QSOX2 Is an E2F1 Target Gene and a Novel Serum Biomarker for Monitoring Tumor Growth and Predicting Survival in Advanced NSCLC

BackgroundQuiescin Q6 sulfhydryl oxidase 2 (QSOX2), an enzyme that can be directly secreted into the extracellular space, is known to be associated with oxidative protein folding. However, whether QSOX2 is abnormally expressed in non-small cell lung cancer (NSCLC) and its role in tumor growth remains unclear.MethodsReal-time quantitative PCR (qPCR), immunohistochemistry (IHC), bioinformatics analyses were applied to analyze the expression pattern and prognostic significance of QSOX2 in NSCLC. Xenografts model, enzyme-linked immunosorbent assays (ELISA), western blot analysis (WB), and IHC were preformed to examine in vivo tumor suppression and intracellular and extracellular expression of QSOX2. Flow cytometry, WB and qPCR analyses were used to elucidate the role of QSOX2 in cell cycle regulation. Chromatin immunoprecipitation assay (ChIP) assay and Dual-Luciferase reporter assay were employed to investigate transcriptional regulation of QSOX2 by E2F Transcription Factor 1 (E2F1).ResultsQuiescin sulfhydryl oxidase 2 was significantly overexpressed in NSCLC and associated with poor survival in advanced-stage patients. The intracellular and extracellular expression of QSOX2 by tumor cells markedly decreased after anti-cancer therapy in vitro, in vivo and in the clinic. Moreover, QSOX2 silencing in NSCLC cell lines resulted in inhibition of cancer cell proliferation, induction of apoptosis, and decreased expression of cell division-related genes (CENPF and NUSAP1) and Wnt pathway activators (PRRX2 and Nuc-β-catenin). Mechanistically, QSOX2 was expressed periodically during cell cycle and directly regulated by E2F1.ConclusionsOur findings demonstrate that QSOX2 is directly regulated by E2F1 in the cell cycle, which is essential for the proliferation of NSCLC cells. Furthermore, QSOX2 is a prognostic indicator for NSCLC and may be developed into a biomarker for monitoring tumor burden and therapeutic progress.

High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.