lncRNA在表阿霉素诱导悬浮乳腺癌细胞发生上皮细胞间质转化中的机制研究

Circulating tumor cells（CTCs） play an important role in the metastasis of breast cancer. lncRNAs (long noncoding RNAs) are known as key regulators of tumor metastasis recently. However, the role of lncRNA in chemotherapy induced EMT in suspension cultured cells remains elusive. The ultra-low attachment plates were recruited in cell culture to mimic the suspension condition of CTCs in circulating system. Significantly differential expressed lncRNAs between the epirubicin treated cells and control cells in suspension culture were screened out by lncRNA microarray. Two lncRNAs，NONHSAT101069 and FR363590 were chosen and validated by Real-time PCR. GDNF and PMEPA1 were distinguished as the target gene of ONHSAT101069 and FR363590 by bioinformatics analysis respectively. We hypothesize that epirubicin induces the EMT of breast cancer cells in suspension culture through upregulation of GDNF and PMEPA1 mediated by NONHSAT101069 and FR363590. We will determine the role of NONHSAT101069 and FR363590 in chemotherapy induced by epirubicin and verify the regulation of GDNF and PMEPA1 by NONHSAT101069 and FR363590. Our study will partly address the mechanism of breast cancer recurrence after adjuvant chemotherapy and provide theoretical basis for the prevention and treatment of breast cancer recurrence after adjuvant chemotherapy.

血行转移是乳腺癌转移的主要途径，循环肿瘤细胞(CTCs)的形成是血行转移的重要步骤。长链非编码RNA (lncRNA)在肿瘤转移中扮演重要角色。本课题以悬浮培养乳腺癌细胞模拟CTCs的状态，通过lncRNA芯片获得与表阿霉素致悬浮乳腺癌细胞EMT相关的差异lncRNA，选择表达差异最大的NONHSAT101069及FR363590并通过生物信息学预测得到其各自的靶基因GDNF和PMEPA1。表阿霉素诱导悬浮培养乳腺癌细胞模拟了CTCs的EMT过程，并提示NONHSAT101069和FR363590的高表达及其后续通路在此过程中的主导作用。本课题将在细胞水平、动物模型及临床标本三个方面探讨目标lncRNA及其下游通路在化疗相关乳腺癌CTCs的EMT过程中的作用、发生条件以及其详细调节机制。本课题的进展将有助于人们理解化疗后乳腺癌复发转移早期事件，为乳腺癌化疗后复发转移的防治提供新的理论基础。

乳腺癌是世界范围女性最常见的恶性肿瘤之一，其发病人数和死亡人数均位于女性恶性肿瘤前位。化疗是乳腺癌手术治疗的重要辅助手段。然而，化疗常常能够引发一些与治疗目的相悖的副反应。既往研究显示：化疗药物可以诱导乳腺癌细胞发生上皮间质转化（epithelial mesenchymal transition, EMT），且增强细胞的迁移和侵袭能力。长链非编码RNA（long non-coding RNA, lncRNA）是一类不编码蛋白质的，长度超过200bp的RNA分子。大量lncRNA被报道在多种肿瘤的化疗耐药和EMT中发挥重要作用。然而，有关lncRNA在乳腺癌表阿霉素耐药和导致乳腺癌细胞在发生EMT改变过程中所发挥的作用尚不清楚。.在本研究中，我们基于乳腺癌细胞MCF-7建立了表阿霉素耐药的细胞系MCF-7/ADR。研究证实MCF-7/ADR侵袭迁移能力增强，且有明显的间质化改变。通过高通量测序MCF-7及MCF-7/ADR，聚类分析显示MCF-7/ADR中有268个表达上调的lncRNA和49个表达下降的lncRNA。其中，位于第5号染色体的lncRNA NONHSAT101069被选择用于后续研究。研究发现：NONHSAT101069在乳腺癌组织及乳腺癌细胞中明显高表达。通过FISH定位及胞浆胞核RNA分离提取的方式，确认NONHSAT101069主要定位于细胞浆。后续实验证实：异常表达的NONHSAT101069通过EMT促进乳腺癌细胞表阿霉素耐药、侵袭迁移。为进一步探索NONHSAT101069的调控机制，运用生物信息学预测，通过荧光素酶报告基因实验证实：NONHSAT101069可作为ceRNA竞争性的结合miR-129-5p从而调控EMT的关键转录因子Twist1。体外回复实验表明：干扰miR-129-5p后，部分逆转了NONHSAT101069沉默诱导的乳腺癌细胞表阿霉素耐药和侵袭迁移能力的抑制。此外，上调Twist1明显提高乳腺癌细胞表阿霉素耐药和侵袭迁移能力，反之抑制。综上，研究表明NONHSAT101069通过NONHSAT101069/miR-129-5p/Twist1网络促进乳腺癌细胞表阿霉素耐药以及侵袭-迁移反应，证明NONHSAT101069在乳腺癌的发生发展中发挥着重要作用。