FENDRR作为ceRNA参与miR-331-3p调控EIF5A2在宫颈癌发生发展中的分子机制研究

Recurrence and metastasis is the major cause of death in cervical cancer, but its moleculor mechanism has not been fully elucidated. In recent years, many studies have shown that long noncoding RNA (lncRNA) can be used as a competitive endogenous RNA interacts with microRNAs, which is involved in regulation of target genes and play an important role in the development of tumor. Our previous study showed that lncRNA FENDRR of eukaryotic translation initiation factor 5A2 (EIF5A2) were overexpression in cervical cancer tissues, and EIF5A2 is correlated with metastasis and poor prognosis, but the mechanism is not clear. In the preliminary experiment, we confirmed the role of miR-331-3p in regulation of EIF5A2, EIF5A2 gene contains the bindingsites of miR-331-3p and miR-331-3p negatively regulates EIF5A2 expression. We predicted that lncRNA FENDRR may act as a ceRNA to competitively inhibit the regulation role of miR-331-3p for EIF5A2, and then influence the invasion and metastasis of cervical cancer. To confirm this speculation, we aim to investigate the relationship between the expression of miR-331-3p, lncRNA FENDRR, EIF5A2 on the level of human, cell and animal model. Then, we will knock-down or overexpression of miR-331-3p and lncRNA FENDRR to observe the changes in the expression of EIF5A2 and observe their role in the invasion and metastasis of cervical cancer. Based on this study, the conclusion will provide a theoretical and experimental basis for clinic to predict the prognosis and make the individualized therapy for patients with cervical cancer.

复发和转移是宫颈癌的主要死亡原因。真核细胞翻译起始因子5A2（EIF5A2）与肿瘤的发生发展密切相关。我们前期研究证实EIF5A2在宫颈癌组织中高表达，且与宫颈癌的侵袭转移和不良预后相关。生物信息软件预测及预实验初步证实miR-331-3p对EIF5A2具有靶向调控作用，长链非编码RNA FENDRR对miR-331-3p具有竞争性抑制作用。由此我们提出：FENDRR可作为ceRNA竞争性抑制miR-331-3p对EIF5A2的调控作用，进而影响宫颈癌的侵袭和转移。本课题将从临床患者-细胞系-动物模型三个层次探讨FENDRR、miR-331-3p与EIF5A2表达相关性，过表达和敲除FENDRR、miR-331-3p对EIF5A2的靶向调控作用以及对宫颈癌侵袭转移能力的影响。从ceRNA网络全新视角，阐明非编码RNA对EIF5A2的调控机制，为临床上靶向EIF5A2治疗宫颈癌提供理论依据。

肿瘤转移是导致宫颈癌死亡率居高不下的原因。我们在临床水平检测到，真核细胞翻译起始因子5A2（EIF5A2）在宫颈癌组织中高表达，且与宫颈癌的不良预后相关。starbase工具生物信息学预测发现，长链非编码RNA FENDRR与miR-331-3p有靶向结合位点，miR-331-3p与EI5A2有靶向结合位点；双荧光素酶实验及RIP验证了FENDRR作为ceRNA竞争抑制miR-331-3p对EIF5A2的调控作用。细胞水平通过以下处理：过表达或敲低EIF5A2，敲低FENDRR，转染miR-331-3p的模拟物和抑制物，以及转染miR-331-3p的模拟物或EIF5A2的保护序列；通过CCK8、克隆形成及周期相关蛋白检测细胞增殖能力，流式细胞术及凋亡相关指标反应细胞凋亡情况，transwell小室和细胞划痕检测细胞侵袭迁移能力，western blot检测EMT相关指标表明细胞上皮间质转化的情况。结果显示：长链非编码RNA FENDRR与EIF5A2促进宫颈癌细胞增殖侵袭迁移及上皮间质转化，miR-331-3p抑制宫颈癌细胞增殖侵袭迁移及上皮间质转化。再在动物水平进行裸鼠异位成瘤实验，验证FENDRR与EIF5A2促进肿瘤组织生长与增殖，抑制凋亡。MiR-331-3p抑制肿瘤组织生长与增殖，促进凋亡。本研究从ceRNA网络的视角，阐明非编码RNA对miR-331-3p/EIF5A2的调控机制，为临床上宫颈癌的诊断和治疗提供了理论依据，具有深远意义。