Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression

T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu

Department of Emergency, Ningbo Medical Center Eastern Hospital, Ningbo, China. achi306@sina.com

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• Oncology

OBJECTIVE: This study aimed to investigate whether long-chain non-coding ANCR is involved in the progression of non-small cell LCa (NSCLC) and its possible molecular mechanisms.

PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to examine ANCR expression in 48 cases of NSCLC and adjacent normal tissues. In addition, ANCR level in patients of different tumor staging was analyzed. The Kaplan-Meier method was applied to analyze the interplay between ANCR expression and the prognosis of patients with NSCLC. Subsequently, qRT-PCR was performed to detect ANCR level in LCa cell lines. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA expression were examined by qRT-PCR, while the expression levels of epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. At the same time, cell viability and migration ability were analyzed through cell counting kit-8 (CCK-8) and cell wound healing assay, respectively. RNA immunoprecipitation (RIP) test was performed to verify the binding of ANCR to EZH2. After knocking down EZH2 in A549 cells, E-Ca messenger ribonucleic acid (mRNA) expression was detected. Additionally, Chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of EZH2 to the E-Ca promoter region. When E-Ca and ANCR were simultaneously knocked down in A549 cells, Western blot investigation was performed to examine the expression of EMT-related proteins, while CCK-8 and wound healing assays were applied to figure out the changes in cell viability and cell migration capacity.

RESULTS: ANCR level was conspicuously higher in NSCLC tissues than that in normal tissues, and that in T3 and T4 tumors was also higher than that in T1 and T2. Meanwhile, ANCR expression in the tissues of patients with lymph node metastasis was conspicuously higher than those without metastasis. Survival analysis revealed that the overall survival of patients with NSCLC with high expression of ANCR was conspicuously lower than patients with low expression of ANCR. The qRT-PCR study verified that ANCR was highly expressed in the LCa cell line A549. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA levels were found conspicuously decreased, and so were the expression levels of EMT-related proteins, as well as the cell viability and migration ability. The RIP assay result indicated that ANCR can indeed bind to EZH2. E-Ca mRNA expression was elevated after the knockdown of EZH2 in A549 cells. In addition, the result of CHIP test demonstrated that EZH2 could combine with E-Ca. Simultaneous down-regulation of ANCR and E-Ca in A549 cells could reverse the influence of knocking down ANCR alone on cell viability and migration ability.

CONCLUSIONS: Long-chain non-coding RNA ANCR was highly expressed in NSCLC tissues and could enhance the viability and malignancy of NSCLC cells by inhibiting the expression of E-Ca, thereby promoting the progression of NSCLC.

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