DataSheet_1_TP53-Activated lncRNA GHRLOS Regulates Cell Proliferation, Invasion, and Apoptosis of Non-Small Cell Lung Cancer by Modulating the miR-346/APC Axis.pdf

Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high mortality worldwide. To improve NSCLC therapy, the exploration of molecular mechanisms involved in NSCLC progression and identification of their potential therapy targeting is important. Long noncoding RNAs (lncRNAs) have shown important roles in regulating various tumors progression, including NSCLC. We found lncRNA GHRLOS was decreased in NSCLC cell lines and tissues which correlated with poor prognosis of NSCLC patients. However, the role and underlying mechanisms of lncRNA GHRLOS in NSCLC progression remains elusive. The expression of lncRNA GHRLOS was examined in NSCLC cell lines and biopsy specimens of patients with NSCLC by quantitative real time polymerase chain reaction (qRT-PCR). The effects of GHRLOS on proliferation, invasion and apoptosis of NSCLC cells were determined by both in vitro and in vivo experiments. The interaction between GHRLOS and TP53 was determined by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) combined with qRT-PCR analysis. RNA immunoprecipitation (RIP) was conducted to validate the binding between GHRLOS and microRNA-346 (miR-346). Dual-luciferase reporter assays were also carried out to reveal the interaction between miR-346 and the 3’ untranslated region (3’UTR) of adenomatous polyposis coli (APC) mRNA.Our data demonstrated that overexpression of lncRNA GHRLOS suppressed cancer cell proliferation and invasion as well as promoted cell apoptosis by regulating the expression of CDK2, PCNA, E-cadherin, N-cadherin, Bax, and Bcl-2 in NSCLC cells. Moreover, lncRNA GHRLOS was upregulated by the binding of TP53 to the GHRLOS promoter. The binding target of lncRNA GHRLOS was identified to be miR-346. Impressively, overexpression of miR-346 promoted cell proliferation and invasion, as well as inhibited cell apoptosis, however, these effects can be blocked by overexpression of lncRNA GHRLOS both in vitro and in vivo. In summary, this study reveals lncRNA GHRLOS, upregulated by TP53, acts as a molecule sponge of miR-346 to cooperatively modulates expression of APC, a miR-346 target, and potentially inhibits NSCLC progression via TP53/lncRNA GHRLOS/miR-346/APC axis, which represents a novel pathway that could be useful in targeted therapy against NSCLC.

非小细胞肺癌（Non-small cell lung cancer, NSCLC）是全球范围内致死率较高的主要肺癌类型。为优化非小细胞肺癌的治疗方案，探究其进展相关的分子机制并鉴定潜在治疗靶点具有重要意义。长链非编码RNA（Long noncoding RNAs, lncRNAs）在包括非小细胞肺癌在内的多种肿瘤的进展调控中发挥关键作用。本研究发现，长链非编码RNA GHRLOS在非小细胞肺癌细胞系及患者组织中表达下调，且与非小细胞肺癌患者的不良预后相关。然而，长链非编码RNA GHRLOS在非小细胞肺癌进展中的作用及潜在分子机制仍不明确。本研究通过实时定量聚合酶链反应（quantitative real time polymerase chain reaction, qRT-PCR）检测了非小细胞肺癌细胞系及患者活检组织中长链非编码RNA GHRLOS的表达水平。通过体外及体内实验，明确了GHRLOS对非小细胞肺癌细胞增殖、侵袭及凋亡的影响。采用双荧光素酶报告基因检测及结合实时定量聚合酶链反应的染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验，验证了GHRLOS与TP53的相互作用。通过RNA免疫沉淀（RNA Immunoprecipitation, RIP）实验，证实了GHRLOS与微小RNA-346（microRNA-346, miR-346）的结合。此外，利用双荧光素酶报告基因检测揭示了miR-346与腺瘤性结肠息肉病蛋白（adenomatous polyposis coli, APC）mRNA的3’非翻译区（3’ untranslated region, 3’UTR）之间的相互作用。研究数据显示，过表达长链非编码RNA GHRLOS可通过调控非小细胞肺癌细胞中CDK2、PCNA、E-钙粘蛋白、N-钙粘蛋白、Bax及Bcl-2的表达，抑制癌细胞增殖与侵袭，并促进细胞凋亡。此外，TP53可结合GHRLOS的启动子区域，从而上调GHRLOS的表达。长链非编码RNA GHRLOS的结合靶点被鉴定为miR-346。值得注意的是，过表达miR-346可促进细胞增殖与侵袭，并抑制细胞凋亡，而该效应可在体外及体内实验中被过表达的长链非编码RNA GHRLOS所阻断。综上，本研究揭示了受TP53调控上调的长链非编码RNA GHRLOS，可作为miR-346的分子海绵，协同调控miR-346的靶基因APC的表达，进而通过TP53/lncRNA GHRLOS/miR-346/APC轴抑制非小细胞肺癌的进展。该通路有望成为非小细胞肺癌靶向治疗的全新潜在靶点。