Upregulation of Long Noncoding RNA (lncRNA) X-Inactive Specific Transcript (XIST) is Associated with Cisplatin Resistance in Non-Small Cell Lung Cancer (NSCLC) by Downregulating MicroRNA-144-3p.

BACKGROUND Patients with advanced non-small cell lung cancer (NSCLC) treated with cisplatin, also termed cis-diamminedichloroplatinum (CDDP) or diamminedichloroplatinum (DDP), may develop chemoresistance. This study aimed to investigate the role of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and multidrug resistance-1 (MDR1) in tumor tissue samples and the chemoresistant human NSCLC cell lines, H460/DDP and A549/DDP, and in a murine A549/DDP tumor xenograft. MATERIAL AND METHODS Tissue samples were from patients with NSCLC who responded cisplatin (DDP-sensitive) (n=24), patients with NSCLC unresponsive to cisplatin (DDP-resistant) (n=30), and normal lung tissue (n=25). In H460/DDP and A549/DDP cells, expression of XIST, microRNA (miR)-144-3p, MDR1, and multidrug resistance-associated protein 1 (MRP1) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The MTT assay measured cell survival and proliferation, a transwell assay evaluated cell migration, and flow cytometry measured apoptosis. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays examined the relationship between XIST and miR-144-3p. Tumor xenografts from A549/DDP cells were studied in BALB/c nude mice. RESULTS In tissue from patients with DDP-resistant NSCLC and the mouse A549/DDP tumor xenograft, lncRNA-XIST expression was upregulated and miR-144-3p expression was inhibited. In A549/DDP and H460/DDP cells, down-regulation of lncRNA-XIST and upregulation of miR-144-3p reduced cell survival, proliferation, migration, induced apoptosis and suppressed MDR1 and MRP1 expression. CONCLUSIONS Upregulation of lncRNA-XIST was associated with cisplatin resistance in NSCLC by downregulating miRNA-144-3p in H460/DDP and A549/DDP cells, a murine A549/DDP tumor xenograft, and human tumor tissues from patients with cisplatin-resistant NSCLC.

[Purification of Taq DNA polymerase expressed in Escherichia coli].

Taq DNA polymerase is one of the most commonly thermostable DNA polymerases in molecular biological researches, which shares its basic characters with others of the family, thereby its purifying strategy could be used not only in itself production but also in the extraction of the others as a reference. At present, the protocols reported for large scale preparation of Taq DNA are high cost, so a cheaper method was described here. In this protocol, by heat denaturation, ammonium sulfate precipitation and cation exchange chromatography of 724 resin, about 18 g powder of Na form resin could recover about 27.07 mg of Taq enzyme. The total activity and specific activity were approximately 2.2 × 105 U and 8131.98 U/mg. The total yield was about 48.92% with 59.35 of purification folds. Analysis of quality of purified enzyme indicated that only one protein 94 kDa was identified against SDS-PAGE and the remnant of DNA nuclease was not detected. For PCR reaction, The amplification ability of purified Taq polymerase was not different from that of the commercially avail-able ones. This method reported in the present study is effective and low cost, making it suitable for general purification in laboratories or business production.