靶向lncRNA HOTAIR的恶性胶质瘤反义显像研究

As a long non-coding RNA, the expression of HOTAIR is strongly increased in many types of tumors, and it is considered as an pro-oncogene. It promotes the formation and metastasis of tumors by epigenetic regulation of gene expression in transcriptional and post-transcriptional levels. It is reported HOTAIR is a predictor of glioma prognosis and biomarker of identifying glioma molecular subtypes. However, its role in promoting the occurrence and progress of glioma remains unclear. We hypothesize that HOTAIR plays an important role in promoting cancer as an pro-oncogene by promoting cell proliferation and epithelial mesenchymal transition (EMT). Combination of nuclide tracing technique and antisense technology, SPECT/CT antisense imaging will display the expression and biodistribution of HOTAIR in tumor tissues. HOTAIR must be a potential molecular marker of early diagnosis of glioma. In previous study, we found HOTAIR was overexpressed in glioma specimens. 99mTc-HOTAIR ASON was radiolabeled and showed a relative higher uptake rate with U87 cells. In this project, we plan to illustrate the molecular mechanism of HOTAIR in promoting glioma by RT-PCR, Western Blot and Flow Cytometry Technology on different types of glioma cell lines, and discuss the targeting efficacy of 99mTc-HOTAIR ASON to HOTAIR by gene and cellular levels; meanwhile, the expression of HOTAIR will be noninvasively visualized in vivo by 99mTc-HOTAIR ASON SPECT/CT imgaging, which should be a new targeting approach for early diagnosis of malignant glioma.

HOTAIR通过表观遗传学、转录及转录后水平等多个层面调控基因表达，扮演着原癌基因的角色。有报道HOTAIR通过调控细胞周期基因、抑制P21和P16表达而促进胶质瘤形成，是判断其分级和预后、识别分子亚型的生物标志物。我们推测，HOTAIR通过促进肿瘤细胞增殖和EMT而发挥促瘤作用；结合核素示踪和反义技术，SPECT/CT反义显像可观察HOTAIR在瘤组织内的分布和数量，可用于胶质瘤早期靶向性诊断。前期工作中，我们已验证HOTAIR在胶质瘤中呈高表达水平，预实验已合成99mTc-HOTAIR ASON探针，但标记率有待提高。本项目中，我们以不同胶质瘤细胞系为对象，拟通过RT-PCR、Western Blot、FCM技术，阐明HOTAIR促瘤作用的分子机制；探讨分子探针在基因、细胞水平上的靶向性；通过SPECT/CT可视化呈现动物体内HOTAIR的表达和分布，为胶质瘤早期诊断提供新的手段。

HOTAIR通过表观遗传学、转录及转录后水平等多个层面调控基因表达，扮演着原癌基因的角色。我们推测结合核素示踪和反义技术，SPECT/CT反义显像可观察HOTAIR在瘤组织内的分布和数量，可用于胶质瘤早期靶向性诊断。前期工作中，我们已验证HOTAIR在患者恶性胶质瘤中呈高表达。本项目中，我们以不同胶质瘤细胞系为对象，拟通过RT-PCR、Western Blot、FCM技术，阐明HOTAIR如何影响细胞增殖，探讨分子探针在基因、细胞水平上的靶向性，通过SPECT/CT可视化呈现动物体内HOTAIR的表达和分布，为胶质瘤早期诊断提供新的手段。结果显示：（1）HOTAIR在U87MG细胞系中的表达丰度高于U251细胞系。转染HOTAIR ASON后，U87MG细胞的迁移能力明显受抑制，且不受是否与99mTc标记、偶联。（2）依据寡核苷酸探针设计原则，结合特异性靶向HOTAIR的siRNA序列，确定分子探针的ASON序列为：5′-CCACGAAGCTAGAGAGAGA-3′，长度为19个碱基，对 ASON 序列进行全程硫代修饰以提高其稳定性，两端2′甲基化修饰等，以提高分子探针稳定性。选择HYNIC作为偶联剂，将99mTc与HOTAIR ASON进行偶联。（3）最优化的反义探针标记条件为： ASON 4OD溶于50μl缓冲液，HYNIC: ASON摩尔比25：1，避光反应1h。反应完毕后调整总体积至500μl，离心超滤10min（体积＜50μl）。随后依次加入100μlTrinine（100mg/ml）， 20μｌ新鲜锝淋洗液（3mCi），4μl新鲜配置的SnCl2.2H2O(1mg/ml)溶液，混匀，避光反应60min。此条件下，最高标记率达到71.3%±5.2%，放射化学纯度约为95%，体外稳定性好，6h血清放射化学纯度仍在90%以上。标记前后、标记物纯化前后，ASON琼脂糖凝胶检测均显示未见核算降解。（4）BALB/c裸鼠右前腋下种植U87MG细胞，成瘤后，病理切片证实造模成功。体内生物分布显示分子探针在胃内摄取最高，双肾及膀胱是排泄通路而摄取也较高，此外小肠摄取也较高。肿瘤部位较软组织本地及血池本底相比，滞留量高，T/NT比值较高，肿瘤部位6h的%ID/g为2.26±0.68。SPECT显像显示腹腔放射性摄取较高、长时间滞留，肿瘤部位显示清楚，仍需进一步改进和优化。