Application of n-dodecane as an oxygen vector to enhance the activity of fumarase in recombinant Escherichia coli: role of intracellular microenvironment

Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.

Mechanisms of miR-31-5p inhibiting biological behavior and radiotherapy resistance of breast cancer cells via regulating TNS1 / 中国肿瘤生物治疗杂志

@#Objective: To investigate the mechanism of miR-31-5p/tension protein 1 gene (TNS1) axis modulating radiotherapy resistance in breast cancer. Methods: The breast cancer tissues and corresponding para-cancerous tissues of 21 patients with breast cancer, who underwent surgical resection at Department of Cancer Radiotherapy of Nanyang Central Hospital from July 2017 to December 2017, were collected for this study; breast cancer cell lines (MCF-7，MDA-MB-23 and SKBR-3) were also collected; qPCR was applied to detect the expression level of miR-31-5p in breast cancer tissues and cell lines. The radiation resistant cell line MCF-7R was constructed by using 6 MV-X ray radiotherapy treatment. Subsequently, the influence of over-expression/kockdown of miR-31-5p on radiation sensitivity of MCF-7 and MCF-7R cells were detected by colony formation assay, Transwell assay and Annexin V-FITC/PI double staining flow cytometry assay, respectively. Moreover, luciferase reporter assay was used to verify whether TNS1 was a target gene of miR-31-5p. Results: Compared with para-cancerous tissues, normal mammary epithelial MCF-10A cells and MCF-7 cells, miR-31-5p was low-expressed in breast cancer, cell lines and MCF-7R (all P<0.01). Over-expression of miR-31-5p resulted in inhibited invasion and promoted apoptosis of MCF-7R cells (P<0.01), whereas miR-31-5p knockdown got opposite results in MCF-7 cells. Moreover, luciferase reporter assay confirmed that TNS1 was a target gene of miR-31-5p. Over-expression of miR-31-5p inhibited invasion and increased radio-sensitivity, apoptosis of MCF-7R cell via targeting TNS1 (P<0.01), whereas knockdown of miR-31-5p significantly promoted the invasion but reduced apoptosis of MCF-7R cells (all P<0.01), and further up-regulated the radio-sensitivity of MCF-7R cells. Conclusion: miR-31-5p/TNS1 axis regulates the radiotherapy resistance of breast cancer, and over-expression of miR-31-5p may reverse the resistance of MCF-7R to radiotherapy.