Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

P-selectin on activated platelets promotes hematogenous metastasis of primary lung cancer via P-selectin glycoprotein ligand-1

AIM: To investigate the relationship between platelets/P-selectin on activated platelets and clinico-pathological features, hematogenous metastasis and prognosis of lung cancer, and to explore the effect of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interaction on the hematogenous metastasis-related integrin β3 (ITGB3). METHODS: The expression of P-selectin on activated platelets was detected by flow cytometry, and its effects on lung cancer and the risk of hematogenous metastasis were analyzed. The expression of PSGL-1 in different types of lung cancer tissues and cell lines was determined by Western blot. By co-culturing platelets and lung cancer cells in vitro, the effects of up- and down-regulation of PSGL-1 on invasion and migration abilities of lung cancer cells were observed. RESULTS: The peripheral blood platelet counts and P-selectin expression on activated platelets in the patients with lung cancer were significantly increased (P<0.05). The P-selectin expression on activated platelets was significantly associated with hematogenous metastasis of lung cancer (r=0.256, P<0.05). The strongest expression of PSGL-1 was found in the lung adenocarcinoma samples, next in the lung squamous-cell carcinoma samples, and the weakest in small-cell lung cancer samples. P-selectin promoted transmembrane invasion of lung cancer cells. Inhibition of P-selectin and its ligand PSGL-1 reduced ITGB3 expression, invasion and migration of lung cancer cells. CONCLUSION: The P-selectin level on activated platelets is significantly associated with hematogenous metastasis of lung cancer, which is related to the binding of P-selectin and its ligand PSGL-1. Up-regulation of ITGB3 level after their binding might be one of the mechanisms of the remodeling of extracellular matrix to facilitate hematogenous metastasis of lung cancer.     

“2012年园艺植物染色体倍性操作与遗传改良学术研讨会”预备通知

自2008年11月在中国农业科学院郑州果树研究所成功召开园艺植物染色体倍性操作与遗传改良研讨会以来,我国科研人员在该领域的研究取得了可喜的成绩。为进一步总结近几年来在该领域的研究进展,促进园艺植物倍性育种研究,同时为该领域的专家、学者和同仁们提供良好的交流平台。中国园艺学会定于2012年4月中旬在重庆召开园艺植物染色体倍性操作与遗传改良学术研讨会。欢迎从事该研究领域及相关研究工作的人员参加。     

MdMYB1转录因子调控红星苹果果实着色的计算机模拟分析及表达验证

 从‘红星’苹果(Malus domestica‘Delicious’) 果实中克隆获得了MdMYB1基因, 通过生物信息学分析( in silico analysis) 和活体细胞融合蛋白荧光观察, 确定其编码蛋白定位于细胞核。同时, 克隆了MdDFR和MdUFGT基因的启动子, 模拟分析表明2个基因的启动子序列中含有MYB结合的顺式作用元件。为了探讨MdMYB1转录因子是否调控MdDFR和MdUFGT基因的表达, 对不同光照条件下果皮花青素含量和基因表达进行了检测, 结果表明光照能够诱导花青素积累, 并迅速启动3个基因表达, 且3个基因表现出高度相似的表达模式, 表明MdMYB1转录因子可能直接调控MdDFR和MdUFGT基因的表达。     

Immunosuppressive and protective effects of human α1-antitrypsin on pancreatic β-cell transplantation

AIM：To study the immunosuppressive and protective effects of human α1-antitrypsin (hAAT) on pancreatic β-cell transplantation. METHODS： An NIT-1 cell line (NIT-hAAT) was constructed, which can stably express the protein of hAAT. The BALB/c mice were intraperitoneally injected with NIT-1 and NIT-hAAT cell lines twice to induce cytotoxic T-lymphocytes (CTL). The apoptotic situation, the cytokine expression, and the mRNA expression of inflammatory factors were examined after mixed culture of CTL with NIT-1or NIT-hAAT cell line pretreated with mitomycin. Both cell lines were transferred into the left renal capsule of the diabetic mice to dynamically observe the changes of blood sugar and body weight, the serum levels of insulin and C-peptide, and the pathological changes of the transplanted sites. RESULTS： The results of extended CTL killing assay showed that the cytotoxic effect on NIT-hAAT cell acceptor mice was significantly reduced compared with NIT-1 cell acceptor mice. hAAT effectively reduced apoptosis, inhibited the mRNA expression of inflammatory factors IL-1β and IL-6, and adjusted the balance of Th1/Th2 cytokine expression. After NIT-hAAT was transplanted into the diabetic mice, blood glucose decreased obviously and maintained for 28 d. The serum levels of insulin and C-peptide increased obviously. The infiltration of the inflammatory cells in the transplanted sites significantly reduced. CONCLUSION：hAAT has the abilities of reducing cytotoxic effect of CTL on the β-cells, inhibiting inflammatory factor expression, and stopping short-term immunological rejection of the acceptor. hAAT has obvious immunosuppressive and protective effects on pancreatic β-cell transplantation for treatment of diabetes.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

Role of damaged mitochondria in Vibrio vulnificus induced dendritic cell apoptosis

AIM： To investigate the role of damaged mitochondria in dendritic cell (DC) apoptosis induced by Vibrio vulnificus (Vv) and its possible mechanism. METHODS：DC2.4 cells were co-cultured with Vv 1.1758 strain. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect reactive oxygen species (ROS) and intracellular Ca2+ concentration in the invaded cells, respectively. The cellular apoptotic rates and mitochondrial membrane potential (Δψm) were measured by flow cytometry. The expression of nuclear factor-kappa B p65 (NF-κB p65) and tumor necrosis factor-alpha (TNF-α) was detected by Western blotting. RESULTS： Vv 1.1758 induced DC2.4 cell apoptosis. Vv 1.1758 bacteria invaded into the DC2.4 cells by binding with cellular membrane though the end of the body. In the invaded DC2.4 cells, the visible mitochondrial damage, elevated ROS and intracellular Ca2+ levels, and declined Δψm were presented. After 1 h of co-culture, NF-κB p65 began to rise and reached the peak at 5 h, and then slightly decreased at 6 h. The TNF-α level increased after 2 h of co-culture and reached the peak at 6 h. CONCLUSION： The damaged mitochondria play an important role in DC apoptosis induced by Vv, and its possible mechanism may associate with the elevation of ROS and intracellular Ca2+ level, and the declined Δψm. Meanwhile, NF-κB p65 and TNF-α are potential critical signaling molecules in the process of apoptosis.