环状RNA circDNAJC16在肺癌中的功能及分子机制研究

CircRNAs are derived from the alternative splicing of the precursor mRNA and are important to the cancer, but the study on the mechanism of lung cancer is still at an initial stage. Our previous studies have shown that circDNAJC16 was specifically down-regulated in lung cancer tissues compared to para-carcinoma tissues, and inhibits lung cancer cell proliferation, promotes its apoptosis. We examined the binding site of miR-93-5p by dual-luciferase reporter system. Therefore, we hypothesized that circDNAJC16 as a competitive endogenous RNA to regulate target genes to participate in cell proliferation or apoptosis signaling pathway through miRNA, thus affecting the occurrence of lung cancer. In addition, we predicted the binding site of PTB protein on circDNAJC16 through bioinformatics prediction and detected relative expression positively correlated with its parental genes. Therefore, we suggested that PTB protein can be used to regulate splicing products in the precursors mRNA splicing events, which can break the expression balance between DNAJC16 and circDNAJC16 in lung cancer, thus affecting the occurrence of lung cancer as well. Through this project, we will elucidate circDNAJC16 down-regulated molecular mechanism underlying lung cancer, through the experiment in vitro and in vivo by experimental method such as quantitative PCR, immunohistochemistry and CLIP-seq. Our study may extend a new layer for the diverse mechanisms throng which the circular RNA function and will shield light on the new diagnose and prevention strategy for lung cancer.

环状RNA来源于RNA前体的可变剪接，对肿瘤有重要作用，可具体对肺癌作用机制研究尚处于起步阶段。我们前期研究显示circDNAJC16在肺癌组织中表达低于癌旁，能抑制肺癌细胞增殖并促进其凋亡，且circDNAJC16与miR-93-5p相互作用；另外，RNA结合蛋白PTB在circDNAJC16上有结合位点，并与DNAJC16基因表达量正相关。因此我们设想PTB蛋白调控DNAJC16前体mRNA剪接引起肺癌细胞中circDNAJC16表达下调，进而circDNAJC16通过miR-93-5p调节靶基因参与肿瘤细胞增殖和凋亡，从而促进肺癌发生发展。本研究将利用定量PCR，免疫组化和CLIP-seq等实验方法通过体外实验及动物模型对circDNAJC16在肺癌中低表达的机制及其调控肺癌细胞增殖、凋亡的机制做深入研究，以揭示circDNAJC16对肺癌发生的作用机制，为肺癌防治提供新思路。

肺癌是全球发病率和死亡率多年高居榜首的恶性肿瘤。2020年中国肿瘤新发病例数和死亡人数均列所有癌症之首。研究调控型非编码RNA来源及作用是调控肺癌发生机制的又一新视野。本研究从环状RNA来源及调控靶基因相关信号通路揭示肺癌发生和转移的机制。研究结果表明：1）芯片法筛选肺癌患者癌与癌旁组织环状RNA差异表达谱，经RT-qPCR检测，结果显示circDNAJC16在肺癌组织中低表达，且表达量与患者的疾病进展及预后相关。2）体外实验和小鼠成瘤实验证实circDNAJC16促肿瘤凋亡，抑制肿瘤增殖、迁移和侵袭。3）通过数据库综合预测荧光探针原位杂交（FISH）及双荧光素酶报告实验结果表明circDNAJC16与miR-93-5p存在相互结合。但过表达或干扰circDNAJC16并不改变miR-93-5p表达量，提示circDNAJC16是通过碱基互补吸附miR-93-5p从而改变其对目标mRNA的调控作用。4）通过在线数据库综合预测及Western Blot验证，在H292中转染miR-93-5p类似物能降低CDKN1A的蛋白表达量；双荧光素酶报告实验验证CDKN1A基因3’UTR区域存在miR-93-5p的特异结合位点。5）在体外细胞克隆、划痕及Transwell实验中可观察到circDNAJC16过表达可降低细胞克隆形成率、迁移和侵袭能力，但同时过表达miR-93-5p或干扰CDKN1A可回复性调高细胞克隆形成率、迁移和侵袭能力。上述实验结果表明circDNAJC16通过靶向调控miR-93-5p/CDKN1A影响肺癌细胞增殖、迁移和侵袭。6）通过体外实验Western Blot检测及肺癌组织免疫组化结果显示E-cadherin表达随circDNAJC16表达升高，Hes1、Notch1、Cleaved Notch1和Vimentin表达降低，说明circDNAJC16调控micR-93-5p/CDKN1A通过Notch1通路影响细胞影响细胞的增殖及上皮-间质转化。本研究揭示了环状RNA circDNAJC16调控肺癌发生及发展的机制，为肺癌的新诊断和预防策略提供了新的靶点。