miR-708/TM4SF1轴调控乳腺癌干性及耐药性的机理研究

microRNAs (miRNAs) are a class of endogenous non-conding small RNAs involved in tumor growth and progression. Transmembrane-4-L-six-family-1 (TM4SF1) is a plasma membrane glycoprotein that can promote the activity of cancer stem cells (CSCs). Our previous work showed that TM4SF1 was a target of breast cancer stemness and drug resistance. Then we screened a larger number of miRNAs and identified that miR-708 had the capacity to repress TM4SF1 expression. Moreover, there was a significant inverse correlation between miR-708 and TM4SF1 expression in breast cancer tissues. Functional studies revealed that miR-708 and TM4SF1 exerted opposite effects on aggressive phenotype of breast cancer cells. These findings encouraged us to hypothesize that miR-708 regulates the stemness of breast cancer likely via targeting TM4SF1, thereby affecting the growth, metastasis, and drug resistance. To test this hypothesis, this project aimed to confirm the biological roles of miR-708 in tumorigenesis and drug resistance of breast cancer cells in vivo, and to explore if overexpression of TM4SF1 reverses the anticancer effects of miR-708 in breast cancer. Also, the mechanisms for the regulation of breast cancer stemness and drug resistance by TM4SF1 will be determined. This project would uncover the roles of miR-708 in the regulation of breast cancer development and progression and associated mechanisms. The results would provide a potential therapeutic target for prevention and treatment of breast cancer.

微小RNA(miRNAs)是一类内源性非编码小RNA，参与调控肿瘤发生及发展。TM4SF1是一个含四个跨膜结构域的表面蛋白，具有促肿瘤干细胞(CSCs)活性的作用。课题组的前期实验结果显示TM4SF1是乳腺癌干性和耐药性的一个诱导因子，并且本课题组通过筛选大量的miRNAs鉴定出miR-708是TM4SF1的一个负调控基因，并且两者的表达水平在乳腺癌组织中呈负相关。因而，我们推测“miR-708通过靶向调控TM4SF1基因影响乳腺癌干性，抑制乳腺癌生长和耐药性”。为了证实该推测，本项目拟在体内外模型确认miR-708对乳腺癌干性及耐药性的调控作用，检测过表达TM4SF1基因对miR-708抗乳腺癌效应的逆转作用；并研究TM4SF1调控乳腺癌干性和耐药性的机制。本课题将揭示miR-708/TM4SF1轴在乳腺癌干性和耐药性中的调控功能和机制，为该病防治提供潜在的靶点。

LOXL1-AS1是一种新近发现的长链非编码RNA (lncRNA)，已被报道在几种类型的癌症中调节肿瘤进展。然而，LOXL1-AS1在乳腺癌中的表达和作用尚不清楚。在本研究中，我们试图识别参与乳腺癌转移的新型lncRNA调控因子。为此，我们在MCF7(低转移潜能)和MDA-MB-231(高转移潜能)细胞之间检测了42个与癌症相关的lncRNA。这些lncRNA已被发现影响多种癌症类型的侵袭性，但在乳腺癌中仍未明确。在42个候选lncRNA中，LOXL1-AS1在MDA-MB-231细胞中明显高于MCF7细胞。我们还发现，与非癌细胞相比，LOXL1-AS1在乳腺癌组织和细胞系中表达上调。LOXL1-AS1表达升高与乳腺癌患者的肿瘤分期和淋巴结转移相关。生物学上，LOXL1-AS1的过表达增强了乳腺癌细胞的迁移和侵袭，而LOXL1-AS1的低表达则抑制了乳腺癌细胞的迁移和侵袭。体内研究表明LOXL1-AS1缺失可抑制乳腺癌转移。机械的，LOXL1-AS1海绵吸附miR-708-5p增加核因子kB (NF-kB)的活性。LOXL1-AS1也可与EZH2蛋白相互作用，增强EZH2介导的miR-708-5p的转录抑制。回补实验表明，miR-708-5p共表达可减弱LOXL1-AS1诱导的乳腺癌侵袭性。此外，在乳腺癌标本中，LOXL1-AS1与miR-708-5p的表达呈负相关。总之，LOXL1-AS1上调通过阻断miR-708-5p的表达和活性促进乳腺癌的侵袭和转移。LOXL1-AS1是乳腺癌治疗的一个潜在靶点。