HOTAIR/miR-326/SP1调控通路对非小细胞肺癌增殖、迁移和侵袭能力的影响及作用机制

microRNA plays a crucial role in development and progression of multiple malignancies. However, the mechanisms of microRNA in cancer are not totally clear. Our previous study showed miR-326 may function as a tumor suppressor in lung cancer. MiR-326 downregulated in lung cancer specimens with lymph node metastasis. Conversely, lncRNA HOTAIR and SP1 upregulated in metastatic lung cancer specimens. In addition, bioinformatics analysis showed miR-326 and SP1 binding sites were found in HOTAIR sequence and in the promoter of HOTAIR, respectively. Therefore, we hypothesize that tumor related lncRNA HOTAIR is a upstream regulator of miR-326, and that SP1 is a downstream target of miR-326 and regulated by miR-326. HOTAIR, miR-326 and SP1 communicates with each other and comprise a miR-326-based regulation network, which is crucial for proliferation, metastasis and invasion of lung cancer. To verify our hypothesis, lung cancer cell lines A549 and H838, nude mince with lung cancer xenograft and lung cancer tissue samples are used. Real time PCR, western blot, adenovirus, RNA interference, luciferase assays and chromatin immunoprecipitation assays (CHIP) are employed to investigate molecular mechanisms of HOTAIR/miR-326/SP1 regulation pathway in lung cancer. Our project will provide new information for lung cancer development and progression, and help establish novel strategy for lung cancer diagnosis and therapy.

microRNA在多种恶性肿瘤的发生发展过程中发挥重要的调控作用，但目前对一些microRNA的作用机制还有待进一步探讨。我们预实验结果提示miR-326可能是肺癌的抑癌基因，其在肺癌组织中表达下调，伴随HOTAIR和SP1表达上调，生物信息学预测HOTAIR与SP1分别有miR-326上游和下游结合位点。故提出假说：HOTAIR能够从上游调控miR-326的表达，同时miR-326可以调控其下游靶基因SP1的表达，从而构成HOTAIR/miR-326/SP1调控通路，影响肺癌细胞增殖、迁移和侵袭。为验证这一假说，我们将利用肺癌细胞系、裸鼠成瘤模型及临床组织样本，采用real time PCR、Western blot、腺病毒载体转染、RNA干扰、荧光素酶报告及CHIP等实验手段，从分子、细胞、组织和动物整体水平阐明该通路的调控功能和作用机制，为肺癌增殖和转移的发生机制奠定新的理论基础。

microRNAs一般通过限制下游靶基因的表达来发挥调控作用。miR－326的失调与恶性肿瘤的发生发展过程有关。HOTAIR是一种与癌症相关的长非编码RNA，能促进多种类型的细胞发生癌变、转移，它能通过直接结合miRNAs负调控miRNAs。SP1则在多种类型的人类癌症中表达上调，在肺癌组织中比邻近正常组织的表达增高。. 本研究显示，转染miR-326可显著降低肿瘤细胞的增殖和迁移，并抑制裸鼠肺癌移植肿瘤生长，说明它在肺癌中起肿瘤抑制作用；miR-326可以与Phox2a的3'UTR结合导致肺癌细胞中Phox2a的表达降低，Phox2a的过表达也可以对肺癌细胞的增殖、侵袭、细胞周期和凋亡发挥作用，因此，Phox2a是miR-326的负调控靶基因；HOTAIR的沉默增加了miR-326在肺癌细胞的表达，相对荧光素酶活性降低也再次证明miR-326是HOTAIR的负调控靶基因；双荧光素酶活性实验揭示Phox2a和SP1可能是miR-326的下游靶基因，而KLF3不是其靶基因，且miR-326对SP1、Phox2a、KLF3三者均起负向调控作用，抑制其生物学功能；阻断SP1后发现KLF3在肺癌细胞中表达下调。荧光素酶、EMSA和ChIP实验均证实KLF3是SP1的一个直接靶基因；抑制KLF3的功能表现为肺癌细胞和裸鼠肿瘤增殖、迁移和侵袭的水平降低，提示它可能起癌基因的作用。临床样本也揭示，KLF3在肺癌组织中表达升高，且其异常高水平与肺癌患者的总体生存不良有关。. 本研究初步阐明了HOTAIR/miR-326/Phox2a和miR-326/SP1/KLF3调控通路的作用机制和对肺癌的生物学功能，即HOTAIR可以通过负调控miR-326，后者靶向作用于Phox2a，抑制其对肺癌增殖、迁移和侵袭的促进作用；miR-326对靶基因SP1发挥负向调控作用，抑制其表达水平，从而间接地降低了下游转录因子KLF3的生物学活性，起到抑制肺癌增殖和侵袭的作用。. 该研究成果为肺癌增殖和转移的发展机制奠定新的理论基础，为肺癌的诊断和治疗提供了新的思路。