miR-375在宫颈上皮内瘤变1级转归中的分子机制及预测意义研究

There is a great controversy on the clinical management of cervical intraepithelial neoplasia grade 1 (CIN1), and over-treatment is quiet common. 2012 updated ASCCP (American Society for colposcopy and cervical pathology) consensus guidelines on the management of CIN1 can reduce the rate of over-treatment. However the long-interval and long-time follow-up put huge psychological pressure on patients, and expends considerable resources of medical and health, which make the exploratory research on new tactics of CIN1 triage significant. In our previous study, we screened the different expressions of miRNAs between cervical normal tissue, CIN tissues and cancer tissues by microarray. We found that miR-375 was progressively down-regulated in CIN2-3 and cancer tissues, compared with normal tissues. The preliminary experiment showed that low expressions of miR-375 was correlated with HR-HPV persistence in CIN1 patients. Further work demonstrated that the expression of miR-375 were negative correlated with HPV16 E6/E7, while the expression of E6/E7 did not significantly altered miR-375 transcripts. Specificity protein 1 (Sp1) and JunD are potential target genes of miR-375 predicted mutually by miRNA TargetScan and Pictar sofeware online. What is more,they are important transcript factors and both have binding site on the early gene promoter P97 of HR-HPV (which regulates the expression of E6/E7). Our previous and preliminary work suggest that miR-375 may influence the biological characteristic and behaviour of CIN1 cells by regulating the expression of E6/E7. In this project, we plan to clarify the molecular mechanism of miR-375 influencing the biological characteristic and behaviour of CIN1 cells. Meanwhile, we intend to confirm the sensitivity and specificity of miR-375 in the prognosis of CIN1 through molecule epidemiology study. This study is conducted to provide scientific evidence for the new tactics of CIN1 triage.

目前对宫颈上皮内瘤变1级（CIN1）的临床处理存在过度治疗倾向。2012年更新的ASCCP临床处理指南可以避免部分CIN1的过度治疗，但是长间隔、长时间的随访给患者带来很大的心理负担，并造成较多医疗卫生资源的消耗，寻求能即刻分流CIN1患者的新策略具有积极的科学意义。我们的前期研究表明，miR-375表达在CIN2-3及宫颈癌组织中呈进行性显著下调，miR-375的异常低表达可能与CIN1向CIN2-3进展有关。本项目在前期研究基础上，拟通过分子生物学技术证实miR-375通过调控HR-HPV癌基因E6/E7的共同启动子序列P97的转录活性，影响E6/E7的表达，进而对CIN1细胞的生物学特征及行为产生影响；并采用分子流行病学研究方法验证miR-375预测CIN1转归的灵敏度和特异度。本研究旨在为探寻CIN1分流处理的新策略提供科学依据。

项目背景: 伴随宫颈癌筛查的广泛开展，大量低度宫颈病变（LSIL/CIN1）被诊断发现。LSIL/CIN1的临床处理需要考量2个因素：1、LSIL/CIN1随访存在疾病进展风险；2、活检局限性存在高级别宫颈病变（（HSIL/CIN2+）漏诊风险。.主要研究内容： 1、队列研究：检测入组病例宫颈脱落细胞miR-375和HPV16/18，以6个月为随访间隔连续随访2年。每一次随访均行宫颈细胞学检查及高危型HPV检测进行初筛，初筛异常者行阴道镜检查，镜检异常者行镜下活检。采用方差分析评估miR-375表达与疾病转归关联性；采用Cox回归模型对HPV16/18分型与疾病转归进行关联分析；2、横断面研究：检测研究对象入组时宫颈脱落细胞10个目标miRNA表达量，与手术病理结果进行关联分析；3、体外试验：将miR-375模拟物转染至LSIL/CIN1细胞株20863，流式细胞仪检测细胞凋亡变化，Cell Counting Kit-WST-8（CCK-8试剂盒）检测细胞增殖改变，并利用Real-time RT-PCR和Western Blot的方法检测Sp1/JUND的mRNA和蛋白水平。.重要结果及关键数据： 1、队列研究：共273例病例完成随访，HPV16/18阳性与疾病的2年累计进展率显著相关（风险比2.708；95%可信区间1.432-5.121；P = 0.002）。分层分析提示HPV16/18的预测价值在细胞学轻度异常且年龄30岁及以上LSIL/CIN1患者中具有显著意义。MiR-375表达水平在疾病进展组与非进展组间无统计学差异。2、横断面调查：miRNA195、miRNA20a、miRNA16-2、miRNA29a表达对宫颈活检LSIL/CIN1漏诊HSIL/CIN2+具有预测价值，其中miRNA29a检测的敏感度和特异度均最高（分别为85.4%，83.5%）；3、体外试验：在20863细胞株中，miR-375高表达可以诱导细胞凋亡，抑制细胞增殖，但是对Sp1/JUND的mRNA和蛋白水平无显著改变。.科学意义： HPV16/18分型对细胞学轻度异常、且30岁及以上的低度宫颈病变患者具有良好的预测价值。miRNA29a对宫颈活检LSIL/CIN1漏诊HSIL/CIN2+具有良好的预测价值。