MiRNA-320c和miRNA-125b调控肺腺癌细胞程序性坏死及其在肺腺癌骨转移中的作用机制研究

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 specimens of lung cancer patients with bone metastasis compared with lung cancer patients without metastasis. Among them, miR-320c、miR-125b was found to regulate necroptosis in lung adenocarcinoma cells. Preliminary data showed that they may target to FADD and PARP-2 genes that are both critical componients in necroptotic signaling pathway. We intend to further study the roles and the mechanisms of miR-320c、miR-125b in regulation of necroptosis, and to validate the targeting relationship between these two miRNAs and their potential target genes. Furthermore, we will investigate the roles of miR-320c and miR-125b in regulation of lung adenocarcinoma cells migrigation, invasion, and metastasis to bones, and reveal whether thesse roles are dependent on necroptosis.

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。本课题组一项miRNA高通量分析结果显示，肺腺癌骨转移患者标本中有21种miRNA表达较无骨转移患者显著上调，有7种miRNA显著下调。其中miR-320c、miR-125b可调控肺腺癌细胞程序性坏死，初步实验显示，它们可能靶向程序性坏死通路的重要基因FADD和PARP-2。本研究拟进一步研究miR-320c、miR-125b调控肺腺癌细胞程序性坏死的作用和分子机制，并确定这两个miRNA与候选基因的直接靶向关系。我们还将在体内外水平上研究miR-320c和miR-125b对肺腺癌细胞迁移、侵袭和骨转移的影响，并确定该作用是否依赖于程序性坏死。

骨转移是肺腺癌患者最严重的并发症之一，深入研究肺腺癌骨转移的发病机制并寻找有效的治疗手段,具有非常重要的临床意义。本项目高通量测序结果显示，miR-320c、miR-125b 在肺腺癌骨转移患者中显著上调，并通过qPCR在临床样本中进行验证。在本项目中，通过体外细胞功能实验、体内动物模型、临床样本，采用qPCR，western blot，Tanswell，双荧光素酶报告实验，透射电镜观察等方法，阐明miR-320c、miR-125b 影响肺腺癌骨转移的分子机制。1.体内外实验证明miR-320c、miR-125b 能够促进肺腺癌细胞侵袭，促进裸鼠肺腺癌骨转移。2.进一步研究发现miR-320c、miR-125b 能够抵抗细胞应对外部刺激诱导的necroptosis，应对细胞死亡。说明miR-320c、miR-125b 调控肺腺癌细胞的necroptosis。通过Rescue实验明确了miR-320c、miR-125b 通过抑制肺腺癌细胞的necroptosis 促进细胞的侵袭及裸鼠肺腺癌骨转移。3.本项目发现miR-320c与necroptosis通路中的重要基因FADD正相关。这说明，miR-320c可能通过FADD调控necroptosis；而miR-125b通过靶向necroptosis通路中的关键基因PARP2调控necroptosis。4.通过检测坏死复合体（即复合体Ⅱb，RIP1-RIP3-MLKL）的形成是否与 miR320c、miR-125b的表达具有相关性，进一步确认了miR-320c、miR-125b抑制坏死复合体的形成从而抑制necroptosis。5.通过Rescue实验明确了miR-320c通过上调FADD抑制necroptosis促进肺腺癌细胞侵袭及骨转移；miR-125b通过靶向PARP2抑制necroptosis促进肺腺癌细胞侵袭及骨转移。综上所述，miR-320c、miR-125b与肺腺癌骨转移密切相关，分子机制涉及到FADD、PARP2参与的necroptosis通路，有助于为发现治疗肺腺癌骨转移新的靶点及研究特异性治疗药物提供新的理论依据。