during wound healing of subconjunctival tissue.

Stable knockdown of ZBTB7A promotes cell proliferation and progression in nasopharyngeal carcinoma

Aims: Although high expression of ZBTB7A is positively relative to metastasis in nasopharyngeal carcinoma (NPC) patients, the association between its low expression and metastasis of NPC remains unclear. The present study aimed to definitely identify the association. Methods: The level of ZBTB7A was effectively knocked down by stable transfection of short hair RNA plasmid in NPC cell lines CNE2 and 5-8F (shRNA-CNE2 and shRNA-5-8F), compared with the cells that stably transfected empty plasmid (NC-CNE2 and NC-5-8F). The levels of ZBTB7A were assessed by real-time polymerase chain reaction and Western blot in the cell lines. MTT assay, colorimetric focus-formation assay, flow cytometry, wound healing assay, transwell assays, and xenograft model were performed to analyze cell vitality, proliferation, cell cycle, migration, invasion, and tumorigenicity. Results: The levels of ZBTB7A were effectively reduced in shRNA-CNE2 and shRNA-5-8F. Their carcinogenicity was stronger separately than the abilities of NC-CNE2 and NC-5-8F. NC-CNE2 and shRNA-CNE2 were selected to establish the xenograft model because of their stronger tumorigenicity than NC-6-10B and shRNA-5-8F. The assay showed that shRNA-CNE2 had stronger tumorigenicity than NC-CNE2. Conclusions: The results demonstrated the reverse association between the expression of ZBTB7A and the tumorigenicity of NPC. We postulate that some oncogenic pathways, which are suppressed by ZBTB7A, will vicariously promote the proliferation and progression of NPC when ZBTB7A is decreased.

Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells

Background/Aims: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. Methods: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated β-galactosidase staining kit was used to assess cell senescence. Results: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G1 phase and fewer cells in the G2/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and β-galactosidase activity compared with the blank and empty plasmid groups. Conclusion: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

Expression ofHelicobacter pylori hspAGene inLactococcus lactisNICE System and Experimental Study on Its Immunoreactivity

Aim. The aim of this study was to develop an oralLactococcus lactis(L. lactis) vaccine againstHelicobacter pylori(H. pylori).Methods. AfterL. lactisNZ3900/pNZ8110-hspAwas constructed, growth curves were plotted to study whether the growth of recombinantL. lactiswas affected afterhspAwas cloned intoL. lactisand whether the growth of empty bacteria, empty plasmid bacteria, and recombinantL. lactiswas affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinantL. lactisand its immunoreactivity.Results. There was no effect observed from the growth curve after exogenous genehspAwas cloned intoL. lactisNZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspAexcept 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity.Conclusion. The growth of recombinantL. lactiswas suppressed even though a small amount of HspA had been induced to express. Therefore recombinantL. lactisonly express HspA which was not suitable to be oral vaccine againstHelicobacter pylori.

Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons

Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%,P<0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons.

Formic Acid Triggers the “Acid Crash” of Acetone-Butanol-Ethanol Fermentation byClostridium acetobutylicum

ABSTRACTSolvent production byClostridium acetobutylicumcollapses when cells are grown in pH-uncontrolled glucose medium, the so-called “acid crash” phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium,C. acetobutylicumDSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation.

Angiogenic and Vasculogenic Effects of Sonic Hedgehog Gene Therapy In An Experimental Model of Peripheral Limb Ischemia

Abstract 563 Sonic hedgehog (Shh) is a morphogen regulating epithelial-mesenchymal interactions during embryogenesis. In post-natal life, Shh is a potent indirect angiogenic agent, able to upregulate different families of angiogenic growth factors. Furthermore, Shh gene transfer enhances the contribution of bone marrow (BM)-derived endothelial progenitor cells to myocardial neovascularization. In this study, we tested the beneficial potentials of Shh gene therapy in an experimental model of peripheral limb ischemia, a disease characterized by occlusion of vessels of the arterial circulation of the legs, often secondary to atherosclerosis, thrombosis, and/or inflammatory processes. Ischemia of the left hindlimb was induced by excising the femoral artery, from its proximal origin as a branch of the external iliac artery till the bifurcation into saphenous and popliteal arteries. Blood flow was measured in ischemic and contralateral hindlimbs by laser Doppler perfusion imaging at days 0, 7, 14, 21, and 28 after ischemia. At day 28, mice were sacrificed and adductor muscles were analyzed for capillary and arteriole density. An additional set of 1-year-old C57BL/6J mice and 8-weeks-old C57BL/6J mice were used to study the effects of Shh gene therapy on mobilization of BM-derived endothelial progenitors in the course of ischemia. Mice were treated with 200 μ g phShh or empty plasmid and hindlimb ischemia was induced 7 days after treatment. Peripheral blood samples were obtained at days 2, 4, and 7 after induction of ischemia. FACS analyses were used to detect cells Lin- and Sca-1+CD34+, Sca-1+CD133+, Sca-1+ Flk-1+, CD14+ Flk-1+, CD14+ CD34+. We also used a BM transplantation model to test the effects of Shh gene therapy on BM-derived cells in the setting of hindlimb ischemia. Wild-type recipient mice received 2×106 BM cells from nls-Ptc1-LacZ mice. Six weeks laster, mice received treatment with 200 μ g phShh or empty plasmid in the hindlimb muscles. Seven days after treatment, ischemia of the left hindlimb was induced. X-gal positive cells were counted on hindlimb muscles 10 days after ischemia. We also measured local expression levels of VEGF165, Ang-1, and SDF-1α proteins in mice treated with 200 μ g phShh or empty plasmid 7 days after ischemia. At day 28 after ischemia, blood perfusion ratio between the ischemic and the contralateral leg was 0.97 ± 0.01 in Shh-treated mice and 0.68 ± 0.03 in control animals (p=0.008) (Figure 1a, b). Capillary and arteriole density were significantly higher in the phShh–treated muscles compared to controls (p=0.02; p=0.03, respectively). In both young and middle-aged animals, phShh treatment resulted in significant increase of the number of circulating CD45-/Sca-1+/Flk-1+ and CD45-/Sca-1+/CD133+ cells (p<0.05). In the BM transplantation model, the number of X-gal positive cells in the ischemic site was significantly higher in phShh-treated animals (p=0.000002). Double fluorescent staining for β-gal and BS-1 lectin demonstrated incorporation of these cells into vascular structures, demonstrating that Shh gene therapy increases the number of cells migrating from the BM to the site of ischemia and contributing to the process of neovascularization. Finally, the expression of VEGF, ang-1, and SDF-1α measured after ischemia was significantly higher in animals treated with phShh than in mice treated with control (p= 0.00001; p= 0.00054; p= 0.00003, respectively). The current standard of care for ischemic diseases of lower extremities relies on direct revascularization, either by endovascular techniques or open surgical approaches. Early preclinical studies and phase I clinical trials achieved promising results with growth factors administered as recombinant proteins or single-agent gene therapies. In this study, we demonstrate that intramuscular administration of a plasmid containing the human Shh gene induces simultaneous activation of angiogenic, arteriogenic, and vasculogenic mechanisms, with beneficial effects in a model of peripheral artery disease. In addition, we showed the ability of Shh gene therapy to increase the number of circulating BM-derived circulating progenitor cells, as well as their incorporation in vascular structures at the level of the ischemic site. Figure 1. (a) Laser Doppler perfusion imaging. (b) Mice treated with phShh displayed significant increase of the ischemic/contralateral leg perfusion ratio. Figure 1. (a) Laser Doppler perfusion imaging. (b) Mice treated with phShh displayed significant increase of the ischemic/contralateral leg perfusion ratio. Disclosures: No relevant conflicts of interest to declare.

Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.

Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase

ABSTRACT We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCSAt ) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCSAt gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCSAt gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(γ-Glu-Cys) n -Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCSAt gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl2 activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd2+/mg (dry weight) of cells. The expression of the PCSAt gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd2+ approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCSAt gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd2+ accumulation in nodules 1.5-fold.