非编码长链RNA-LOC102723323调控乳腺癌细胞ERBB3表达的分子机制研究

Molecular targeting drugs against ERBB2,such as trastuzumab or lapatinib have been widely used in breast cancer patients with a good clinical response. However,during the treatment, part of these cancer patients show tumor progress because of drug resistance. The mechanism of this process is not very clear. ERBB3, as the only member lacking catalytic kinase function in ERBB family, can be activated by the formation of heterodimers with ERBB2 and other members. Furthermore, ERBB3 has been proved to play important roles during the process of the initiation and progression in breast cancer cells. Therefore, investigation of the molecular mechanism of ERBB3 expression in breast cancer cells has great potential clinical significance to explain the reasons of carcinogensis and drug resistance in breast cancer. Based on other research reports and our previous data, our hypothesis is that long non-coding RNA LncRNA-LOC102723323 can bind with Micro-RNA miR-450b-3p in breast cancer cells. And this binding will unblock the inhibition of the ERBB3 expression regulated by miR-450b-3p. Then the ERBB3 expression levels will be up-regulated in these cells. Meanwhile ERBB3 downstream signals will be activated.Thereby, these biological processes will promote the initiation, progression and drug resistance in breast cancer. With the applications of bioinformatics and cell biology tools, our project will be performed in cancer cells, animal model and human tissue samples. Our project will explore the further mechanisms of Loc102723323 in the regulation of the ERBB3 expression in breast cancer cells.Moreover, this study may can provide the theory of more personalized target therapy in breast cancer patients.

目前针对ERBB2的分子靶向药物，如曲妥珠单抗、拉帕替尼等在乳腺癌中取得了良好的临床疗效。部分患者治疗过程中出现耐药，目前认为引起耐药可能与ERBB3有关。ERBB3本身没有激酶活性，不是激酶抑制剂的直接靶标，但却能传递与肿瘤增殖密切相关的信号。因此研究ERBB3表达的分子机制对乳腺癌发生发展及药物耐受具有重要的临床价值。根据文献及我们的前期工作，我们推断lncRNA-LOC102723323可通过结合miR-450b-3p，解除对ERBB3的抑制，上调ERBB3的表达，进而激活ERBB3下游信号，促进乳腺癌发生发展及药物抵抗。本课题拟采用生物信息学和细胞生物学等技术，在动物水平、临床组织标本及细胞水平展开研究。本研究的完成不仅可阐明lncRNA-LOC102723323在乳腺癌发生发展中调控ERBB3表达的分子机制，而且可为乳腺癌的防治提供新思路和新靶标。

临床上乳腺癌患者抗ERBB2治疗中，部分患者中出现耐药，耐药可能与ERBB3有关。ERBB3本身没有激酶活性，不是激酶抑制剂的直接靶标，但却能传递与肿瘤增殖密切相关的信号。研究ERBB3表达的分子机制对乳腺癌发生发展及药物耐受具有重要的临床价值。本课题关注在ERBB2阳性乳腺癌中LncRNA-LOC102723323和miR-450b-5p的分子生物学作用。.研究结果显示：与癌旁正常腺体组织相比，ERBB2阳性乳腺癌中ERBB3和LncRNA-LOC102723323表达均显著升高，而miR-450b-5p明显下调。测序分析LncRNA-LOC102723323高表达的癌组织中肿瘤增殖相关信号通路因子上调。此外，ZR-75-1细胞中上调LOC102723323后ERBB2和ERBB3表达显著升高，而沉默LncRNA后两者表达则显著下降。沉默表达LOC102723323，细胞迁移和侵袭能力明显下降，且磷酸化AKT和磷酸化ERK表达明显下调。过表达LncRNA则磷酸化AKT和磷酸化ERK表达明显升高。ERBB2阳性乳腺癌细胞株给与抗ERBB2药物梯度浓度加压培养，传代细胞中：LOC102723323高表明显上调。结果提示LncRNA-LOC102723323和 ERBB3可能与ERBB2阳性乳腺癌的发生发展相关。经生物信息序列比对显示LOC102723323与miR-450b-5p存在可能结合位点，进行荧光素酶实验，结果显示：LOC102723323能与miR-450b-5p结合，且结合后信号改变具有统计学意义。进一步分析ERBB3也有与miR-450b-5p结合位点，同样荧光酶素实验证实ERBB3可与miR-450b-5p相结合。通过构建载体包装慢病毒感染ERBB2阳性乳腺癌细胞株筛选获得LOC102723323稳定表达细胞株，使用高度免疫缺陷NSG小鼠构建负瘤模型。动物实验结果：LOC102723323稳定表达肿瘤体积及重量明显高于对照组，说明LOC102723323稳定表达能明显加速肿瘤细胞增殖生长。本研究结果阐明ERBB2阳性乳腺癌细胞中：lncRNA-OC102723323可能通过结合miR-450b-5p，解除对ERBB3的抑制，上调ERBB3的表达，进而激活ERBB3下游信号，促进乳腺发生发展。课题结果基本符合预期课题科学假想。

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