Linc-ROR/ATG9B诱导自噬调控宫颈癌放疗敏感性的机制研究

Radio-resistance is the main reason for failure of cervical cancer after therapy.Long intergenic noncoding RNA, regulator ofreprogramming, linc-ROR was shown to play an important part intumorigenic, invasion, metastasis, drug resistanceand others.We observe that there was high expression of linc-ROR and autophagy 9B in cervical cancer patients after local control with radiotherapy by gene chip.ATG9B expression was decreased when linc-ROR was knockout in heLa cell line. According to reviews andbioinformatics analysis, we will do some researchon how linc-ROR regulating in cervical cancer affect radio-sensitivity.Firstly, we will observe the relation between linc-ROR and radio-resistance of cervical cancer from the cell, animal and clinical samples by qRT-PCR, in-situ hybridization and others. Secondly，we study the relationship between linc-ROR and autophagyof cervical cancer though knockout or overexpressionof linc-ROR gene and lastly, we will reveal the mechanismthat linc-ROR may compete withmiR-145 to regulate ATG9B and by raising the autophagy pathway to produce radio-resistance of cervical cancer by luciferase reporter geneassay,RIP and others.Our objective is to explore new ideas and provide theoretical basis for thepredictive marks of radio-resistance and effective targets of radio-sensitizationfor cervical cancer from lncRNA angles.

放射抵抗是宫颈癌治疗失败的主要原因。长链非编码RNA ROR（linc-ROR）与肿瘤的发生、侵袭、转移及耐药等密切相关。课题组前期研究发现linc-ROR及自噬相关蛋白9B（ATG9B）的表达与放疗抵抗正相关，且敲除linc-ROR后细胞中ATG9B的表达下调，结合文献报道及生物信息学分析，本课题拟通过：1）采用qRT-PCR、原位杂交等方法，从细胞、动物、临床标本等不同水平观察linc-ROR的表达与宫颈癌放射抵抗的关系；2）敲除或过表达linc-ROR，观察linc-ROR与辐射诱导宫颈癌细胞自噬的关系；3）进一步应用siRNA、荧光素酶报告基因、RIP等技术，揭示linc-ROR通过miR-145调控靶基因ATG9B的作用机制以及影响宫颈癌放射敏感性的机制。预期阐明linc-ROR可能靶向调节ATG9B诱导自噬导致宫颈癌产生放射抵抗的机制，为宫颈癌的放疗增敏研究提供新的靶点。

宫颈癌是女性最常见的恶性肿瘤之一，其发病率及病死率均较高，严重危害妇女健康，放射治疗是主要治疗手段。目前研究表明影响放射治疗效果的主要原因在于肿瘤组织内在的放射敏感性差异。因此，探讨宫颈癌放射抵抗生物学机制，对于提高放疗疗效，改善患者预后具有十分重要的意义。最新研究发现，LincROR与肝癌和结直肠肿瘤放射敏感性密切相关。本课题首先应用qRT-PCR、流失细胞仪、MTT以及细胞克隆等方法检测宫颈癌组织样本及宫颈癌细胞系，结果发现linc-ROR的表达与细胞增殖呈正相关，与凋亡及放射敏感性呈负相关；并且发现linc-ROR的表达与miRNA145呈负相关而与ATG9B的表达呈正相关。电镜及MDC染色检测linc-ROR上调或抑制的转染细胞经照射后，结果发现overROR-Siha细胞中的自噬水平明显升高，而siROR-SihaR细胞自噬水平降低。共转染细胞系mimics NC LincROR siha；miRNA145 mimics LincROR siha；inhibitor NC shROR siha-R；miR145 inhibitor shROR siha-R，上述细胞系经4Gy照射后，细胞克隆结果表明miRNA145 mimics LincROR siha较mimics NC LincROR siha明显减少；而miR145 inhibitor shROR siha-R较inhibitor NC shROR siha-R明显增多。应用电镜及MDC染色检测上述转染细胞的自噬水平，结果表明miRNA145 mimics LincROR siha细胞中的自噬水平较mimics NC LincROR siha下降，而miR145 inhibitor shROR siha-R较inhibitor NC shROR siha-R细胞的自噬水平升高。体内实验结果进一步验证上述实验结果。以上研究结果说明linc-ROR调控宫颈癌放射敏感性是通过改变宫颈癌细胞中linc-ROR及miR-145的表达，证实了linc-ROR通过miR-145调控其靶基因ATG9B调节自噬影响宫颈癌的放射敏感性。该课题探讨了宫颈癌放射抵抗的生物学机制、为宫颈癌放射敏感性的预测指标及放射增敏提供了新的实验数据，对于提高肿瘤局控率，改善患者预后以及实施个体化治疗具有十分重要的意义。