PO.TB07.01 · 肿瘤生物学
研究细胞迁移和转移过程中LIN28/let-7/SOX2/SOX9发育反馈环路
Investigation of developmental LIN28/ let-7 /SOX2/SOX9 feedback loop during cell migration and metastasis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
RNA结合蛋白LIN28和微小RNA（miRNA）let-7家族代表一个进化上保守的发育反馈环路，在后生动物中作为发育时序的调控因子。我们最近发表的数据表明，在发育中的肺和脑中，Sox2和Sox9转录调控Lin28a/b。此外，我们实验室先前的工作表明，Lin28a结合Sox2和Sox9的mRNA并调控其稳定性和翻译。我们的假设是，由SOX9和SOX2调控的早期发育通路的重激活，导致在肿瘤发生的不同时期LIN28A/B的转录上调。我们的目标是理解为何发育通路在肿瘤发生过程中被重新表达，以及找到特异性靶向这些通路以抑制转移的方法。我们在肺和胰腺的正常及癌细胞系中，采用siRNA建立了瞬时SOX9敲低（KD），并采用慢病毒shRNA构建体建立了稳定SOX9 KD。我们在对照和SOX9 KD细胞中进行迁移试验，采用免疫印迹和免疫荧光实验，在不同迁移阶段检测SOX9、SOX2、LIN28A和LIN28B的蛋白表达。我们采用Incucyte SX5活细胞成像仪通过高通量迁移试验进行迁移/侵袭试验，以检测对照和SOX9 KD细胞的迁移潜能。我们发现，在正常和癌细胞的迁移阶段，SOX9表达变为核内定位，提示细胞迁移过程中可能存在转录激活。我们注意到SOX9表达在不同迁移阶段增加，但在伤口闭合后降低。有趣的是，当SOX9表达较低时，SOX2表达在伤口闭合后的癌细胞系中较高。LIN28A和LIN28B表达在肺癌和胰腺癌细胞系的不同迁移阶段增加，但在正常细胞系中则不然。重要的是，我们观察到在肺癌和胰腺癌细胞中，SOX9 KD后迁移、侵袭和增殖减缓。我们现将研究在非小细胞肺癌（NSCLC）和胰腺癌的病理生理过程中，LIN28A/B是否为SOX9/SOX2轴的功能性下游靶点，重点关注迁移/侵袭和转移。
查看英文原文 English abstract
The RNA binding protein LIN28, and the microRNA (miRNA) let-7 family represent an evolutionarily conserved developmental feedback loop that acts as a regulator of developmental timing in metazoans. We have recently published data demonstrating that in the developing lung and brain, Sox2 and Sox9 transcriptionally regulate Lin28a/b . Moreover, previous work from our lab show that Lin28a binds Sox2 and Sox9 mRNAs and regulates their stability and translation. Our hypothesis is that reactivation of an early developmental pathway governed by SOX9 and SOX2 lead to transcriptional upregulation of LIN28A/B at various times during tumorigenesis. Our goal is to understand why developmental pathways are re-expressed during tumorigenesis as well as find ways to specifically target these pathways to inhibit metastasis. We established transient SOX9 knockdown (KD) using siRNA and stable SOX9 KD using lenti-viral shRNA construct in normal and cancer cell lines of lung and pancreas. We performed migration assays in control and SOX9 KD cells to test protein expression of SOX9, SOX2, LIN28A, and LIN28B using immunoblot and immunofluorescence experiments at different migratory stages. We performed migration/invasion assays using the Incucyte SX5 live-cell imager via high throughput migration assays to test migratory potential of control and SOX9 KD cells. We have found that SOX9 expression becomes nuclear during migratory stages in normal and cancer cells indicating a potential transcriptional activation during cell migration. We noticed that SOX9 expression is increased at different migratory stages but decreased after wound closure. Interestingly, SOX2 expression is higher in cancer cell lines after wound closure when SOX9 expression is lower. LIN28A and LIN28B expression is increased at different migratory stages in lung and pancreatic cancer cell lines, but not in the normal cell lines. Importantly, we observed that in lung and pancreatic cancer cells migration, invasion and proliferation are slow after SOX9 KD. We will now investigate whether LIN28A/B are functional downstream targets of the SOX9/SOX2 axis during the pathophysiology of non-small cell lung cancer (NSCLC) and pancreatic cancer with specific focus on migration/invasion and metastasis.
利益披露 Disclosure
I. Thevarajan, None..
M. Osuna, None..
B. Kim, None..
J. K. Osborne, None.