【作者】 常亮；

【导师】 崔有斌；

【作者基本信息】 吉林大学 ， 外科学， 2016， 博士

【摘要】 背景:肺癌是目前发病率和死亡率最高的恶性肿瘤,且发病率呈持续增长趋势,严重威胁人类健康,其中以非小细胞肺癌(Non-small cell lung cancer,NSCLC)流行最广,约占肺癌总数的80%,其发病和致死的主要原因是癌细胞转移,但目前对NSCLC转移机制尚不清楚,使得对NSCLC的预防和治疗更加困难。目前对NSCLC的治疗主要采用手术、放疗和化疗,但多数患者确诊时已是晚期,失去了手术治疗的最佳时机而以化疗为主。化疗虽然能改善晚期NSCLC患者生存期限,但由于其没有细胞选择性,对正常细胞的杀伤同样严重,会导致患者免疫力下降并产生严重并发症。因此,研究潜在NSCLC治疗靶点和治疗策略迫在眉睫。近几年对分子靶向治疗的研究日益深入,并逐渐应用于癌症的临床治疗,取得了较好的疗效,为NSCLC的治疗提供了新的方向。去整合素和金属蛋白酶家族(A disintegrin and metalloproteinases,ADAMs)是一类锌依赖性I型跨膜蛋白,具有蛋白水解酶活性和细胞粘附功能,能水解多种蛋白的胞外功能区,使之脱落,从而参与调节相关信号转导途径。此外,ADAMs还参与降解胞外基质、细胞间及细胞与基质间粘连、细胞融合等过程,并在精卵结合、伤口愈合及肿瘤发生和转移等病理生理过程中发挥重要作用。ADAM9作为ADAMs家族重要成员,可高表达于多种癌变组织和细胞中,并参与肿瘤的发生发展和转移过程。研究发现,ADAM9显著表达于NSCLC患者中,并可导致患者生存期缩短;ADAM9过表达可增加NSCLC细胞粘附和侵袭能力;通过RNA干扰(RNA interference,RNAi)技术使ADAM9基因沉默可抑制腺样囊性癌细胞生长和转移;RNAi介导ADAM9基因沉默可抑制A549细胞增殖、迁移和侵袭,诱导细胞凋亡。研究表明,ADAM9参与表皮生长因子受体(epidermal growth factor receptor,EGFR)配体外功能区酶解脱落及裂解Delta-likel等过程。以上研究结果揭示,ADAM9可作为NSCLC治疗的潜在靶点。信号转导与转录激活因子3(signal transducer and activator of transcription,Stat3)是STAT家族重要成员,多种细胞因子、生长因子均可激活Stat3,并参与相关信号转导。研究发现,Stat3活化后可参与调节细胞生长、凋亡及细胞周期等;Stat3参与多个癌症相关的酪氨酸激酶信号途径,并高表达于多种肿瘤组织和细胞中,如前列腺癌、乳腺癌、NSCLC和卵巢癌等。Stat3的活化有利于诱导肿瘤细胞增殖、抑制细胞凋亡,从而使癌症恶化;而通过RNAi或小分子抑制剂抑制Stat3的表达,可抑制肿瘤细胞增殖、侵袭,诱导细胞凋亡;且在对NSCLC的研究中发现,下调Stat3的表达可抑制肿瘤生长。揭示Stat3可能是NSCLC治疗的潜在靶点。EGFR具有酪氨酸蛋白激酶活性,研究表明,EGFR过表达可导致细胞过度生长和恶性肿瘤,Stat3是STAT家族肿瘤细胞和癌组织中表达最活跃的转录因子。研究证明,应用EGFR单克隆抗体治疗后,抑制Stat3的磷酸化,细胞增殖、侵袭能力降低,促进细胞凋亡,这些结果表明EGFR-STAT3信号转导途径作用于肺癌细胞增殖过程中起到重要作用。目前实验研究表明JAK-STAT3信号转导通路和ADAM9-EGFR信号转导通路对NSCLC发生发展起到促进作用,两条信号转导通路在肿瘤的发生发展中存在信号对答。目的:探讨JAK-STAT3与ADAM9-EGFR信号转导通路在NSCLC中的关系方法:构建ADAM9 sh RNA、Stat3 sh RNA以及对照组的慢病毒载体,并转染至293T细胞,从而分别获得表达ADAM9 sh RNA慢病毒(Lv/sh-ADAM9)、Stat3 sh RNA慢病毒(Lv/sh-Stat3)或非沉默sh RNA慢病毒(Lv/sh-NC),用慢病毒Lv/sh-Stat3、Lv/sh-ADAM9、Lv/sh-NC单独感染A549细胞,或Lv/sh-Stat3和Lv/sh-ADAM9联合感染细胞。分别采用RT-q PCR技术和WB方法检测细胞中Stat3和ADAM9 m RNA和蛋白表达水平。通过CCK-8方法检测A549细胞增殖水平。TUNEL方法检测细胞凋亡;检测细胞caspase-3、caspase-8和caspase-9活性。细胞划痕实验检测细胞迁移能力。Transwell方法分析细胞侵袭水平;检测细胞MMP-2和MMP-9蛋白表达水平。雄性BALB/c裸鼠皮下注射A549细胞以构建NSCLC荷瘤裸鼠模型,并对荷瘤小鼠注射慢病毒Lv/sh-NC、Lv/sh-ADAM9、Lv/sh-Stat3或Lv/sh-ADAM9和Lv/sh-Stat3联合进行治疗,对肿瘤体积及重量进行测量;通过TUNEL方法检测肿瘤细胞凋亡水平。结果:所构建慢病毒载体经PCR和测序鉴定无误;重组质粒可成功转染至293T细胞中;慢病毒Lv/sh Stat3和Lv/sh ADAM9可分别或者共同感染A549细胞。RNAi介导的Stat3或ADAM9基因沉默可分别抑制A549细胞Stat3或ADAM9 m RNA和蛋白表达,二者基因共沉默同样可抑制Stat3和ADAM9 m RNA和蛋白表达。RNAi介导的Stat3或ADAM9基因沉默均可抑制A549细胞的体外增殖,而二者基因共沉默在抑制细胞增殖上显示出协同效应。RNAi介导的Stat3或ADAM9基因沉默均可诱导A549细胞的体外凋亡,二者基因共沉默对细胞凋亡的诱导作用更为显著。RNAi介导的Stat3或ADAM9基因沉默均可抑制A549细胞体外迁移和侵袭,而二者基因共沉默对细胞侵袭显示出协同抑制作用。RNAi介导的Stat3和ADAM9基因共沉默可抑制MMP-2和MMP-9蛋白表达,与细胞迁移和侵袭能力的变化一致,揭示MMP-2和MMP-9参与A549细胞的体外迁移和侵袭。RNAi介导的体内Stat3或ADAM9基因沉默可抑制NSCLC肿瘤组织生长,二者基因共沉默对肿瘤组织生长的抑制作用更强。结论:ADAM9和Stat3基因共沉默可协同抑制体外NSCLC增殖、迁移和侵袭能力,并诱导细胞凋亡,能够协同抑制体内肿瘤生长。JAK-STAT3与ADAM9-EGFR信号转导通路在NSCLC发生发展过程中起协同作用。Stat3不是JAK-STAT3与ADAM9-EGFR两条信号转导通路下游的共同关键节点。更多还原

【Abstract】 Background:The incidence rate of lung cancer, a therioma with the highest morbidity and mortality, shows a sustained growth trend, seriously threatening human health. Non-small cell lung cancer(NSCLC) is the most popular and accounts for ~80% of all type of lung cancer. Metastasis is a major cause of morbidity and mortality in NSCLC cancer. However,the mechanism of such metastasis is unclear, which worsens the prevention and treatment of NSCLC. Surgery, radiotherapy and chemotherapy are the primary treatment of NSCLC. However, most patients have been diagnosed at the advanced stage, a stage is not the best time for treatment, and treated by chemotherapy. Although chemotherapy would effectively improves the life quality and symptoms of patients with advanced NSCLC, it also causes serious damage to normal cells because of the lack of selectivity, impairs the patient’s immune system and induces serious complications. Thus, the effective targets and therapeutic strategies for the treatment of NSCLC are required for development.In recent years, targeted molecular therapy gradually applied to the clinical treatment of cancer. This approach has received good curative effect and provided a new strategy for NSCLC.A disintegrin and metalloproteinases(ADAMs) comprise a family of zinc dependent type I transmembrane protein with proteolysis and cell adhesion activity, which hydrolysize a variety of protein sextracellular domain and induce cell signal transduction. Further, ADAMs have a variety of functions including the degradation of extracellular matrix,cell-cell or cell-matrix adhesion, cell fusion, sperm-egg fusion, wound healing and cancer metastasis, formation and progression.ADAM9, an important member of the ADAM protein family, is frequently upregulated in various types of cancer which triggering tumorigenesis and metastasis of tumor cell. Consistent with these results, a previous study demonstrated that ADAM9 is highly expressed in NSCLC and the increasing levels of ADAM9 correlates with a shortened survival time.Additionally, ADAM9 overexpression increases cell adhesion and invasion of NSCLC cells; RNA interference(RNAi)-mediated downregulation or knockout of endogenous ADAM9 inhibit adenoid cystic carcinoma cell growth, metastasis, proliferation, migration and invasion of A549 cells. The above studies indicating that ADAM9 is an ideal target for the treatment of NSCLC.Signal transducer and activator of transcription(Stat3) is one of the most important members of the STAT family and could be activated by many cytokines and growth factors, which is involved in signal transduction. It is has been demonstrated that activated Stat3 is capable of regulating cell growth, cell apoptosis and cell cycle. Increased expression of Stat3 is observed in the cancer including prostate cancer, breast cancer,NSCLC and ovarian cancer, which inhibit cell apoptosis and facilitate the development of cancer. Inhibition of the expression of Stat3 by RNAi attenuates tumor cell proliferation and invasion and induce cell apoptosis.Therefore, Stat3 can represent an attractive target for therapeutic intervention against NSCLC.Epidermal growth factor receptor(EGFR) is a multifunctional membrane glycoprotein found in a variety of tissue types. The overexpression of EGFR leads to excessive cell growth and malignancy.Constitutively activated Stats proteins are found in many tumor cells and cancer tissues, which show that after treatment with anti-EGFR monoclonal antibodies lung cancer cells are inhibited Stat3 phesphorylation and result in slowed proliferation, accelerates apoptosis,weakened invasiveness, and arrested cell cycles. These results suggest a key role for EGFR-STAT3 signaling in the proliferation of lung cancer cells.By now, the research indicate that ADAM9-EGFR and JAK-STAT3 signaling pathway promote the development of non-small cell lung cancer,but the relationship in tumorigenesis development between the two signal pathway is no clear.Objective:To investigate the relation between JAK-STAT3 and ADAM9-EGFR signaling pathway in NSCLCMethods:To recombinant lentiviral small hairpin RNA expression vector carrying ADAM9 and Stat3, then transfect into 293 T cells for ADAM9 sh RNA-expressing lentivirus(Lv/sh ADAM9), Stat3 sh RNA-expressing lentivirus(Lv/sh-Stat3) or non-targeting sh RNA-expressing lentivirus(Lv/sh NC). The m RNA and protein levels expression in A549 cells were determined by RT-q PCR and western blot after the cells were infected with the lentivirus carrying the ADAM9 si RNA, Stat3 si RNA, negative control si RNA or Combined ADAN9. The viability level of A549 cells was determined by CCK-8. The cell apoptosis was determined by TUNEL assay and caspase(caspase-, caspase-8 and caspase-9) activity assay. The cell migration and invasion determined by wound healing assay and transwell system assay. The MMP-2 and MMP-9 level were determined by western blot. A male BALB/c mice model bearing A549 tumor cell following treated with sh RNA-expressing lentivirus(Lv/sh-ADAM9, Lv/sh-Stat3 and Lv/sh NC) was constructed, and determined by TUNEL assay.Results:A recombinant lentiviral small hairpin RNA expression vector carrying ADAM9, Stat3 and control was constructed and then transfected into 293 T cells to generate sh RNA-expressing lentivirus(Lv/sh ADAM9,Lv/sh-Stat3 and Lv/sh NC). The expression level of ADAM9 or Stat3 m RNA in Lv/sh-ADAM9-infected or Lv/sh- Stat3-infected A549 cells were decreased compared with that in untreated cells or Ad/sh-NC-infected cells. The results from CCK-8 assays further reveal that the control had no cytotoxic effect on A549 cells, while the viability of A549 cells was markedly inhibited by ADAM9 and Stat3 silencing.RNAi-mediated ADAM9 and Stat3 silencing significantly induce cell apoptosis. The level of cell migration and cell invasion were inhibited in the ADAM9 and Stat3 silencing group. The tumor growth in male BALB/c mice bearing A549 tumor cells following treated ADAM9 and Stat3 silencing was significantly inhibited.Conclusions:Silencing of ADAM9 and Stat3 reduces proliferation,migration and invasion of NSCLC,and increases apoptosis in vitro and in vivo.Therefore, silencing of ADAM9 and STAT3 inhibits the growth of NSCLC. Our results further demonstrates JAK-STAT3 and ADAM9-EGFR signaling pathways have synergistic effect on the growth of NSCLC. Moreover,Stat3 is not the key downstream mediator in the two signaling pathways.更多还原