PYGO2 increases proliferation and migration capacities through critical signaling pathways in esophageal squamous cell carcinoma.

Esophageal cancer, an increasingly prevalent malignancy, is a major concern for global health. The development of esophageal squamous cell carcinoma (ESCC) involves various genetic abnormalities that affect key cell signaling pathways, including Wnt, Hh, Apoptosis, MAPK, EGFR, AKT, Notch, and EMT. Additionally, this malignancy involves some changes in the expression of long noncoding RNAs (LncRNAs). The present study examines the relationship between PYGO2 gene expression and the activity of cell signaling pathways in KYSE-30 and YM-1ESCC cell lines. To this end, several cellular and molecular tests were performed, including cell migration, cell cycle, and apoptosis. Also, expression levels of CD133 and CD44 markers, real-time PCR, and western blot were analyzed after inducing PYGO2 protein expression in the cells. Overexpression of the PYGO2 protein resulted in the upregulation of Wnt pathway-related genes, leading to enhanced cell migration and proliferation and reduced apoptosis in both cell lines. Furthermore, PYGO2 gene expression induction analysis showed the correlation of several involved genes in Wnt, Hh, Apoptosis, MAPK, EGFR, AKT, and EMT pathways with various LncRNAs.

Apoptosis Induction by ZnFe2O4-Ag Biosynthesized by Chlorella vulgaris in MCF-7 Breast Cancer Cell Line.

The incidence and mortality of breast cancer are growing which indicates the inefficiency of the current chemotherapy drugs. Due to the anticancer potential of Zn and Ag and the magnetic feature of iron oxide, in this work, we synthesized ZnFe2O4-Ag nanocomposite using Chlorella vulgaris and investigated its anticancer effect on breast cancer cell line. Physicochemical characterization was performed by FT-IR, XRD, SEM, TEM, VSM, EDS mapping, UV, and zeta potential assays. Cell cytotoxicity and apoptosis frequency were studied by the MTT and flow cytometry assays. Also, cell cycle analysis, Hoechst staining, and measuring ROS (reactive oxygen species) level were performed. The synthesized particles were almost spherical with a size range of 14-52 nm. The FT-IR and XRD assays confirmed the proper synthesis of the particles and VSM analysis showed that particles had magnetic property and the maximum saturation magnetization was 0.8 Emu/g. Also, the EDS mapping of the nanocomposite showed the Zn, Fe, O, and Ag elements. The MTT assay showed that the 50% inhibitory concentration (IC50) of ZnFe2O4-Ag for breast cancer and normal cells were 28 and 154 µg/mL, respectively, and the nanocomposite had stronger anticancer activity than cisplatin (IC50 = 84 µg/mL). Flow cytometry analysis showed that the exposure to the nanocomposite induced cell apoptosis by 77.5% and significantly induced ROS generation. Also, treating breast cancer cells with the nanocomposite induced cell cycle arrest and apoptotic features, including chromatin condensation and fragmentation. In conclusion, ZnFe2O4-Ag nanocomposite synthesized by C. vulgaris could suppress the proliferation of breast cancer cells by the generation of oxidative stress, apoptosis induction, and cell cycle arrest.

A simple and efficient method for cytoplasmic production of human enterokinase light chain in E. coli.

Human enterokinase light chain (hEKL) cDNA sequence was designed with the help of codon optimization towards Escherichia coli codon preference and ribosome binding site design and artificially synthesized with a thioredoxin fusion tag at the N-terminal and a five his-tag peptide at the C-terminal. The synthetic hEKL gene was cloned into the pET-15 expression vector and transferred into the three different expression strains of E. coli BL21(DE3), NiCo21, and SHuffle T7 Express. Different growth and induction conditions were studied using a statistical response surface methodology (RSM). Recombinant hEKL protein was expressed at high levels in soluble form with 0.71 mM IPTG after 4 h of induction at 25 °C. Autocatalytic process cleaved TRX tag with enterokinase recognition site by the impure hEKL and yielded the mature enzyme. The target protein was then purified to homogeneity (> 95%) by affinity chromatography. The activity of hEKL was comparable to the commercial enzyme. From 1 L culture, 80 mg pure active hEKL was obtained with the specific activity of 6.25 × 102 U/mg. Three main parameters that help us to produce the enzyme in the folded and active form are the type of strain, SHuffle T7 strain, TRX and histidine fusion tags, and growth conditions including the increase of OD of induction and IPTG concentration and the decrease of induction temperature.

EZH2 regulates oncomiR-200c and EMT markers in esophageal squamous cell carcinomas.

EZH2, as a histone methyltransferase, has been associated with cancer development and metastasis possibly through the regulation of microRNAs and cellular pathways such as EMT. In this study, the effect of EZH2 expression on miR-200c and important genes of the EMT pathway was investigated in esophageal squamous cell carcinoma (ESCC). Comparative qRT-PCR was used to examine EZH2 expression in ESCC lines (YM-1 and KYSE-30) following the separately transfected silencing and ectopic expressional EZH2 vectors in ESCC. Subsequently, expression of miR-200c and EMT markers was also assessed using qRT-PCR, western blotting and immunocytochemistry. Underexpression of Mir200c was detected in YM-1 and KYSE-30 cells after EZH2 silencing, while its overexpression was observed after EZH2 induced expression. Following EZH2 silencing, downregulation of mesenchymal markers and upregulation of epithelial markers were detected in the ESCCs. Our results demonstrate that EZH2 regulates the expression of miR-200c and critical EMT genes, implying that overexpression of Zeb2, Fibronectin, N-cadherin, and Vimentin lead to a mesenchymal phenotype and morphology while underexpression of epithelial genes, enhance cell migration after enforced expression of EZH2 in ESCCs. EZH2 gene can be a beneficial treatment marker for patients with esophageal cancer through decrease invasiveness of the disease and efficient response to neoadjuvant therapy.

A Signature of Three microRNAs Is a Potential Diagnostic Biomarker for Glioblastoma

Background: Glioblastoma is the most common primary malignant neoplasm of the central nervous system. Despite progress in diagnosis and treatment, glioblastoma still has a poor prognosis. This study aimed to examine whether a signature of three candidate miRNAs (i.e. hsa-let-7c-5p, hsa-miR-206-5p, and hsa-miR-1909-5p) can be used as a diagnostic biomarker for distinguishing glioblastoma from healthy brain tissues. Methods: In this study, 50 formalin­fixed paraffin­embedded (FFPE) glioblastoma tissue samples and 50 healthy tissue samples adjacent to tumor were included. The expression of each candidate miRNA (i.e. hsa-let-7c-5p, hsa-miR-206-5p, and hsa-miR-1909-5p) was measured using quantitative reverse transcription PCR. To show the roles of each miRNA and their biological effects on glioblastoma development and clinicopathological characteristics, in silico tools were used. ROC curves were performed to assess the diagnostic accuracy of each miRNA. Results: Based on the results, hsa-let-7c-5p and hsa-miR-206-5p were downregulated, while hsa-miR-1909-5p was upregulated in glioblastoma tumors compared to healthy samples. No association was detected between the expression of each candidate miRNA and sex. Except for hsa-let-7c-5p, other miRNAs did not correlate with age status. ROC curve analysis indicated that the signature of candidate miRNAs is a potential biomarker distinguishing between glioblastoma and healthy samples. Only hsa-miR-206-5p suggested the association with poor prognosis in glioblastoma patients. Conclusions: Our findings revealed that the signature of three miRNAs is capable of distinguishing glioblastoma tumor and healthy tissues. These results are beneficial for the clinical management of glioblastoma patients.

Expression pattern of neurotrophins and their receptors during neuronal differentiation of adipose-derived stem cells in simulated microgravity condition.

OBJECTIVES: Studies have confirmed that microgravity, as a mechanical factor, influences both differentiation and function of mesenchymal stem cells. Here we investigated the effects of simulated microgravity on neural differentiation of human adipose-derived stem cells (ADSCs). MATERIALS AND METHODS: We have used a fast rotating clinostat (clinorotation) to simulate microgravity condition. Real-time PCR and flow cytometry analysis were used to evaluate the regulation of neurotrophins, their receptors, and neural markers by simulated microgravity and their impact on neural differentiation of cells. RESULTS: Our data revealed that simulation microgravity up-regulated the expression of MAP-2, BDNF, TrkB, NT-3, and TrkC both before and after neural differentiation. Also, the neural cells derived from ADSCs in microgravity condition expressed more MAP-2, GFAP, and synaptophysin protein in comparison to the 1G control. CONCLUSION: We showed that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons. Our findings provide a new strategy for differentiation of ADSCs to neural-like cells and probably other cell lineages. Meanwhile, microgravity simulation had no adverse effects on the viability of the cells and could be used as a new environment to successfully manipulate cells.