Role of translationally controlled tumor protein in PRL-3-promoted metastasis of colon cancer cells

AIM: To study the role of translationally controlled tumor protein (TCTP) in the proliferation, migration and invasion of phosphatase of regenerating liver-3(PRL-3)-promoted colon cancer cells.METHODS: The vectors pAcGFP-C3 and pAcGFP-C3-PRL-3 were constructed and transfected into the colon cancer cell line LoVo.LoVo-PRL-3 cells stably expressing PRL-3 and LoVo-control cells were established. The expression levels of PRL-3 and TCTP in both cells were detected by Western blotting and real-time PCR. The specific siRNA sequence for TCTP mRNA and control-siRNA were synthesized and transfected into the LoVo-PRL-3 cells. TCTP expression at mRNA and protein levels in LoVo-PRL-3 was detected by Western blotting and real-time PCR 24 h, 48 h and 72 h after transfection. The proliferation, migration and invasion abilities of LoVo-control cells, LoVo-PRL-3 cells, TCTP-siRNA and control-siRNA cells were detected by CCK-8 assay and the method of Transwell cell culture chambers.RESULTS: The expression of TCTP at mRNA and protein levels in LoVo cells was significantly increased after PRL-3 transfection (P<0.05). TCTP mRNA was significantly inhibited 24 h, 48 h and 72 h after transfection of TCTP-siRNA (P<0.01). TCTP protein was also significantly inhibited 48 h and 72 h after transfection (P<0.01). Compared with LoVo-control cells, the proliferation, migration and invasion abilities of LoVo-PRL-3 cells were significantly enhanced (P<0.05). However, lowering the up-regulated expression of TCTP in LoVo-PRL-3 cells inhibited the proliferation, migration and invasion abilities (P<0.05). CONCLUSION: PRL-3 promotes proliferation, migration and invasion of colon cancer cells by up-regulating the TCTP expression. siRNA targeting TCTP may be an effective method for prevention and treatment of colon cancer cell metastasis.     

High expression level of TAL1 gene in newly diagnosed acute myeloid leukemia

AIM: To investigate the characteristic of T-cell acute lymphocytic leukemia 1 (TAL1) gene expression in acute myeloid leukemia (AML) cell lines and in primary AML cells from de novo AML patients with different subtypes. METHODS: Real-time PCR was used to determine the expression of TAL1 mRNA in acute leukemia cell lines (Jurkat, CCRF-CEM, HL-60 and NB4 cell lines) and peripheral blood mononuclear cells from 47 newly diagnosed AML patients. Twelve healthy individuals were served as healthy control group. RESULTS: A significantly increased level in TAL1 mRNA was found in AML cell lines (HL-60 and NB4), T-cell acute lymphacytic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and primary AML cells compared with the healthy controls. Over-expression of TAL1 was found in all detected AML subtypes, the highest level of TAL-1 mRNA was found in AML-M1 and AML-M5 subtype (P<0.05). CONCLUSION: High expression of TAL1 in AML might influence the differentiation and proliferation of myeloid cells, further investigation needs to confirm whether it would be as a biomarker for pathogenesis of AML.     

Inhibition of c-Myc by 10058-F4 overcomes imatinib resistance in chronic myeloid leukemia cells

AIM：To investigate the effect of c-Myc inhibitor 10058-F4 on human chronic myeloid leukemia (CML) K562 cells and imatinib-resistant K562/G cells. METHODS：The protein expression of c-Myc was detected by Western blotting. Cell proliferation was evaluated by MTT assay and colony formation assay. PI staining was used to determine the cell cycle distribution. Annexin V-PI staining was applied for apoptosis detection. RESULTS：Imatinib-resistant K562/G cells displayed lower sensitivity to imatinib than K562 cells with high expression of c-Myc. Treatment with specific c-Myc inhibitor 10058-F4 inhibited the cell proliferation in a dose- and time-dependent manner, and K562/G displayed more sensitivity to 10058-F4 than K562 cells. 10058-F4 also induced cell cycle arrest in G0/G1 phase and induced apoptotic cell death in the 2 cells. Importantly, 10058-F4 suppressed the colony formation ability in K562 and K562/G cells. CONCLUSION：c-Myc is a novel target to overcome imatinib-induced drug resistance, and c-Myc inhibitor provides a new approach in CML therapy.     

Jagged1 regulates STAT3 signaling pathway and affects growth and apoptosis of multiple myeloma cells

AIM:To investigate the effect of Jagged1 on the growth and apoptosis of multiple myeloma cells. METHODS:Transfection with small interfering RNA targeting Jagged1 and negative control was carried out in multiple myeloma cell line U266, and the mRNA and protein levels of Jagged1 in the cells were determined by RT-qPCR and Wes-tern blot. The cells without transfection were used as blank control. Trypan blue staining was used to draw the cell growth curve. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and Bax in the cells were determined by Western blot. STAT3 signaling pathway inhibitor AG490 was used to detect the activation level of STAT3 signaling in the cells. RESULTS:Compared with the U266 cells without transfection, the expression of Jagged1 at mRNA and protein levels decreased in the U266 cells transfeced with small interfering RNA targeting Jagged1 (P<0.05). However, the expression of Jagged1 at mRNA and protein levels did not change in the U266 cells transfected with small interfering RNA negative control. Knockdown of Jagged1 expression decreased the cell viability, increased the apoptotic rate, increased Bax levels, and decreased the protein level of p-STAT3 in the U266 cells (P<0.05). AG490 treatment decreased the protein level of p-STAT3, blocked the activation of STAT3 signaling pathway, promoted the cell apoptosis induced by Jagged1 knockdown, and inhibited the viability of the U266 cells. CONCLUSION:Knock-down of Jagged1 expression promotes the apoptosis of multiple myeloma cells by inhibiting STAT3 signaling pathway, thus suppressing cell growth.     

Effect of cytarabine on curcumin-induced autophagy in human acute myeloid leukemia cells

AIM:To observe whether autophagy occurs in curcumin-induced human acute myeloid leukemia KG1a cells in the presence of chemotherapeutic drug cytarabine and the possible mechanism. METHODS:KG1a cells were cultured in vitro. The ultrastructural changes of the cells were observed under transmission electron microscope. Autophagy was detected by acridine orange staining. The cell viability was measured by MTT assay. The cell cycle distribution was analyzed by flow cytometry. The expression of autophagy-related molecules beclin-1 and LC3 at mRNA and protein le-vels was determined by RT-qPCR and Western blot. RESULTS:Curcumin dose-dependently inhibited the viability of KG1a cells (P<0.05). The growth inhibition rate in combination group was significantly higher than that in single reagent group and control group (P<0.01). Electron microscopical observation showed that curcumin induced the occurrence of autophagosomes, and cytarabine increased curcumin-induced autophagosomes. Acridine orange staining showed that the combined treatment with cytarabine increased the autophagy induced by curcumin, and the number of autophagic acid vesicles and cells containing autophagic acid vesicles were increased. Curcumin blocked the cell cycle in the G₀/G₁ phase. The mRNA expression levels of beclin-1 and LC3 in combination group were significantly higher than those in single reagent group and control group(P<0.01). The results of Western blot showed that the protein expression of beclin-1 was significantly up-regulated in combination group (P<0.05), and the ratio of LC3-Ⅱ/LC3-I was higher than that in control group (P<0.01). CONCLUSION:Curcumin inhibits the viability of KG1a cells and induces autophagy. Cytarabine promotes autophagy, which is superior to curcumin alone. It may be related to the up-regulation of beclin-1 and LC3-Ⅱ by the two reagents.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Effect of Qingyi decoction on p-STAT3 expression in pancreas of mice with severe acute pancreatitis induced by L-arginine

AIM: To explore the role of STAT3 signaling pathway in acute pancreatitis induced by L-arginine, and the mechanisms of Qingyi decoction(QYD) treatment on severe acute pancreatitis (SAP). METHODS: The Kunming mice were randomly divided into 3 groups (n=10): control group, SAP group and QYD treatment group (SAP+QYD). The mice in SAP group and SAP+QYD group were intraperitoneally injected with 20% L-arginine (3 g/kg, bid). The mice in SAP+QYD group were also administered intragastrically with QYD(10 mL/kg) 30 min after the second injection of 20% L-arginine and twice a day for the following 2 days. The mice were anesthetized and sacrificed 72 h after SAP induction. The activity of amylase was measured in serum, the relative pancreatic weight was assayed, and the activity of myeloperoxidase (MPO) was analyzed to evaluate the neutrophil infiltration in lung tissues. The morphology of pancreas and lung was observed. The protein of pancreas was extracted to detect the expression of p-STAT3 by Western blotting. The mRNA expression of monocyte chemoattractant protein-1(MCP-1) was determined by real-time PCR. RESULTS: Compared with control group at 72 h, L-arginine induced SAP with increased serum amylase activity, pancreatic wet weight ratio and MPO activity in lung tissues (P<0.05). In SAP+QYD group, the activity of amylase, pancreatic wet weight ratio and MPO levels were significantly lower than those in SAP group (P<0.01). Compared with control group at 72 h, the pancreas and lung were obvious injured, the protein level of p-STAT3 and mRNA expression of MCP-1 in pancreas tissues increased significantly in SAP group. Compared with SAP group, the pathologic damage of the pancreas and lung tissues, the protein level of p-STAT3 and mRNA expression of MCP-1 in pancreas were significantly reduced in SAP+QYD group. CONCLUSION: The expression of p-STAT3 in pancreas increases in the mice with SAP induced by L-arginine. The activation of STAT3 may take part in the development of SAP. Inhibition of STAT3 activation in pancreas is one of the mechanisms of QYD treatment for SAP.     

NLRP3 inflammasome in pathogenesis of MS/EAE and its targeted therapy

Inflammasomes, the important component of innate immune system, play an important role in inflammatory diseases. NLRP3, the most studied inflammasome, is activated after recognizing danger-associated molecular patterns and pathogen-associated molecular patterns. The activated NLRP3 inflammasome promotes inflammation by maturation and release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Involvement of the NLRP3 inflammasome in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) was suggested in a number of studies. Therefore, targeting on NLRP3 inflammasome is one of the promising methods for treatment of related diseases. In this review, we summarize the main ways by which the NLRP3 inflammasome is activated in the cytosol. We also discuss the development and treatment of NLRP3 inflammasome in MS and EAE, and expect to provide reference for the treatment of MS.     

Single nucleotide polymorphism and mutation of miRNA binding sites at BCL11B-3'UTR in healthy individuals and patients with T-ALL

AIM:To analyze the microRNA (miRNA) binding sites at B-cell lymphoma/leukemia 11B gene 3'-untranslated region (BCL11B-3'UTR), and to establish the method of identifying the single nucleotide polymorphism (SNP) and mutation of these miRNA binding sites in healthy individuals and patients with T-cell acute lymphoid leukemia (T-ALL). METHODS:TargetScan was used for screening and predicting the miRNA binding sites at BCL11B-3'UTR. PCR and sequencing were used to identify the miRNA binding sites at BCL11B-3'UTR. The polymorphisms in DNA sample of peripheral blood mononuclear cells from 20 healthy individuals and 21 patients with T-ALL at this region were analyzed. RESULTS:Twenty-four highly conserved miRNA binding sites were screened according to the criteria of context ++ score and seed match categories. The nucleotide exchange (T>C) located at site 2 402 of BCL11B-3'UTR was detected in one case out of 21 cases of T-ALL samples, which had been registered as SNP (rs184678181) in dbSNP. No polymorphism or mutation in BCL11B-3'UTR miRNA binding sites was identified in the samples from the healthy individuals. CONCLUSION:Polymorphism or mutation in BCL11B-3'UTR is rare in healthy individuals and T-ALL patients. To our best knowledge, it is the first identification of BCL11B-3'UTR SNP (T>C at site 2 402), which is involved in hsa-miR-6814-5p binding site in a patient with T-ALL. Further investigation will focus on its effect for BCL11B expression regulation.     

Advances in experimental research in HCV cell model and anti-HCV effect of As2O3

Hepatitis C virus (HCV) is a human pathogen responsible for liver diseases including acute and chronic hepatitis and hepatocellular carcinoma. However, the high prevalence, the absence of antiviral drugs and vaccines for prevention and treatment are the difficult medical problems. Lacking appropriate culture method and small animal model have severely limited investigation of the HCV infection mechanism and the development of the therapeutic strategy. Recently, the in vitro culture system develops rapidly, and provides a powerful tool for HCV related research. Despite the well known toxicity of the chemical, arsenic trioxide (As₂O₃) is effective for treating the patients with refractory or relapsed acute promyelocytic leukemia and many other cancers. Interestingly, As₂O₃ shows the ability of inhibiting HCV RNA replication and infection. This review describes the different types of in vitro HCV models developed, and many studies of the potent effect of As₂O₃ against HCV and its associated molecular mechanisms.     

Role of PI3K/Nrf2 signaling pathway in endotoxin-induced acute kidney injury in rabbits

AIM: To evaluate the role of phosphatidylinositol 3-kinase/nuclear factor E2-related factor 2 (PI3K/Nrf2) signaling pathway in endotoxin-induced acute kidney injury in rabbits. METHODS: Healthy male New Zealand white rabbits were randomly divided into 5 groups: control group (group C), LPS group (group L), wortmannin+LPS group (group WL), wortmannin group (group W) and dimthyl sulfoxide (DMSO) group (group D). Wortmannin at dose of 0.6 mg/kg was injected via the auricular vein in groups W and WL, DMSO at concentration of 0.08 mL/kg was injected in group D, while normal saline (0.08 mL/kg) was injected in groups C and L. LPS at dose of 5 mg/kg was injected via the auricular vein in groups L and WL 30 min later, and equal volume of normal saline was injected in group C, D and W for control. The rabbits were sacrificed 6 h after LPS or normal saline administration. The kidneys were removed for microscopic examination and the determination of histological scores of kidney (HSK). The concentrations of blood urea nitrogen (BUN) and creatinine (Cr), urinary α1-microglobulin (α1-MG), MDA content, SOD activity, the mRNA expression of Nrf2 and HO-1, and the protein levels of total Akt, p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were also detected. RESULTS: Compared with groups C, D and W, the concentrations of BUN and Cr, urinary α1-MG concentration, MDA content and HSK were significantly increased, while SOD activity was significantly decreased (P<0.05). The mRNA expression of Nrf2 and HO-1, and the protein levels of p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were significantly increased in groups L and WL. No significant change among groups C, D and W was observed. Compared with group L, the concentrations of BUN and Cr, urinary α1-MG concentration, MDA content and HSK were significantly increased, while SOD activity, the mRNA expression of Nrf2 and HO-1, and the protein levels of p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were significantly decreased in group WL. CONCLUSION: Activation of PI3K/Nrf2 signaling pathway may be one of the regulatory mechanisms of the body adapting to the endotoxin-induced acute kidney injury in rabbits.     

Establishment of a method to quantitatively detect FLT3 internal tandem duplication in acute myeloid leukemia with denaturing high-performance liquid chromatography

AIM: To establish a relatively-quantitative method to detect the internal tandem duplication (ITD) mutation of Fms-like tyrosine kinase 3( FLT3 )gene in acute myeloid leukemia (AML) patients using denaturing high-performance liquid chromatography (DHPLC).METHODS: According to the fact that much more FLT3 -ITD mutations are located in exon 14, we designed the primers, and use the method of polymerase chain reaction (PCR) to specifically amplify FLT3 -ITD mutation gene in 121 cases of AML, and relatively quantified the situation of mutant allelic gene of FLT3- ITD by the method of DHPLC.The effectiveness of DHPLC was verified by the method of capillary electrophoresis (CE).The sequenced results from PCR amplified products of 121 samples were compared.RESULTS: A characteristic of elution peak was detected by DHPLC with 10.7% overall positive rate (13/121) and varied in the proportion of mutant alleles,with a single duplicated insert fragment from 21 bp to 87 bp.The median range of mutant alleles was 34.5% (11.4%-80.2%).No significant difference of the positive rates and mutation proportions between the results with DHPLC and the results with CE method was observed.The results of FLT3 -ITD mutant gene of 121 samples were consistent with the results using sequencing method.CONCLUSION: A relatively-quantitative method to analyze AML patients with FLT3 -ITD mutation by DHPLC is successfully established.     

Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

中国水仙R2R3-MYB基因NtMYB5的克隆和功能研究

为研究中国水仙（Narcissus tazetta var. chinensis）中类黄酮代谢途径的调控网络,从其转录组中筛选出1条R2R3-MYB基因并从花瓣cDNA中克隆其编码区全长序列,命名为NtMYB5。NtMYB5开放阅读框为681 bp,编码226个氨基酸。蛋白多重序列比对分析发现NtMYB5含有R2和R3结构域以及1个pdLNLD/ELxiG/S氨基酸基序;系统进化树分析表明,NtMYB5与花青素合成抑制因子亲缘关系最近;通过对NtMYB5在中国水仙中的表达检测发现,其表达量在花器官中较高,且随花开放逐渐上升;在烟草瞬时表达中,NtMYB5显著抑制花青素合成促进因子StMYB的效果;转NtMYB5烟草花瓣颜色变浅,qPCR检测表明NtMYB5抑制类黄酮代谢途径大部分结构基因表达。NtMYB5为中国水仙中花青素合成抑制因子。     

GA3 对宿根福禄考组培苗在北京冬季移栽成活率以及光合速率的影响

 本文对北京地区宿根福禄考组培苗冬季移栽成活率低的问题进行了研究。不同浓度的GA₃ 处理可显著提高组培苗移栽成活率并促进生长。20μmol·mol^{- 1}的GA₃ 处理后组培苗移栽成活率由68.18%提高到100%; 与对照相比较, 喷施GA₃ 的小苗在冬季两个月内株高增至约300% , 功能叶单叶面积增至约200%。各梯度GA₃ 对宿根福禄考功能叶片光合色素含量以及单位面积的叶片净光合速率影响不显著, 但显著提高了株高和冠幅, 因此移栽苗总的同化产物增加。