miR-424-5p在细胞周期进程中的网络调控作用对食管鳞癌细胞增殖影响的机制研究

Deregulation of cell cycle plays an important role in tumor cell proliferation. We found that miR-424-5p was overexpressed and might function as an oncogene in esophageal squamous cell carcinomas (ESCCs), but the specific mechanism has not been determined. Preliminary experiments showed that expression of miR-424-5p was up-regulated in ESCCs and varied along with cell cycle progression, which further down-regulated the expression of targeted genes Npm1 and Wee1 and promoted cell cycle progression and cell proliferation. However, the reason why miR-424-5p was up-regulated in specific cell cycle of ESCC cells has not been determined, which might be due to transcriptional regulation and post-transcriptional modification of miR-424-5p per se. And miR-424-5p exhibited heterogeneity in regulating target genes’ expression, probably because of the effect of other non-coding RNAs as competing endogenous RNAs (ceRNAs). In order to reveal the cell cycle regulation network composed of transcription factor, miR-424-5p, target coding genes, and non-coding RNAs functioning as ceRNAs in this study, we plan to observe the effect of miR-424-5p in ESCC proliferation in vitro and in vivo, identify factors inducing high miR-424-5p expression in ESCCs, and illuminate the specific role of miR-424-5p and ceRNA in regulating target genes’ expression and cell cycle progression. The study would provide an alternative mechanism to understand the ESCC cell cycle regulation, and lay foundation for the identification of useful ESCC prognostic molecular markers.

细胞周期失调控影响肿瘤细胞增殖。我们首次发现miR-424-5p在食管鳞癌中高表达，且具有促癌作用。预实验表明：食管鳞癌中，miR-424-5p表达随细胞周期进程改变；通过调控靶基因Npm1及Wee1表达，促进细胞跨越细胞周期检测点，加速细胞增殖。然而，食管鳞癌中miR-424-5p高表达，且表达随细胞周期进程变化原因未明，推测与转录调控及转录后修饰相关；其对靶基因的调控存在细胞异质性，推测有其他非编码RNA作为ceRNA影响其功能。为了验证这一以miR-424-5p为核心，由转录因子、靶基因和ceRNA等构成的细胞周期调控网络，我们将从动物-细胞-分子三个层次，体内外观察miR-424-5p表达对食管鳞癌增殖的影响；阐明其在食管鳞癌中高表达的原因；明确miR-424-5p及ceRNA对靶基因表达和细胞周期进程的调控。本课题将揭示细胞周期调控的新机制，为寻找食管鳞癌预后分子标志物奠定基础。

背景：细胞周期失调控参与食管鳞癌发生发展。本研究目的是探索miR-424在食管鳞癌细胞周期调控及增殖中的作用。.方法：通过体外及体内实验探索miR-424在食管鳞癌增殖中的作用。运用染色质免疫沉淀探索miR-424的转录激活。 运用miRNA-核蛋白免疫共沉淀验证mi-424与靶基因的结合。.结果：食管鳞癌中miR-424表达较正常食管黏膜上皮显著上调，并且与食管鳞癌患者预后显著相关。抑制或过表达miR-424可在体内和体内分别减弱或增强食管鳞癌增殖。miR-424表达受E2F1转录调控，并且在G1/S期表达增高。敲除或过表达miR-424可分别抑制或促销食管鳞癌细胞的G1/S期和G2/M期细胞周期进程，并且这一作用是通过两个新发现的miR-424靶基因PRKCD和WEE1实现的。因此，miR-424敲除，一方面上调PRKCD表达，通过PRKCD及下游p38 MAPK, JNK信号通路的激活，增强P21蛋白稳定性，从而抑制CDK2激活和G1/S期转化；另一方面，可上调WEE1表达，抑制CDC2活性从而抑制G2/M期转化。.结论：miR-424通过对食管鳞癌细胞周期的多层次调控促进食管鳞癌增殖，有望作为新的食管鳞癌预后分子标志物。