Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527.

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in ß-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.

Re-sensitization of 5-FU resistance by SPARC through negative regulation of glucose metabolism in hepatocellular carcinoma.

Secreted protein, acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. Currently, there is growing evidence for important functions of SPARC in a variety of cancers and its role in cancer depends on tumor types. In this study, we reported SPARC negatively regulated glucose metabolism in hepatocellular carcinoma (HCC). Overexpression of SPARC inhibited glucose uptake and lactate product through downregulation of key enzymes of glucose metabolism. On the other hand, knock down of SPARC reversed the phenotypes. Meanwhile, exogenous expression of SPARC in HepG2 cells resulted in tolerance to low glucose and was correlated with AMPK pathway. Interestingly, the 5-fluorouracil (5-FU)-resistant HepG2 cells showed increased glucose metabolism and downregulated SPARC levels. Finally, we reported the overexpression of SPARC re-sensitize 5-FU-resistant cells to 5-FU through inhibition of glycolysis both in vitro and in vivo. Our study proposed a novel function of SPARC in the regulation of glucose metabolism in hepatocellular carcinoma and will facilitate the development of therapeutic strategies for the treatments of liver tumor patients.

HDAC6 promotes hepatocellular carcinoma progression by inhibiting P53 transcriptional activity.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. HDAC6 is a transcriptional regulator of the histone deacetylase family, subfamily 2. Previous studies have shown that HDAC6 plays critical roles in transcription regulation, cell cycle progression and developmental events. However, its biological roles in the development of HCC remain largely unexplored. In the present study, we found that mRNA and protein levels of HDAC6 were up-regulated in HCC tissues and cell lines. The proinflammatory cytokines, which were up-regulated in the human HCC microenvironment, increased HDAC6 expression through a proximal NF-kappaB binding site on the HDAC6 gene promoter. Furthermore, overexpression of HDAC6 could promote cell proliferation in HCC cell lines. In contrast, HDAC6 knockdown using small interfering RNA inhibited cell proliferation. At the molecular level, we demonstrated that HDAC6 could interact with p53 and attenuate its transcriptional activity through promotion of its degradation. Therefore, our results suggest a previously unknown HDAC6-p53 molecular network controlling HCC development.

[Transcriptional regulation of genes involved in liver-selective cell communication].

OBJECTIVE: To explore the mechanism of transcription regulation of the liver-selective genes responsible for cell communication. METHODS: Tissue-selective Affymetrix probe sets (3919 probes in total) were clustered by functional categories. Liver-selective cell communication (LSCC) genes were selected for further analysis. The 500-bp upstream sequences of all the LSCC genes were extracted for predicting the transcription factor binding sites (TFBS) of known transcription factors (TFs) using 3 programs; literature mining was then performed for these LSCC genes and TFs, and the transcription regulatory network were constructed. RESULTS: The binding sites of 50 and 72 transcription factors were predicted from the upstream sequences of 23 LSCC genes by two programs respectively. Among them, 18 transcription factors were found in common. The top 10 TFBS sequences were basically consistent to the predicted TFs. Literature mining indicated that LSCC genes and TFs were closely related to such terms as albumin, diabetes, glucose, lipid, metabolism, and JNK, in addition to those associated with hepatic tissue and TFs. These observations suggested that LSCC genes and TFs were involved in the regulation of glucose and lipid metabolism, binding and transport, coagulation signal cascades, inflammatory response, etc. PPP2R1B, which was out of the network, showed a partial functional similarity to DUSP10 in the network. CONCLUSIONS: LSCC genes and the predicted TFs may be involved in the regulation of many important functions of the liver, which are integrated into a sophisticated transcription regulatory network. JUN may be the key target for regulation, and PPP2R1B is presumed to participate in the regulation of JUN.

[Effect of Velcade on the gene expression profiles of K562 cells: study of its molecular mechanism].

OBJECTIVE: To analyze alterations in the gene expression profiles of Velcade-treated K562 cells using bioinformatics methods. METHODS: The total RNAs of Velcade-treated and control K562 cells were amplified and labeled with fluorescent dyes. The labeled RNAs were hybridized to Agilent Human 1A Microarray, and the raw expression data were processed with Agilent Feature Extraction Software. GeneSifter and GATHER were used for data analysis of the differentially expressed genes to perform gene ontology classification, KEGG pathway analysis, functional protein association network construction and literature mining. RESULTS: Totally 228 differentially expressed genes were identified in the Velcade-treated K562 cells. including 84 up-regulated and 144 down-regulated genes. Chymase 1 gene had the greatest down-regulation by 10.80 folds (log ratio), and interferon alpha-21 gene was also down-regulated by 2.31 folds. Gene ontology classification suggested enhanced aging and leukocyte activity. KEGG pathway analysis showed significant impact of Velcade on JAK-STAT signaling pathway, cytotoxicity mediated by natural killer cells, and antigen processing and presentation pathways. Protein-protein interaction analysis revealed that ubiquitin-dependent protein catabolism, antigen presentation and immune response, as well as JAK-STAT signaling pathway were the major elements of the protein network. Literature mining showed that the differentially expressed genes were strongly associated with terms such as leukemia, apoptosis, cell cycle, proteasome, inhibitor, aging and IkappaB, etc. CONCLUSIONS: Velcade may inhibit the cell survival pathways such as NF-kappaB and JAK-STAT signaling pathways to enhance the cytotoxicity and inducing tumor cell apoptosis. Velcade might also be involved in antigen processing and presentation, immune response and inflammation. Chymase 1 gene is probably the key target of Velcade.

[Effect of Epstein-Barr virus reactivation on gene expression profile of nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) infection plays a key role in the pathogenesis of nasopharyngeal carcinoma (NPC). This study was to explore the effects of the recurrent infection by EBV reactivation on the genomic expression profile of NPC. METHODS: The microarray expression data from different cell lines subjected to primary infection of EBV+ vs. EBV- targets in NPC and recurrent EBV reactivation were collected from public data depository. Cross comparison, t-test analysis as well as filtering by flag, expression level and fold change were used to analyze the data and identify differential genes. Moreover, a set of web-based applications, such as DAVID (database for annotation, visualization and integrated discovery), pSTIING (protein, signaling, transcriptional interactions and inflammation networks gateway), GATHER (gene annotation tool to help explain relationships) and TELiS (transcription element listening system), were used to analyze and predict the probable expression profile of the differential genes. RESULTS: As compared with the genes expressed during primary infection of EBV, 25 genes, including DUSP1, TOP1, HOXA9, DEK, PABPC1 and IMPDH2, were differentially expressed during EBV reactivation. Many of them were oncogenic. The differential genes together with related transcriptional factors were interacted mainly through 2 mechanisms: one mainly included TOP1, DUSP1, DUSP6, and RPS28; the other one was a circuit of PITX1, CD9, HOXA9 and IMPDH2. CONCLUSION: The differential genes might participate in EBV reactivation by changing their expression level through two mechanisms, which contributes to the final development of NPC.