PSMA7 promotes the malignant proliferation of esophageal cancer.

Background: It is important to explore novel molecules that play a key role in esophageal cancer (ESCA) progression. Methods: Two ESCA tissue expression profile microarrays (GSE92396 and GSE17351) data from GEO were downloaded, and differentially expressed genes (DEGs) were analyzed using GEO2R. The DEGs common to both microarrays were analyzed for protein-protein interactions, KEGG and GO. The altered expression of proteasome 20S subunit α 7 (PSMA7) in ESCA tissues was analyzed using information from publicly available databases (GEO, TCGA, TNMplot). PSMA7 was overexpressed or knocked down in Eca109 and KYSE150 cells using transfection, and the effects on cell proliferation, migration, invasion and apoptosis were examined using CCK-8, Transwell, and flow cytometry experiments. Results: 284 common DEGs were identified, and 10 core proteins, HSP90AA1, AURKA, CDC6, PCNA, MCM5, KAT2B, GRB2, MYBL2, PSMA7, and CKAP5, involved in ESCA progression were identified. PSMA7 mRNA level was significantly increased in ESCA tissues. PSMA7 overexpression significantly promoted the proliferation, migration and invasion of Eca109 and KYSE150 cells, and significantly promoted apoptosis. In contrast, PSMA7 knockdown inhibited their proliferation and motility, and significantly suppressed apoptosis. Conclusion: This study analyzed multiple proteins that may play a key role in ESCA progression, and identified the pro-cancer role of PSMA7.

[Expression of a thermostable a-amylase mutant into Escherichia coli and Pichia pastoris].

Alpha-amylase are of considerable commercial value. It can be produced by a wide variety of microorganisma. The alpha-amylase gene (amyE) from Bacillus licheniformis, which is widely used for the industrial hydrolysis of starch, was mutated (amyEM), then amplified by PCR and inserted into pBV220 and pPIC9k to obtain the recombinant vector pBV220-amyEM and pPIC9k-amyEM. These recombinant vectors were transformed into corresponding competent cell E. coli DH5alpha and P. pastoris GS115 respectively. The resulting recombinant strains, DH5alpha/pBV220-amyEM and GS115/ pPIC9k-amyEM, were then screened by measuring the enzymatic activity and SDS-PAGE. DH5alpha/pBV220-amyEM was induced by temperature and GS115/pPIC9k-amyEM by methanol. In contrast to the parent cells, the a-amylases were expressed in both the recombinant strains. In E. coli the molecular weight was approximately 55kDa; optimal temperature and pH of the recombinant a-amylase were 80 degrees C - 90 degrees C and 6.0 respectively. The recombinant amylase had high activity in pH 5.0 - 5.5 compared to wild type. In Pichia pastoris, the recombinant amylase was secreted to the medium; molecular weight was 60kDa for the putative post-translational modifications; optimal pH shifted to 5.5. The specific activities of alpha-amylase produced by E. coli and P. pastoris were 8.1U/mg and 102U/mg respectively. This result indicated that the alpha-amylase were secreted into the culture medium with high efficiency in the recombinant P. pastoris High activity in high temperature and lower pH properties impart the recombinant amylase potential applications in industry.

[Molecular cloning and expression of extremely thermostable and acid-stable amylase gene in Pichia pastoris].

The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.