骨转移是肺癌患者最严重的并发症之一，阐明肺癌骨转移的发病机制是当前医学研究的迫切需要。microRNA(miRNA)在多种肿瘤的生长、转移中发挥重要功能，但miRNA在肺癌骨转移中的作用尚不清楚。本实验室一项miRNA高通量分析结果显示，肺腺癌骨转移患者肿瘤标本中有21种miRNA表达较无骨转移患者显著上调，有7种miRNA显著下调，这提示miRNA在肺腺癌骨转移中可能发挥重要功能。同时，实验室前期研究还表明细胞自噬与肺腺癌骨转移密切相关。本研究拟从上述差异表达的miRNA中选取5种可能调控自噬的miRNA在肺腺癌细胞中进行验证，对可调控自噬的miRNA选择1-2个进行重点研究，确定其直接靶向的自噬相关基因。在此基础上，在细胞和动物两个水平上研究候选miRNA对肺腺癌细胞迁移、侵袭及骨转移的影响，并用自噬抑制剂或自噬缺陷小鼠研究miRNA对肺腺癌细胞迁移、侵袭及骨转移的作用是否由自噬引起。

Lung cancer bone metastasis is one of the most serious complications of lung cancer patients. To elucidate the pathogenesis of lung cancer bone metastasis is an urgent need for medical research. MicroRNA (miRNA) plays an important role in the growth, metastasis of many kinds of tumors, but the role of miRNA in bone metastasis of lung cancer is not clear. A miRNA HiSeq high-throughput sequencing by our laboratory showed that 21 miRNAs expression are markedly upregulated and 7 miRNAs expression are markedly down-regulated in tumor specimens of lung cancer patients with bone metastasis compared with lung cancer patients without metastasis, thus suggesting that miRNA may play an important role in lung cancer bone metastasis. Besides, our previous study also showed that autophagy is closely correlated to bone metastasis of lung adenocarcinoma. In this study we intend to choose 5 miRNAs that may correlate with autophagy from the above differentially expressed miRNAs and validate whether they truly affect autophagy in lung adenocarcinoma cells. Next, we will focus on one or two miRNAs that can regulate autophagy and determine their directly targeted autophagy related genes. On this basis, we intend to investigate the role of candidate miRNA in lung adenocarcinoma cells migration, invasion and bone metastasis and further determine whether these effects are mediated by autophagy via use of autophagy inhibitors or autophagy-defective mice. This study should add to understanding of pathogenesis of lung adenocarcinoma bone metastasis and provide new therapeutic targets for prevention and treatment of this disease.

肺癌骨转移是肺癌患者最严重的并发症之一。阐明肺癌骨转移的发病机制是医学研究的迫切需要。目前，自噬是肿瘤转移过程中的研究热点，miRNA也参与到该过程中。在本研究项目中，我们以自噬相关基因为靶标，通过肺癌骨转移miRNA高通量测序结果和生物信息学的挖掘，发现调控自噬的一批与肺腺癌骨转移相关的miRNAs。但这些miRNAs参与自噬的分子机制是什么？我们首次提出这些miRNA通过调节靶基因参与自噬是肺腺癌骨转移的关键。我们在此项目以miRNAs调控自噬为切入点，过表达miRNAs、通过双荧光素酶报告、蛋白组学等方法，获得肺腺癌骨转移相关miRNAs调控自噬的可靠证据；进而以体外细胞功能学实验、动物模型、临床样本免疫组化等方法阐明miRNA调控自噬影响肺腺癌骨转移的分子机制。1，我们筛查到了miR-21、miR-34a、miR-196a、miR-106a、miR-365等在肺腺癌骨转移和非骨转移样本中差异性表达，并进一步发现miR-21和miR-106a能够促进自噬；miR-34a和miR-365能够抑制自噬。2，我们发现了miR-34a能够靶向调控自噬相关基因SIRT1和Atg9a，其中Atg9a在肺腺癌骨转移患者中的阳性表达率显著增高，进一步验证发现抑制Atg9a能够抑制自噬水平，回补实验进一步证实，Atg9a在miR-34a抑制肺腺癌细胞迁移和侵袭的过程中起到节点作用。另外，miR-34a/Atg9a同转移过程中的EMT密切相关。3，同时，我们发现了过表达miR-365能够抑制肺腺癌细胞的增殖、迁移和侵袭。但当自噬激活时，miR-365失去了抑癌作用，提示自噬在miR-365的抑癌过程扮演重要角色。进一步的研究发现了RICTOR是miR-365的靶基因，是miR-365调控自噬的媒介，其在肺腺癌骨转移患者中高表达。我们最后发现miR-365/RICTOR可能通过mTORC2/AKT/P70S6信号通路发挥作用。4，我们也对miR-106a进行了初步研究，确认了miRNAs能够促进肺腺癌细胞的迁移和增殖，自噬缺陷后，miR-106a的促进作用受到抑制。miR-106a的靶基因SIRT7和SQSTM1可能参与到该过程中。综上所述，以上miRNAs均和肺腺癌骨转移密切相关，有望为确立自噬作为抗肿瘤骨转移治疗提供更充分的科学依据和治疗靶点。