Effect of Egr-1 gene transfection on renal inflammation in diabetic mice

AIM: To observe the effects of Egr-1 gene transfection on the expression of tumor necrosis factor-α(TNF-α) and intercellular adhesion molecule-1(ICAM-1), and to investigate the role of Egr-1 in the pathogenesis of diabetic nephropathy.METHODS: The diabetic mouse model was established. Ten mice were randomly selected as the diabetic group. The remaining 40 mice were injected with empty plasmid, Egr-1 expression plasmid or Egr-1 siRNA plasmid via the tail vein once a week. The normal control group was also set up. The animals were sacrificed at the end of the 4th week. The renal tissues were harvested. The expressions of Egr-1, TNF-α and ICAM-1 were detected by immunohistochemistry and Western blot. The pathological changes were observed under electron microscope.RESULTS: In diabetic mouse kidney, the expression of Egr-1, TNF-α and ICAM-1 was increased, and irregular thickening of glomerular basement membrane, mesangial expansion and fusion of foot were observed. The change trend was more significant in Egr-1 gene transfection group, and these changes in siRNA plasmid transfection group were obviously reduced compared with diabetes group.CONCLUSION: Egr-1 up-regulates the expression of TNF-α and ICAM-1, and induces mesangial cell proliferation and mesangial extracellular matrix accumulation, which is probably one of the mechanisms of accelerating glomerulosclerosis.     

Reduction of inflammatory-related factor expression in experimental acute pancreatitis in Egr-1 knockout mice

AIM: To observe the effects of Egr-1 gene knockout on the expression of inflammatory-related factors in pancreatic tissue in a mouse acute pancreatitis model. METHODS: The experimental pancreatitis was induced by high-dose of cearulein in wildtype mice and Egr-1 knockout mice. The pancreatitis indexes, such as serum amylase, pancreata edema, and myeloperoxidase(MPO) levels in pancreata and lungs were recorded. The mRNA levels of tissue factor(TF), plasminogen activator inhibitor(PAI-1), monocyte chemoattractant protein(MCP-1), Gro-1, IL-6 and ICAM-1 were measured by quantitative PCR. RESULTS: Contrary to wildtype mice, typical pancreatitis was not induced by high-dose cearulein in the Egr-1 knockout mice, not only markedly reduced edema in pancreata and lungs, but decreased MPO levels in lungs as well were found. Furthermore, the mRNA of TF, PAI, MCAP, ICAM-1 and IL-6 in pancreata were significantly decreased in Egr-1 knockout mice. CONCLUSION: The severity of pancreatitis and lung damage is ameliorated in Egr-1 knockout mice stimulated by high-dosage of cearulein, which was probably mediated by decreasing expression of inflammatory-related factors in pancreata, such as TF, PAI, MCP-1, ICAM-1 and IL-6.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

Radiation-induced expression of IL-18 and B7.1 genes in transfected B16 cells and antitumor effect of Egr-IL18-B7.1 in vivo

AIM: Plasmid Egr-IL18-B7.1 was constructed to explore its expression characteristics induced by different doses of radiation and its suppressive effect on melanoma under radiotherapy. METHODS: The plasmid containing both IL-18 and B7.1 genes downstream of Egr-1 was constructed using gene recombination technique. The in vitro expression of IL-18 and B7.1 induced by ionizing radiation was measured with ELISA and flow cytometry, respectively. The effect of gene radiotherapy on malignant melanoma was assayed by observing the growth rate of B16 cells implanted into C57BL/6J mice. RESULTS: Effective expression of IL-18 and B7.1 by plasmid Egr-IL18-B7.1 treated with different doses of X-irradiation were observed and in vivo experiments showed significant inhibition of tumor growth after combined gene-radiotherapy. CONCLUSION: Data presented in this paper implicate that gene radiotherapy with plasmid containing double genes might be one of the effective anticancer therapeutic measures.     

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells

AIM and METHODS:To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION:The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression.     

NET-1 promotes metastasis of lung squamous-cell carcinoma by activating EMT

AIM: To explore the effect of neuroepithelial cell transforming gene-1 (NET-1) expression on the metastasis of lung squamous-cell carcinoma (LSC) and the underlying molecular mechanism. METHODS: Immunohistochemistry was used to detect the expression of NET-1 protein in 53 cases of lung squamous-cell carcinoma (LSC group), 24 cases of normal lung epithelium (NLE group) and 27 cases of lung squamous intraepithelial lesions (SIL group). The correlation of clinical and pathological factors was analyzed. The protein expression of NET-1 in human lung squamous-cell carcinoma cell lines H226, H1703, H2170, SK-MES-1, H520 and YTMLC-90 was determined by Western blot. The RNA interference recombinant adenovirus against NET-1 gene (Ad-NET-siRNA) and Ad-control with control sequence were constructed and infected with human lung squamous cell carcinoma cell YTMLC-90 to silence the expression of NET-1 gene. The protein expression of NET-1, E-cadherin, vimentin and Snail1 in the BEAS-2B cells and the YTMLC-90 cells was determined by Western blot. The mRNA expression of E-cadherin and vimentin in each group of the cells was detected by qPCR. The invasive ability of the cells in each group was detected by Transwell chamber assay. RESULTS: The positive expression rate of NET-1 in LSC group was significantly higher than that in NLE group and SIL group(P<0.05). The distribution of NET-1 protein positive expression population was correlated with histological grade, lymph node metastasis, and TNM stage. The NET-1 expression rate of LSC with lymph node metastasis was significantly higher than that without lymph node metastasis. Over-expression of NET-1 protein in YTMLC-90 cells was observed. The expression of E-cadherin was decreased, and the protein expression of vimentin and Snail1 was increased in YTMLC-90 cells. Knock-down of NET-1 expression increased the expression of E-cadherin, and decreased the expression of vimentin and Snail1 in the YTMLC-90 cells. CONCLUSION: The expression of NET-1 promotes the lymphatic metastasis of lung squamous-cell carcinoma. This promotion may be achieved through the activation of epithelial-mesenchymal transition (EMT) by NET-1 expression.     

The expression and significance of Apaf-1 in papillary thyroid carcinoma

AIM: To investigate the mRNA and protein expression of apoptotic protease-activating factor 1 (Apaf-1) in papillary thyroid carcinoma (PTC) and its relationship with cell proliferation.METHODS: The methods of real-time PCR, Western blot and immunohistochemistry were used to detect the mRNA and protein expression of Apaf-1 in PTC and tumor-adjacent tissues. The relationship between Apaf-1 expression and clinicopathological characteristics was analyzed. The effect of Apaf-1 on cell proliferation was verified by down-regulating the expression of Apaf-1 in CGTHW-3 cells.RESULTS: The mRNA and protein expression levels of Apaf-1 in the PTC tissue were significantly lower than those in the tumor-adjacent tissue (P<0.05). Down-regulation of Apaf-1 expression enhanced the proliferation of CGTHW-3 cells (P<0.05).CONCLUSION: The expression of Apaf-1 is low in PTC, and inhibition of its expression enhances the proliferation of CGTHW-3 cells. Apaf-1 may play a tumor suppressor role in PTC.     

Long noncoding RNA ZFAS1/miR-150/ROCK1 axis regulates proliferation and migration of vascular smooth muscle cells

AIM: To investigate whether long noncoding RNA ZNFX1 (zinc finger NFX1-type containing 1) antisense RNA 1 (ZFAS1) promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) by regulating microRNA-150 (miR-150)/ROCK1, and the involving mechanism of atherosclerosis. METHODS: Platelet-derived growth factor-BB (PDGF-BB) was used to induce proliferation and migration of VSMCs. Real-time PCR was used to detect the content of ZFAS1 in the VSMCs. After further down-regulating the expression of ZFAS1 by siRNA, the viability of VSMCs was detected by MTT assay, and the proliferation was measured by EdU staining. The migration ability of VSMCs was detected by Transwell method. The expression levels of miR-150 and ROCK1 were detected by RT-qPCR, and the protein level of ROCK1 was determined by Western blot. Luciferase reporter assay was used to confirm that ROCK1 was the target gene of miR-150. Finally, miR-150 expression was inhibited, and the proliferation and migration ability of VSMCs and expression of ROCK1 after down-regulation of ZFAS1 expression were examined. RESULTS: PDGF-BB up-regulated the expression of ZFAS1 in the VSMCs. After down-regulating the expression of ZFAS1, the proliferation and migration abilities of VSMCs were inhibited (P<0.05), the expression level of miR-150 was increased (P<0.05), and the expression level of ROCK1 was decreased (P<0.05). The results of luciferase reporter assay showed that miR-150 directly targeted ROCK1. Inhibition of miR-150 expression attenuated the inhibition of proliferation and migration of VSMCs by ZFAS1 expression knock-down (P<0.05) and up-regulated the expression level of ROCK1 (P<0.05). CONCLUSION: ZFAS1 promotes the proliferation and migration of VSMCs induced by PDGF-BB by regulating miR-150/ROCK1.     

Silencing of TKTL1 gene by siRNA down-regulates HIF-1α expression and activity of glycolytic key enzymes in human cervical cancer cells

AIM：To explore the relationship between the expression of transketolase-like gene 1 (TKTL1) and glycolysis metabolism in human cervical cells. METHODS：The changes of hypoxia-inducible factor 1α (HIF-1α) expression and the activity of glycolytic key enzymes, hexokinase Ⅱ (HK-Ⅱ) and lactate dehydrogenase (LDH), under hypoxia in human cervical cell line HeLa were observed after TKTL1 was knockdown by siRNA. The specific siRNA expression vector targeting TKTL1 gene was constructed, and the recombinant plasmid was transfected into HeLa cells. The effects of TKTL1 silencing were evaluated by detecting transketolase (TKT) activity and TKTL1 mRNA expression using RT-PCR. The changes of HIF-1α expression and HK-Ⅱ expression, and HK-Ⅱ and LDH activity were also observed in transfected HeLa cells. RESULTS：The mRNA expression of TKTL1 and the activity of TKT decreased significantly (P<0.01) after TKTL1 silencing. Meanwhile, all HIF-1α expression, HK-Ⅱexpression, and HK-Ⅱ and LDH activity decreased significantly compared with the untransfected cells (P<0.01). CONCLUSION：Silencing of TKTL1 gene in human cervical cancer cells by siRNA down-regulates HIF-1α expression and the activity of glycolytic key enzymes, thus changing the malignant phenotype of carcinoma cells.     

Effects of Ku70 on protein expression of HTLV-1 in HTLV-1 positive T cells

AIM:To explore the effects of Ku70 on the protein expression of human T-lymphotrophic virus 1 (HTLV-1) in HTLV-1 positive T cells. METHODS:The expression level of Ku70 in HTLV-1 positive T cells was exa-mined by Western blot. The siRNA targeting Ku70 was constructed and the effect of the siRNA on knockdown of Ku70 expression was determined by Western blot. After knockdown of Ku70 expression in the HTLV-1 positive T cells by siRNA, the expression of HTLV-1-related proteins at mRNA and protein levels was examined by real-time PCR and Western blot, and the expression levels of interferons and proinflammatory cytokines were examined by real-time PCR. RESULTS:The HTLV-1 positive T cells, including MT2, MT4 and C8199 cells, displayed a higher expression level of Ku70. The protein expression of HTLV-1 was increased in Ku70-silencing MT2 cells and MT4 cells. After knockdown of Ku70 expression in the MT2 cells and MT4 cells, the production of interferon (IFN)-α, IFN-γ and tumor necrosis factor-α was reduced.CONCLUSION:The HTLV-1 positive T cells have a higher expression level of Ku70. In HTLV-1 positive T cells, Ku70 promotes the production of interferons and proinflammatory cytokines and inhibits HTLV-1-related protein expression.     

Knock-down of NEDD1 expression inhibits lung adenocarcinoma cell proliferation and migration

AIM: To explore the role of neural precursor cell expression developmentally down-regulated protein 1 (NEDD1) in the development and progression of lung cancer. METHODS: The differences of NEDD1 expression levels between lung cancer tissues and tumor-adjacent tissues were analyzed by the method of immunohistochemistry and TCGA database. Kaplan-Meier curve was used to analyze the correlation between lung cancer prognosis and the expression level of NEDD1. The proliferation of A549 cells was tested by plate colony formation experiment after knock-down of NEDD1 expression. The apoptosis rate and cell cycle distribution were examined by flow cytometry. The migration ability of the A549 cells was detected by Transwell assay. The protein levels of cell cycle-related molecules were determined by Western blot. Database analysis was performed to evaluate the relationship between the expression of NEDD1 and cyclin-dependent kinases 2 (CDK2). RESULTS: Compared with the tumor-adjacent tissues, the expression level of NEDD1 in the lung cancer tissues was increased, so as the database analysis, and the higher expression of NEDD1 showed a poorer prognosis. Under light microscope, the A549 cells showed a low proliferation rate after silencing the NEDD1 expression, and the colony formation ability of the cells was also reduced; knock-down of NEDD1 expression induced the apoptosis and inhibited the cell migration; knock-down of NEDD1 expression blocked the cells in G₁/S phase, and the protein levels of p-Rb and cyclinD1 were decreased, while the protein levels of p-Chk1, p-Chk2 and p-p53 were increased (P<0.05). A positive correlation between the expression of NEDD1 and CDK2 was noted by database analysis. CONCLUSION: NEDD1 plays an crucial role in promoting cell proliferation via inhibiting apoptosis and accelerating cell cycle, high expression of NEDD1 in lung adenocarcinoma tissue is related to poor prognosis, thus NEDD1 may be used as a candidate marker molecule for the diagnosis and prognosis of lung cancer.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

Relationship between phosphoglycerate kinase 1 and clinical features of prostate cancer and its role in prognosis

AIM: To study the expression and prognostic functions of phosphoglycerate kinase 1 (PGK1) in prostate cancer. METHODS: The prostatic samples were collected from the patients with prostate cancer and benign prostatic hyperplasia (BPH) in TCM-Integrated Hospital of Southern Medical University from Jan 2013 to Dec 2013. The protein expression of PGK1 in the prostate specimens was detected by immunohistochemical analysis and Western blot. Furthermore, the correlations of PGK1 expression with the clinicopathological features and prognosis of prostate cancer were also evaluate. RESULTS: The expression of PGK1 in the prostate specimens was significantly up-regulated compared with the BPH individuals. In addition, the expression of PGK1 was significantly correlated with the local infiltration, Gleason score, TNM grade, bone metastasis, and serum prostate-specific antigen (PSA) concentration. Finally, bone metastasis, serum PSA level and PGK1 expression were independent risk factors for prostate cancer illustrated by Cox analysis, and high expression of PGK1 was correlated with poor prognosis. CONCLUSION: PGK1 expression is an independent risk factor for prostate cancer, and it might act as a prognostic biomarker for prostate cancer.     

Over-expression of SATB1 mediates invasion and metastasis of nasopharyngeal carcinoma cells through epithelial-mesenchymal transition

AIM:To analyze the high expression of special AT-rich sequence-binding protein 1 (SATB1) in nasopharyngeal carcinoma (NPC) and its role in tumor invasion and metastasis. METHODS:The method of immunohistochemistry was used to detect the expression of SATB1 and epithelial-mesenchymal transition (EMT)-related molecules E-cadherin and vimentin in 76 cases of NPC and 61 cases of nasopharyngeal chronic inflammation (NPI), and the correlations of over-expression of SATB1 with NPC patients' clinical parameters as well as the expression of E-cadherin and vi-mentin were analyzed. Variously differentiated NPC cell lines CNE1, CNE2Z and C666-1 were cultured in vitro, and then SATB1-overexpressing cell line was screened. After interfering with SATB1 expression by siRNA, the expression of EMT-related molecules and the change of cell invasiveness were analyzed. RESULTS:The expression of SATB1 in the nasopharyngeal tissue was dominantly localized in the nuclei. The positive rate of SATB1 in NPC group was significantly higher than that in NPI group (P<0.01). E-cadherin was membrane-positive in NPI epithelial cells, while membrane E-cadherin in NPC was decreased but cytoplasmic expression was increased. The positive expression rate of membrane E-cadherin in NPI was significantly higher than that of NPC (P<0.01). Vimentin was localized in cytoplasm and negative in NPI epithelial cells, but the positive rate in NPC parenchymal cells was significant higher than that in NPI (P<0.01). The high expression of SATB1 in NPC was not related to the patents' sex, age, clinical classification and N classification, but positively correlated with T and M classification (P<0.05). Besides, high expression of SATB1 was positively correlated with vi-mentin in NPC tissues (r=0.358, P=0.009). SATB1 expression in NPC cell lines was negatively correlated with the levels of cell differentiation. Knockdown of SATB1 expression in C666-1 cells with siRNA was accompanied by an increase in E-cadherin and a decrease in vimentin levels, as well as a decrease in cell invasiveness. CONCLUSION:High expression of SATB1 promotes the clinical progress of NPC through EMT mechanism.     

Expression of telomerase inhibitor Pinx1 in leukemia cells and its correlation with telomerase activity

AIM:To study the expression of telomerase inhibitor Pinx1 in acute leukemia cells and during the differentiation of acute promyelocytic leukemia cells,and to realize its effect on telomerase activity.METHODS:Realtime quantitative PCR with fluorescence probe hybridization was used to measure the expression of Pinx1 and hTERT mRNA in acute leukemia cells and during differentiation of NB4 cells induced by ATRA.The correlations between Pinx1 and hTERT expression were also analyzed.RESULTS:Pinx1 mRNA expression in acute leukemia samples (0.00312,5.42×10-4-0.024) was significantly higher than that in normal bone marrow mononuclear cells (7.89×10-4,0-0.00863,P<0.01).The expression of Pinx1 mRNA had significant positive correlation with hTERT mRNA expression (r=0.296,P<0.05).Pinx1 mRNA expression decreased during differentiation,its expression was positive correlated with hTERT mRNA expression (r=0.900,P<0.05).CONCLUSIONS:As an inhibitor of telomerase,however,Pinx1 also had the same direction of regulation with telomerase activity in acute leukemia cells,suggesting its expression variation may be a subsequent reaction induced by that of hTERT to stabilize telomerase activity.The exact mechanisms remained to be verified.     

Caveolin-1 expression is downregulated by shear stress and TNF-α in human aortic endothelial cells

AIM: To observe effects of shear stress and TNF-α on caveolin-1 expression. METHODS: Cultured human aortic endothelial cells (HAECs) of passage 3-5 were used in the experiment. Cells were exposed to a laminar flow (shear stress 1.0 Pa) by using a parallel rectangular flow chamber for different time. Caveolin-1 mRNA and protein expression were measured by RT-PCR and Western blot, respectively. Caveolin-1 expression of the cells stimulated by TNF-α were also studied to elucidate the influence of this inflammatory factor. RESULTS: After 24 h of exposure to 1.0 Pa shear stress, both of caveolin-1 protein and mRNA expression decreased in HAECs, especially caveolin-1 mRNA expression (P<0.05). No significant decrease in caveolin-1 protein expression was found after 4 h exposure to the shear flow, although there was a decrease in caveolin-1 mRNA expression (P<0.05). TNF-α induced decreases in caveolin-1 protein and mRNA expression in the cells stimulated for 24 h (P<0.05). CONCLUSION: Laminar flow of 1.0 Pa and TNF-α induce decreases in caveolin-1 protein and mRNA expression, which could play an important role in atherogenesis.     

Effect of Epstein-Barr virus latent membrane protein 1 on oncomiRs expression profile in nasopharyngeal carcinoma cell line CNE1

AIM: To investigate the differential expression profile between nasopharyngeal carcinoma cell line CNE1 and its steady EBV-LMP1-transfected cell line CNE1-LMP1, and to explore the regulatory effect of LMP1 on oncomiRs expression in CNE1 cell line. METHODS: A microRNA array that targets 132 of the most well studied oncomiRs was used to detect the expression profile of CNE1 and CNE1-LMP1. qRT-PCR assay were used to verify the expression data detected by microarray. RESULTS: Among the restricted 132 miRNAs, 30 were detectable. Among which, 30 were expressed in CNE1-LMP1, 19 in CNE1 and 11 were specifically expressed in CNE1-LMP1. Among the 19 shared miRNAs, the expression level of 6 miRNAs (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150 and hsa-miR-188) elevated over two folds in CNE1-LMP1. No decrease in miRNA expression more than two folds was observed. qRT-PCR confirmed the expression difference of these six miRNAs (P<0.01). Among the 11 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a showed the highest expression level surpassing the internal control sample. CONCLUSION: Our data suggest that LMP1 may play an important role in regulating the expression of miRNAs in tumor, which may be another important pathway employed by LMP1 in the development of nasopharyngeal carcinoma.     

香蕉WAT1基因的鉴定及表达分析

【目的】基于香蕉基因组数据筛选Walls are thin 1(WAT1)基因,分析它们的序列及表达特性。【方法】以拟南芥WAT1为参考序列,通过本地Blast筛选获得香蕉WAT1基因,分析其核苷酸、启动子及编码蛋白特性,并利用实时定量PCR技术研究其在不同组织部位、不同激素和逆境胁迫处理下的表达情况。【结果】筛选获得5个香蕉WAT1基因(命名为MaWAT1-1~5)。蛋白亚细胞定位预测结果显示,MaWAT1-1、MaWAT1-2、MaWAT1-4主要定位在液泡和细胞膜上,MaWAT1-3主要定位在细胞质和细胞膜,MaWAT1-5定位在细胞膜和叶绿体。基因结构分析和系统进化树分析均将MaWAT1s分为两组,MaWAT1-1、MaWAT1-2、MaWAT1-4聚为一组(含6个外显子和5个内含子),MaWAT1-3和MaWAT1-5归为一组(含7个外显子和6个内含子)。启动子顺式作用元件分析结果显示:MaWAT1s启动子含有大量激素和胁迫响应相关元件。实时定量PCR结果显示,MaWAT1-4在叶片中表达量最高,MaWAT1-1在根和假茎中表达量最高,其余均在根中表达量最高;大多数MaWAT1s的表达受GA3、SA、盐胁迫和干旱等显著诱导,受高温显著抑制,同时部分成员的表达受IAA、ABA、JA、低温、机械损伤和枯萎病影响显著。【结论】MaWAT1s的表达受多种激素和逆境影响显著,可能在香蕉生长发育和抗逆防御反应中发挥着重要作用。     

Expression and prognostic significance of cyclin D1, RBL2/p130 and MCM7 in hepatocellular carcinoma

AIM: To investigate the expression and prognostic significance of cyclin D1, retinoblastoma-like protein 2(RBL2/p130) and minichromosome maintenance protein 7(MCM7) in hepatocellular carcinoma(HCC). METHODS: The expression of cyclin D1, RBL2/p130 and MCM7 in 44 HCC specimens, 26 adjacent noncancerous cirrhotic liver specimens and 18 normal liver specimens were detected by the method of immunohistochemistry. The correlations of cyclin D1, RBL2/p130 and MCM7 with the clinical parameters of HCC patients were analyzed. RESULTS: The positive expression rates of cyclin D1 and MCM7 in HCC were 68.2% and 72.7%, higher than those in normal livers and adjacent noncancerous cirrhotic livers(P<0.01). The positive expression rate of RBL2/p130 in HCC was 34.1%, lower than that in normal livers and adjacent noncancerous cirrhotic livers(P<0.01). The expression of MCM7 in HCC was positively correlated with the expression of cyclin D1(r=0.349, P<0.05), and negatively correlated with the expression of RBL2/p130(r=-0.421, P<0.01). The expression of cyclin D1 in HCC was negatively correlated with the expression of RBL2/p130(r=-0.435, P<0.01). The expression of MCM7 and cyclin D1 was associated with the integrity of tumor capsule, differentiation degree and TNM stage(P<0.05). The expression of MCM7 was also associated with tumor size and the value of alpha fetoprotein(AFP). The tumor size, the expression levels of MCM7 and cyclin D1 were correlated with the prognosis determined by multivariable analysis. The patients with positive expression of MCM7 and cyclin D1 had lower survival rate than those with negative expression(P<0.05). The patients with positive expression of RBL2/p130 had higher survival rate than those with negative expression(P<0.05). CONCLUSION: The abnormal expression of cyclin D1, RBL2/ p130 and MCM7 plays an important role in the development of HCC, indicating that monitoring their expression in HCC patients may be helpful to the judgment of prognosis.