Tongxinluo improves anti-hypoxia ability of vascular endothelial cells via PI-3K/Akt/HIF pathway

AIM: To investigate the effects of PI-3K/Akt/HIF pathway on anti-hypoxia ability of vascular endothelial cells influenced by Tongxinluo under hypoxic condition. METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into the following groups: control group, Tongxinluo (100 mg/L) group, hypoxia group and hypoxia+Tongxinluo (100 mg/L) group. The CCK-8 assay were used to detect the viability and proliferation rate of the cells in each group. The protein levels of HIF-1α, Bcl-2, Mcl-1, Bax and phosphorylated Akt were studied by immunoblotting analysis. The HUVECs were transiently transfected with the dominant negative mutant of HIF-1α (DN-HIF). The apoptotic rates were analyzed by flow cytometry (FCM). The HUVECs were transiently transfected with the dominant negative mutant of PI-3K (Δp85) or Akt (DN-Akt) to investigate the role of PI-3K/Akt signal pathway in the anti-hypoxia ability of Tongxinluo on endothelial cells. RESULTS: Under hypoxic condition, although the proliferation rate increased significantly in Tongxinluo group compared with hypoxia group, the degree was notably weak compared with control group. The protein levels of HIF-1α, Bcl-2, Mcl-1 and phosphorylated Akt were up-regulated by Tongxinluo. Meanwhile, the expression of Bax was down-regulated. Inhibition of HIF-1α activation by DN-HIF and inhibition of the PI-3K/Akt pathway by Δp85 or DN-Akt attenuated the increase in HIF-1α expression and HUVEC viability induced by Tongxinluo. The percentage of apoptotic HUVECs was down-regulated to a certain extent by Tongxinluo. CONCLUSION: Tongxinluo improves the anti-hypoxia ability of vascular endothelial cells by up-regulating the protein level of HIF-1α, promoting the expression of anti-apoptotic factors, improving the cell viability and eventually reducing the apoptotic rate.These effects of Tongxinluo depend on PI-3K/Akt signal pathway.     

Effects of lovastatin on differentially expressed genes in HepG2 cells

AIM: To analyze the lovastatin-induced differential gene expression in HepG2 cells using a cDNA microarray assay. METHODS: Total RNA was extracted from the lovastatin-treated HepG2 cells and control group. cDNA was synthesized from RNA with Cy3/Cy5-labelled dCTP. Then the hybridization was conducted. The result was analyzed using Imagene and Genespring software. RT-PCR was carried to confirm the hybridization results. RESULTS: 30 genes were up-regulated while 11 genes were down-regulated in lovastatin-treated HepG2 cells, involved in some major functional areas including signal transduction, cell cycle regulation, tumor immunity, and so on. CONCLUSION: The analysis of differentially expressed genes in lovastatin-treated HepG2 cells is helpful to explore the mechanism of the anti-tumor activity of statins.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Effect of NP9 on gene expression profile of NPC cell line CNE1

AIM: To identify the gene expression profiles of CNE1 cells stably transfected with NP9 expressing plasmid, and to explore the potential molecular function of NP9 gene.METHODS: CNE1 cells stably expressing NP9 protein and CNE1 cells transfected with empty vector were used as test and control. Differentially expressed genes were screened with high- throughout human genome array. Differential expression of 6 genes was analyzed with quantitative RT-PCR. RESULTS: Of all the 14 500 human genes in array, 266 genes were revealed differential expression between test and control , of which 82 genes (RA>1)were up-regulated in test and 184 genes (RA<1) were down-regulated. 34 genes and 75 genes were found distinctively up-regulation (RA>1.5) and down-regulation (RA<1.5),respectively.CONCLUSION: NP9 expression in CNE1 cells leads to changes of some genes involved in regulation of cell cycle, cell proliferation and differentiation, cell signal transduction, cell adhesion. Some new clues may be provided for further studying the potential function and molecular mechanism of NP9 gene.     

Preliminary comparison of inflammatory differential gene expression during atherosclerosis in ApoE-/- mice

AIM: To screen the expression of inflammatory genes associated with atherosclerosis (AS) in different weeks of ApoE^-/- mice using Agilent gene expression profile chip (AGEPC). METHODS: Male ApoE^-/- mice (n=60) were randomly divided into 3 groups:initial phase of AS (10 weeks old), early phase of AS (15 weeks old), and late phase of AS (25 weeks old). Homologous wild-type C57BL/6J mice were used for the control. The RNA samples of the arcus aortae from these mice were isolated. Total RNA from each sample was labeled with Cy3 and hybridized with AGEPC, and microarray detection was conducted. After washing, scaning, acquiring data, and standardized analysis, the expressed genes with default threshold of statistical significance of P≤0.05 and fold change ≥ 2.0 were selected. The expression of these genes were further verified by RT-qPCR. RESULTS: Compared with the control group, there were 895 differential genes in 10 weeks of ApoE^-/- mice, while 540 genes in 15 weeks, and 591 genes in 25 weeks, respectively. KEGG pathway and gene ontology (GO) analyses revealed that those diversely expressed genes related to inflammation were particularly arresting. Several selected genes including interleukin-12a (IL-12a), matrix metallopeptidase-12 (MMP-12), IL-1β, growth differentiation factor-15 (GDF-15) and interferon-γ (IFN-γ) were validated by RT-qPCR. Compared with the control group, the expression levels of IL-12a and MMP-12 were up-regulated while IL-1β was down-regulated in 10 weeks, the expression level of GDF-15 was up-regulated while the IL-12a and IL-1β levels were down-regulated in 15 weeks, and the levels of IL-12a, MMP-12 and GDF-15 were up-regulated in 25 weeks (P<0.05). Moreover, the increased level of IL-12a in 10 weeks, decreased level of IL-1β in 15 weeks, and increased levels of MMP-12 and GDF-15 in 25 weeks were even more statistically significant (P<0.01). CONCLUSION: The changes of inflammatory gene expression in different phases of AS suggest an important direction for medical intervention of AS.     

Gene microarray analysis of ovarian serous cystadenocarcinoma-related genes based on TCGA

AIM: To detect the differentially expressed genes associated with ovarian serous cystadenocarcinoma (OV) by microarray and to analyze the participated signaling pathway. METHODS: We analyzed 16 datasets of Affymetrix GeneChip Human Exon 1.0 ST Arrays from The Cancer Genome Atlas (TCGA), including 8 OV and 8 normal ovary samples. The function of differential genes was determined by pathway and gene ontology (GO) analysis. The probable functions of the key genes were predicted according to intergenic signal transduction network. RESULTS: The 1 144 genes were identified as distinctively expressed in OV (P<0.05), 747 of which were up-regulated and 397 were down-regulated. The GO analysis results showed that the altered genes were involved in 362 up-regulated and 160 down-regulated significant functions (P<0.05) related to cell cycle, DNA replication, cell proliferation, cell apoptosis, cell adhesion, etc. The pathways of the different genes were involved in the 59 enrichment-related pathways (P<0.05), 45 of which were up-regulated and 14 were down-regulated. Among the 59 pathways, cell cycle, P53 signaling pathway, DNA replication, pathways in cancer, PI3K-Akt signaling pathway, ECM-receptor signaling pathway, cell adhesion molecules and cell apoptosis were related to tumor genesis, development and metastasis. As a result, 229 genes with significant functions and pathways in GO and pathway analysis were selected to construct signal transduction network (Signal-Net), 4 of which, CDK1, PLK1, MCM3 and PGK1, were found to play key roles in OV signal regulation network. CONCLUSION: The OV shows abundant differentially expressed genes that play key roles in cancer-related signal pathways.     

Effects of miR-30c on viability and migratory ability of HUVECs by targeting PAI-1

AIM: To investigate the effect of microRNA (miR)-30c on the viability and migratory ability of human umbilical vein endothelial cells (HUVECs) by targeting plasminogen activator inhibitor-1 (PAI-1).METHODS: The HUVECs were transfected with miR-30c mimic and inhibitor or negative control (NC), and then the expression levels of miR-30c, PAI-1 mRNA and protein were detected by RT-qPCR and Western blot. The viability and migratory ability of HUVECs were measured by CCK-8 assay and wound healing test. After bioinformatic analysis, the assessment of miR-30c binding to PAI-1 3'-UTR was carried out using a luciferase reporter gene assay. RESULTS: miR-30c directly down-regulated PAI-1 levels by binding to the 3' UTR seed sequence of PAI-1 mRNA. Furthermore, transfection of a miR-30c mimic down-regulated the expression of PAI-1 at mRNA and protein levels, leading to enhanced migratory ability and viability of the HUVECs. However, transfection of a miR-30c inhibitor up-regulated the expression of PAI-1 at mRNA and protein le-vels, leading to decreased migratory ability and viability. CONCLUSION: Regulation of miR-30c level changes the migratory ability and viability of HUVECs by affecting the PAI-1 expression, indicating the involvement of miR-30c in modulating endothelial function.     

Gene expression pattern of periphery blood mononuclear cells in patients with active lupus nephritis

AIM: To find new gene function associate with active lupus nephritis (LN) through study on the difference in gene expression of peripheral blood mononuclear cells between LN patients and healthy controls by gene chip. METHODS: The CSC-GE-80 chip containing 8 000 spots of cDNAs were used to investigate the difference of the expression. Both the total RNA from peripheral blood mononuclear cells of active LN patients and healthy donors were reversely transcribed to cDNA with the incorporation of fluorescent( cy3 and cy5) labeled dCTP to prepare the hybridization probes. After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. We repeated that in three groups of LN patients and healthy controls, respectively, and only the genes that have differential expression in all three chips were considered associated with LN. RESULTS: 75 genes were identified to be differently expressed in all three groups of LN patients as compared with healthy controls, including 42 up-regulated genes and 33 down-regulated ones. CONCLUSION: The present study represents a global view of gene expression of LN and provides important clues for further study of LN related genes. And it also suggests defensin α₁, S100A8, S100A9 may be involved in the pathogenesis of LN.     

Autophagy plays a protective role in advanced glycation end product-induced apoptosis of vascular endothelial cells

AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Changes and significance of Fas and cFLIP in apoptosis of human rhabdomyosarcoma cells induced by combination of TRAIL and cisplatin

AIM: To investigate the synergistic induction of apoptosis in rhabdomyosarcoma cells by the combination of TRAIL or TRAIL gene with cisplatin. METHODS: Rhabdomyosarcoma cells were treated with TRAIL, Ad／GT-TRAIL, cisplatin, respectively or the combination for 3 days. The cytotoxicity was observed by MTT assay. The apoptotic rates and the expression rates of Fas protein were measured by flow cytometry (FCM). The expression of cFLIP mRNA was determined by RT-PCR. RESULTS: Rhabdomyosarcoma cells were treated with Ad／ GT-TRAIL and TRAIL (100.0 μg/L), the cytotoxicity index were 52.5% and 43.5%, the percentage of apoptotic cells were 12.95% and 10.26%, respectively. Combined with cisplatin, the cytotoxicity index and the percentage of apoptotic cells were increased significantly (P<0.05). The expression of Fas protein in rhabdomyosarcoma cells was up-regulated and the expression of cFLIP was down-regulated with cisplatin, which were paralleled by the apoptotic rates. CONCLUSION: Combinatiion of Ad／GT-TRAIL or TRAIL and cisplatin has synergistic apoptosis-inducing effects on rhabdomyosacoma cells.     

Effect of miRNAs on apoptotic leukemia cells induced by demethylcantharidin

AIM: To explore the role of miRNA expression in the process of antitumor drug demethylcantharidin (DMC)-induced leukemia cell apoptosis. METHODS: K562 cells were treated with DMC to induce apoptosis. Microarray assessment was performed to detect the changes of miRNAs. The expression of miRNAs was further detected by RT-PCR and real-time PCR. The miRNA-relative genes were analyzed by gene information softwares,and their expression was determined by ELISA analysis. RESULTS: The results of microarray showed that more than 290 miRNAs were differentially expressed after DMC treatment. The expression of miR-16, miR-34 and miR-125 increased at more than 1.5 folds in DMC treatment group determined by RT-PCR and real-time PCR analysis, while both miR-106 and miR-150 were down-expressed over 60％. Using microRNA TargetScan and miRanda analysis software, we found that the expression of oncogenes ( bcl-2, E2F1, E2F3 ) and tumor suppressor genes ( RB1, p53 ) may be regulated by the above miRNAs. The expression of RB1 and P53 proteins significantly increased, while Bcl-2, E2F1 and E2F3 proteins were obviously down-regulated after DMC treatment.CONCLUSION: DMC induces K562 cell apoptosis by regulating the expression of miRNAs and their relative genes.     

Microarray analysis of tumor metastasis-related gene expression in PSMA-silencing prostate cancer cells

AIM: To investigate the functions of prostate-specific membrane antigen (PSMA) in prostate cancer metastasis. METHODS: Specific siRNA to knock down PSMA expression was designed and transfected into LNCaP cells. The tumor metastasis gene chip was also used to analyze the differential expression of 84 genes related to cancer metastasis. RESULTS: Specific siRNA was successfully designed and constructed and the gene expression of PSMA in LNCaP cells was knocked down. The RNAi efficiency was more than 75% at mRNA level and more than 68% at protein level. The results of the tumor metastasis gene chip indicated that 10 genes were up-regulated (such as CDH6 and CXCL12) and 4 genes were down-regulated (such as CCL7 and MDM2) in the LNCaP cells treated with PSMA siRNA. CONCLUSION: The PSMA is involved in the regulatory pathways in prostate cancer metastasis.     

Influence of ovariectomy and estrogen replacement on gene expression profile of myocardium in rats

AIM:To investigate the influence of ovariectomy and estrogen replacement treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham (Ⅰ),ovariectomy (Ⅱ,OVX) and estrogen replacement treatment (Ⅲ,OVX+E2) group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters (18),cell receptors (9),intracellular transducers/effectors/modulator (7) and metabolism (6).Most of the genes (45) were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/ modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.