ABSTRACT
OBJECTIVE: Recent reports have suggested that long non-coding RNA LBX2 antisense RNA 1 (LBX2-AS1) acts as an important regulator in cancer progression. This study aimed to investigate the clinical significance of LBX2-AS1 in non-small cell lung cancer (NSCLC) patients and its biological functions. PATIENTS AND METHODS: The expressions of LBX2-AS1 were examined in 165 paired NSCLC tissues and adjacent normal tissues from NSCLC patients by qRT-PCR. The clinical significance of LBX2-AS1 was determined using a series of statistical methods. The effects of LBX2-AS1 knockdown on NSCLC cell proliferation, migration, and invasion were investigated by CCK-8 assays, colony formation assays, EdU proliferation assays, Wound healing assays, and transwell assays. The promotive roles of LBX2-AS1 on Notch1 signal were determined using RT-PCR and Western blot. RESULTS: We found that LBX2-AS1 was highly expressed in NSCLC tissues and cell lines. The increased levels of LBX2-AS1 were observed to be positively correlated with TNM stage, histological grade, and lymph node metastasis. Furthermore, the Kaplan-Meier survival curves indicated that patients with higher expressions of LBX2-AS1 had unfavorable overall survival. Lost-of-functions assays revealed that the knockdown of LBX2-AS1 in H1299 and A549 cells inhibited cell proliferation, migration, and invasion. Mechanistic studies revealed that the suppression of LBX2-AS1 resulted in the reduced expressions of Notch1, p21, and Hes1, suggesting that LBX2-AS1 might promote the activation of the Notch pathway. CONCLUSIONS: Our study identified a novel NSCLC-related lncRNA LBX2-AS1, which may represent a novel prognostic biomarker and a potential therapeutic target for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , RNA, Long Noncoding/genetics , Signal Transduction , A549 Cells , Adult , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , Receptors, Notch/metabolism , Survival AnalysisABSTRACT
The glutathione-S-transferase A1 cDNA was amplified from human liver total RNAs by RT-PCR and was cloned into a Escherichia coli expression vector pET23b, then the recombinant plasmid pET23bhgst was introduced into E. coli BL21 (DE3) and induced by IPTG, the high-level expression of hGSTA1 appeared in the E. coli cells. The cDNA encoding hGSTA1 was subcloned into pMG36e, a lactococcal expression vector, and introduced into Lactococcus lactis MG1363 by electroporation. In the positive transformants, the hGSTA1 was expressed as a fusion protein which was verified by SDS-PAGE and Western blot. The hGSTA1 from both E. coli and L. lactis was purified by affinity chromatography on glutathione-agarose and all showed enzymatic activity. The potential application of the recombinant Lactococcus lactis in functional food was also discussed.
