Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Effect of xeroderma pigmentosum group D gene on proliferation of human umbilical arterial smooth muscle cells induced by Ox-LDL

AIM: To investigate the effects of xeroderma pigmentosum group D (XPD) gene on the proliferation of human umbilical arterial smooth muscle cells (HUASMCs) induced by oxidized low-density lipoprotein (Ox-LDL). METHODS: The recombinant plasmid pEGFP-N2/XPD was transfected into HUASMCs by liposome. The cells were divided into blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, Ox-LDL group, Ox-LDL+pEGFP-N2 group and Ox-LDL+pEGFP-N2/XPD group. The proliferation rate of the cells was detected by MTT and EdU assays. The apoptotic rate and cell cycle distribution were analyzed by flow cytometry. The protein levels of XPD, caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with blank control group, the expression of XPD was increased in pEGFP-N2/XPD group (P<0.05). According to the results of MTT and EdU assays, the cell proliferation in pEGFP-N2/XPD group was reduced compared with blank control group (P<0.05). Compared with Ox-LDL group, the cell proliferation in Ox-LDL+pEGFP-N2/XPD group was significantly inhibited (P<0.05). According to the results of flow cytometry, the cell proportion of S phase decreased and the G₀/G₁-phase cell proportion increased significantly in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group compared with blank control group and Ox-LDL group, repectively (P<0.05). Compared with blank control group and Ox-LDL group, the protein level of Bcl-2 decreased and the protein levels of Bax and cleaved caspase-3 increased in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group, respectively (P<0.05). CONCLUSION: XPD inhibits the proliferation of HUASMCs and promotes their apoptosis, and reduces the promoting effect of Ox-LDL on the proliferation of HUVSMCs. XPD may be the target for treatment of atherosclerosis.     

Correlation between Mnk2 protein expression and prognosis of patients with esophageal squamous cell carcinoma

AIM: To investigate the protein expression of mitogen-activated protein kinase-interacting kinase-2 (Mnk2) and its prognostic effect in the patients with resected esophageal squamous cell carcinoma (ESCC). METHODS: A total of 86 informative patients with surgically resected ESCC and 54 normal esophageal tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess the protein expression of Mnk2, and its correlation with prognosis was statistically analyzed by the methods of Kaplan-Meier curve and Cox proportional hazard mode. RESULTS: The protein expression of Mnk2 was elevated in most of tumor tissues compared with the adjacent tissues. Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with the TNM stage (P<0.05). Both disease-free survival (DFS) and overall survival (OS) of Mnk2 over-expression patients were shorter than those in Mnk2 negative expression group. Multivariate analysis confirmed that Mnk2 expression, as an independent and significant factor for both DFS and OS, predicted a poor prognosis of the patients with resected ESCC (P<0.05). CONCLUSION: The expression of Mnk2 was significantly related to the TNM stages, and might be a novel predictor for prognosis in ESCC.     

甜瓜白粉病抗病基因的定位及候选基因分析

以甜瓜高抗白粉病自交系MR-1为母本,易感白粉病自交系Top Mark为父本,构建BC_1P_2和F_2群体,经13个国际通用的甜瓜白粉病生理小种鉴别寄主鉴定,2015年东北农业大学设施园艺工程中心的白粉病发病病菌为P.xanthii生理小种1,甜瓜MR-1对P.xanthii生理小种1的抗性由单显性基因控制。通过对266个F2分离群体的抗病性鉴定和CAPS标记分析,采用复合区间作图法对甜瓜抗白粉病性状进行QTL分析,最终构建了1张包含203个CAPS标记的甜瓜遗传连锁图谱,并将抗病基因PXR定位在第12号染色体上M12-GH和M12-TE 2个标记之间,该基因与两侧翼标记间的距离分别为0.63 c M和0.42 c M,两侧翼标记间在甜瓜参考基因组上对应的物理距离为303 kb,该区域内含有60个预测基因,其中10个基因含有非同义单碱基突变。10个基因荧光定量分析结果显示,在抗病与感病甜瓜表达量存在较大差异的4个基因MELO3C002434、MELO3C002437、MELO3C002441、MELO3C002457为甜瓜白粉病抗病候选基因。     

野生番茄LA2157中热稳定抗根结线虫基因Mi-9候选基因的分离与过表达载体构建

根结线虫是番茄上的重要土传病害,选育抗根结线虫品种是最有效的防治方法,但是目前转育到栽培番茄中的抗根结线虫基因Mi-1在土温高于28℃时就丧失了抗性。本试验利用热稳定抗根结线虫野生番茄材料LA2157,根据Mi-1基因的序列信息,对其中热稳定抗根结线虫Mi-9基因进行同源克隆,在LA2157中共获得2个候选基因片段;通过In-Fusion克隆技术,将候选基因与过表达载体p BI121进行连接,经电泳检测和测序分析,最终构建Mi-9候选基因的过表达重组载体。     

Effect of antisense locked nucleic acid blocking translation initiation region of c-myc exon 2 on apoptosis of hepatocellular carcinoma cells

AIM: To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS: The anti-LNA that was complementary to the translation initiation region of c-myc exon 2 was designed, synthesized, and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry, and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS: Five days after transfection, the mRNA level of c-Myc in anti-LNA group was 0.335±0.016, and the protein level was 0.448±0.037, significantly lower than those in control group (both P<0.05). The ratio of apoptotic cells in anti-LNA group was 32%±6%, which was higher than that in control group (P<0.05).CONCLUSION: Antisense locked nucleic acid targeting at the translation initiation region of c-myc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

Identification of S-haplotypes of European cultivars of sour cherry

The sour cherry is known to exhibit the phenomenon of gametophytic self-incompatibility which prevents self-fertilization. In sour cherry, besides self-incompatible cultivars, there often occur self-compatible cultivars. This is due to the occurrence of natural mutations of the S-RNase or SFB genes and, consequently, loss of functionality of S-alleles. Here we present the results of the identification of S-haplotypes of 21 cultivars of sour cherry from various regions of Europe. The analyses were performed using methods based on the amplification of intron I and intron II of the S-RNase gene and fragments specific to the individual alleles of the S-RNase or SFB genes. The tested cultivars were found to contain 15 S-haplotypes: S₁, S_1?, S₄, S₆, S_6m, S_6m2, S₉, S₁₂, S₁₃, S_13?, S₂₆, S₃₅, S_36a, S_36b, and S_36b2. The most frequently occurring S-haplotypes were S_13? (61.9%), S_36a (57.1%), and S₂₆ (47.6%). On the basis of the results, 17 of the 21 cultivars were deduced to be self-compatible. The results will be of use in the production of sour cherry fruit by facilitating the selection of suitable pollinating cultivars. The results are also expected to be useful in the breeding of new cultivars of sour cherry when selecting genotypes for crosses.