lncRNA RP11-264F23.3抑制宫颈癌细胞增殖的分子机制研究

lncRNA RP11-264F23.3 (RP11), a novel long noncoding RNA (lncRNA) of unknown function, which is down-regulated significantly in cervical cancer tissues, is identified by lncRNA sequencing. We previously found that overexpression of RP11 inhibited cell proliferation in Hela and SiHa cell lines, via promoting G1/S phase arrest. Furthermore, bioinformatic analyses and experiments suggested that RP11 could bind with EZH2 and reduce expression of the target CCND2. Based on above results, we therefore hypothesized that RP11 might suppress cervical cancer cell proliferation by recruiting EZH2 and subsequently decreasing the expression of CCND2. To achieve this goal, we design to over-express and knock-down RP11 and evaluate the impact of RP11 on the abilities of cell proliferation. Highlighted by the relationship between CCND2 gene and RP11, we design to investigate the interaction between CCND2 and RP11 via ChIP, luciferase reporter assay, RIP, RNA pull down and chromosome conformation capture. Our study is the original source of innovation and can provide new potential target and clue for the treatment of cervical cancer.

lncRNA RP11-264F23.3（简称RP11）是我们采用高通量测序技术筛选获得的、显著低表达于宫颈癌组织、功能未知的一条lncRNA。前期发现，过表达RP11能显著抑制Hela和SiHa细胞增殖，细胞周期阻滞在G1/S期；生物信息学分析与实验均提示RP11与EZH2相互结合；过表达RP11后EZH2靶基因CCND2表达显著下降。因此我们提出RP11通过招募EZH2特异性结合于CCND2启动子抑制宫颈癌细胞增殖的科学假说。本研究拟采用过表达/沉默策略，论证RP11在宫颈癌增殖中的作用；应用ChIP、荧光素酶活性分析、RIP、RNA pull-down、染色体构象捕获等技术，充分论证RP11招募EZH2结合于CCND2启动子表观调控CCND2表达的分子机制。研究具有源头创新性，有助于阐明宫颈癌发生发展的机制，为宫颈癌的治疗提供新线索和潜在靶标。

宫颈癌是女性生殖系统最常见的肿瘤之一，严重危害女性健康。世界范围内，宫颈癌的死亡率位居女性恶性肿瘤的第三位。虽然有手术切除子宫、化疗、放疗等方法治疗宫颈癌，但是会使年轻患者丧失后续生育能力，严重危害后代的数量和质量，已经成为重大的社会问题。近年来，lncRNA不断被证实参与了肿瘤的增殖、分化、转移等过程。我们采用高通量测序技术筛选获得了一条显著低表达于宫颈癌组织、功能未知的lncRNA: RP11-264F23.3（简称 CCND2 AS1）。通过实时定量PCR技术验证了46例组织样本中CCND2 AS1的低表达情况，且与病人的年龄及肿瘤的大小显著相关；CCK-8技术检测了CCND2 AS1抑制宫颈癌细胞增殖的作用，流式细胞仪发现 CCND2 AS1通过调节细胞周期G1/S期转变来影响宫颈癌细胞增殖，裸鼠皮下成瘤实验揭示了CCND2 AS1对宫颈癌的抑瘤作用；CCND2 AS1通过调控细胞周期蛋白CDK4、CCND1及CCND2的表达抑制宫颈癌细胞的增殖；甲基化特异性PCR和亚硫酸盐测序法检测了CCND2 AS1启动子区的甲基化状态，揭示了CCND2 AS1在宫颈癌中的低表达受启动子区甲基化调控的可能机制。研究具有源头创新性，有助于阐明宫颈癌发生发展的机制，为宫颈癌的治疗提供新线索和潜在靶标。