一个功能未知的环状RNAcirc-0036602促进HPV16阳性宫颈癌侵袭转移的机制研究

The mechanism of circRNA involving in HPV16 E7 promoting the progression of cervical cancer is still unknown. Analysis of our previous microarray data with the RNA-seq, we identified circ-0036602 is highly over-expressed in HPV 16 positive cervical cancer. Also, the expression level of circ-0036602 is positively correlated with the expression of E7,the differentiation and lymph node metastasis, and negatively correlated with the long-term survival time of patients significantly. siRNA-mediated silence of circ-0036602 significantly suppressed the capacity of migration and invasion. Bioinformatics analysis and previous experiments indicated that circ-0036602 could regulate HMGB1 expression by competive binding miR-34a-5p and miR-431-5p. We therefore hypothesized that circ-0036602 could regulate HMGB1 expression to promote cervical cancer progression by acting as sponge of miR-34a-5p and miR-431-5p. The current project aims to validate that circ-0036602 could promote the migration and invasion of HPV 16 positive cervical cancer in vitro and in vivo at first. Secondly, the mechanism of competing endogenous RNA between circ-0036602, miR-34a-5p/ miR-431-5p, and HMGB1 will be deeply validated by the experiments of RIP and dual-luciferase reporter gene system. Finally, the correlation between circ-0036602/miR-34a-5p,miR-431-5p/ HMGB1 ceRNA pathway and HPV16 positive cervical cancer progression will be further validated in animal experiment and samples of clinical cervical cancer patients. There is no report about function and mechanism of circ-0036602 in HPV16 positive cervical cancer, our study offers a new target for the diagnosis and treatment of cervical cancer.

环状RNA参与HPV16E7促进宫颈癌恶性进展的机制未明。circ-0036602（简称CI）是前期基于宫颈癌细胞芯片及组织筛选验证获得的一条敲减E7后下调、高表达于HPV16+宫颈癌的环状RNA。CI与E7表达和淋巴结转移正相关，表达越高预后越差；沉默CI可抑制HPV16+宫颈癌细胞侵袭转移。预实验及生信分析提示CI可吸附miR-34a-5p/431-5p转录后共同上调促癌基因HMGB1。故提出“CI通过竞争性内源RNA（ceRNA）机制吸附miR-34a/431上调HMGB1，促进HPV16+宫颈癌侵袭转移”的科学假说。本研究拟体内外实验明确CI促进HPV16+宫颈癌侵袭转移的生物学功能；设计拯救实验，阐明CI/miR-34a、431/HMGB1调控宫颈癌侵袭转移的ceRNA机制；并在动物及临床水平验证该分子机制。有关CI功能和机制未见报道，本研究可为HPV16+宫颈癌诊疗提供新靶标。

circ-0036602是项目组前期基于宫颈癌细胞芯片及组织筛选验证获得的一条敲减E7后下调、高表达于HPV16阳性宫颈癌的环状RNA。通过聚合酶链式反应和组织学验证，结果表明circ-0036602与E7癌蛋白的表达密切相关，且circ-0036602在HPV16阳性宫颈癌组织中和细胞中显著高表达，与肿瘤的病理分级和淋巴结转移相关，且其表达越高患者预后越差。沉默circ-0036602后可显著抑制HPV16阳性宫颈癌细胞株的增殖、侵袭和迁移，促进HPV16阳性宫颈癌细胞株凋亡；抑制裸鼠皮下荷瘤动物模型肿瘤生长。circ-0036602位于人类15号染色体的长臂25.3区，由宿主基因ZNF592的5至10号外显子环化形成，属于外显子来源的circRNA，核质分离试验提示其大多定位在细胞质中。进一步采用免疫共沉淀（RIP）、PITA、miRanda在线数据库和荧光素酶报告基因实验证实circ-0036602可与miR-34a-5p和miR-431-5p结合，在HPV16阳性宫颈癌组织中行qRT-PCR验证发现，miR-34a-5p和miR-431-5p表达显著下调，提示circ-0036602能够吸附抑癌基因miR-34a-5p及miR-431-5p、通过ceRNA机制促进HPV16阳性宫颈癌的侵袭转移。接下来RNAseq检测沉默circ-0036602后Caski细胞差异表达基因，qRT-PCR和western blot实验验证发现，HMGB1与circ-0036602表达正相关，且与miR-34a-5p、miR-431-5p表达负相关，证实了circ-0036602通过竞争性结合miR-34a-5p、miR-431-5p上调HMGB1表达促进了HPV16阳性宫颈癌侵袭转移。有关circ-0036602促进HPV16阳性宫颈癌恶性进展的功能和机制尚未见报道，本研究将可为HPV16阳性宫颈癌的防治提供新靶标。