口腔白斑癌变过程中DNA损伤信号通路相关lncRNA的筛选及其调控机制的研究

Leukoplakia is the most common premalignant or potentially malignant lesion of the oral mucosa. The percentages of malignant transformation rates of oral leukoplakia have been reported to range from 10 up to 36%. The mechanism of carcinogenesis still remains unknown. It’s now confirmed that the DNA damage response takes part in the cancer occurrence and progression. Cells would activate the DNA damage signaling pathway for stopping the cell cycle to repair the DNA damage when DNA damage occurs.The applicant have investigated that the histoneγ-H2AX、chk2 of activated DNA damage signaling pathway had different expression in oral normal mucosal and oral leukoplasia (mild-moderate-severe dysplasia), which indicated that DNA damage signaling pathway had been activated in the oral precancerous lesions, but the regulatory mechanism was not clear. Recently, scholars found that lncRNA as a kind of regulatory RNA, detection and repair of DNA damage in a complex network involving DNA damage, plays an important role in maintaining the stability and integrity of the human genome..Until now there has been no report on the research of relationship between oral precancerous lesions, DNA damage signaling pathway and it’s regulatory mechanism then. Therefore, we plan to observe dynamically the activation of DNA damage signaling pathway in oral normal mucosal and oral leukoplasia (mild-moderate-severe dysplasia) and oral squamous cell carcinoma. We also plan to conceive the lncRNAs expression profile related to DNA damage signaling pathway of OLK carcinogenesis and select several lncRNAs serve as biological tags to investigate the impact of the lncRNAs and their target molecules on in DNA damage signaling pathway of OLK carcinogenesis and the biological functional effect. The expression profile of lncRNAs of OLK tissue is expected. It’s also anticipated that the mechanism of the lncRNAs’ regulation on DNA damage signaling pathway and the relation between the lncRNAs and OLK carcinogenesis, which could be a basis for lncRNAs in biological treatment on OLK.

口腔白斑（OLK）是一种有潜在不良预后的难治性癌前病变，癌变率10－36％，癌变机制尚不明确。现已证实DNA损伤与人类肿瘤的发生发展密切相关。当DNA损伤发生时生物体会激活DNA损伤信号通路让细胞周期停下来对损伤进行修复。申请人前期研究发现DNA损伤组蛋白γ-H2AX、chk2在正常组织、OLK中表达存在差异，但机制尚不明了。最近研究发现lncRNA在检测和修复复杂的DNA损伤网络，维持人类基因组的稳定和完整中发挥着重要的作用。因此申请人拟动态观察从口腔黏膜正常组织-轻度异常增生OLK-中重度异常增生OLK-口腔鳞癌这一过程中DNA损伤信号通路；筛选调控OLK癌变的DNA损伤信号通路相关lncRNAs；进一步验证这些lncRNAs在DNA损伤信号通路和OLK癌变过程不同阶段的功能及相关靶基因。通过本项目的研究，有助于进一步揭示OLK的癌变机制，并为不同病理阶段的口腔白斑生物学治疗提供依据。

口腔白斑（OLK）是一种有潜在不良预后的难治性癌前病变，癌变率10－36％，癌变机制尚不明确。口腔白斑（OLK）的发展经由多个阶段：由轻-中-重度异常增生到原位癌，最后发展成浸润癌，对其治疗和癌变的预防是降低口腔癌发病率的关键问题。课题组采用 Affymetrix GeneChip® Human Transcriptome Array 2.0 芯片检测和分析了正常口腔黏膜、低风险口腔白斑、高风险口腔白斑和早期舌鳞癌组织标本，利用KEGG数据库对差异基因进行pathway分析，得到口腔白斑癌变过程中DNA损伤γH2AX-ATM-Chk2信号通路。为了验证该结果课题组调取了口腔病理科61例OLK（分为低风险异常增生组OL low-risk 和高风险异常增生组OL high-risk ，分别为41例和20例），33例口腔鳞状细胞癌（OSCC，均由口腔白斑癌变而来）的组织蜡块，15例正常口腔黏膜作为对照。结果显示ATM与γH2AX表达正相关，与口腔白斑异常增生程度正相关。对26例口腔白斑癌变患者（7例失访）的回顾性研究发现ATM在口腔白斑预后评价中的价值可以和病变部位、病理异常增生程度相等。在以上基础上课题组再进一步采用western blot、RT-PCR技术检测发现伴有中重度异常增生OLK组织的γH2AX和Chk2蛋白、mRNA表达显著增高。这些研究结果均提示：在口腔白斑阶段已经发生DNA损伤，γH2AX-ATM-Chk2信号通路参与口腔白斑不同发展阶段和癌变过程中的DNA损伤修复。课题组进一步通过lncRNA芯片分析了DNA损伤相关lncRNAs，通过富集分析和深入数据分析，得到γH2AX-ATM-Chk2信号通路相关lncRNAs。通过lncRNA与转录因子关联性分析验证lncRNA的功能和对应的靶基因。.通过以上实验内容课题组研究了口腔黏膜正常组织-轻度异常增生OLK-中重度异常增生OLK-口腔鳞癌这一过程中DNA损伤信号传导通路；筛选了调控OLK癌变过程中DNA损伤信号通路相关lncRNA；进一步验证这些lncRNAs在OLK癌变过程中的功能及相关靶基因。通过本项目的研究，对于阐明OLK癌变的分子机制，寻找预防口腔白斑癌变的新靶点有重要意义。