结合99mTc-TP1623靶向HER2显像研究环状RNA circITCH特异结合miR-944促进HER2阳性乳腺癌转移及分子机制

HER2 positive breast cancer is characterized by strong invasiveness，easy metastasis and low survival rate，which is closely related to the overexpression of HER2 gene，but the mechanism of HER2 up-regulated was not clear. Our preliminary study suggested that in HER2 positive breast cancer tissues，circle RNA circITCH expressed high level and was found to be significantly related to the metastasis of HER2 positive breast cancer and miR-944 expressed low level. We also found that circITCH had a response element for miR-944，while miR-944 potentially targeted and regulated TFAP2C，a transcription factor for HER2. We therefore speculated that circITCH promotes HER2 positive breast cancer metastasis through a specific combination of miR-944. We have prepared the HER2 imaging agent 99mTc-TP1623 to monitor the heterogeneity expression of tumor and metastatic HER2 in real time. The present project on the base of the relationship of circITCH with clinical prognostic parameters of positive HER2 breast cancer aims to clarify the molecular mechanism of circITCH/miR-944/TFAP2C signaling axis regulates positive HER2 breast cancer metastasis in cell and overall levels and screening other related circRNAs，providing basis for discovering new HER2 therapeutic targets and building the related regulatory network.

HER2阳性乳腺癌侵袭性强、易转移及存活率低的特点与HER2高表达水平密切相关，但HER2表达上调确切机制尚不清楚。我们前期研究发现HER2 阳性乳腺癌组织环状 RNAcircITCH高表达且与转移显著相关，而miR-944表达降低；circITCH有miR-944应答元件，而 miR-944有靶向HER2 转录因子TFAP2C潜能。由此我们假设circITCH 通过特异结合 miR-944促进HER2阳性乳腺癌转移。我们已制备HER显像剂99mTc-TP1623，可活体监测HER2异质性表达水平。本项目拟在探究circITCH 与HER2阳性乳腺癌临床预后关系基础上，从细胞和整体水平阐明circITCH/miR-944/TFAP2C信号轴调控HER2阳性乳腺癌转移及分子机制，芯片筛选其它相关circRNA，为构建相关circRNA调控网络及发现新HER2治疗靶点提供依据。

HER2是乳腺癌诊断和治疗的重要靶点。.一、探究microRNA对HER2阳性乳腺癌转移的调控作用机制。研究发现miR-136-5p和miR-198在HER2阳性乳腺癌细胞中表达下调，且miR-136-5p和miR-198均可通过靶向结合TFAP2C并抑制其表达；circ-ERBB2在HER2阳性乳腺癌中高表达，进一步研究发现circ-ERBB2与miR-136-5p、miR-198存在相互作用。功能研究表明，干扰circ-ERBB2通过上调miR-136-5p、miR-198的表达，抑制TFAP2C的表达，抑制HER2阳性乳腺癌细胞增殖、迁移、侵袭，促进细胞凋亡。进一步的体内研究证实，干扰circ-ERBB2对HER2阳性乳腺癌的抑制作用。随后为了更深入的探究microRNA对HER2阳性乳腺癌转移的调控作用机制，我们进一步明确了miR-1283表达在HER2阳性乳腺癌组织中显著降低，且miR-1283通过靶向TFAP2C 抑制其表达；KLF14在HER2阳性乳腺癌中显著下调，进一步研究发现KLF14能够与miR-1283结合并增强其表达。功能研究表明，过表达KLF14通过促进miR-1283的表达，下调TFAP2C水平，抑制HER2阳性乳腺癌细胞增殖，促进细胞凋亡，进而抑制肿瘤生长。综上，干扰circ-ERBB2/过表达KLF14通过上调miR-136-5pmiR-198/miR-1283的表达，下调TFAP2C的表达，进而抑制HER2阳性乳腺癌。.二、探究了99mTc、68Ga及18F标记的HER2 高亲和力与高特异性的小分子模拟肽靶向显像和定量评价HER2的作用。Micro SPECT/CT 显像示：99Tcm-TP1623 在荷瘤裸鼠体内HER2 高表达肿瘤摄取明显，低表达肿瘤明显低或无摄取；肿瘤摄取值与HER2 表达正相关（rs=0.76）。提示该探针可活体靶向定量HER2表达。Micro PET 显像示：18F-NFP-TP1296 在荷瘤鼠体内60min时肿瘤摄取值达高峰（6.26 ±0.27% ID/g），2h 肿瘤摄取值仍较高为 5.83 ±0.44% ID/g，阻断显像提示高度特异结合。68Ga-TP1580 在荷瘤鼠体内各时相肿瘤摄取值均明显低。综上，18F-NFP-TP1296 对HER2阳性乳腺癌诊断的灵敏度和特异性高，值得进一步研究。