【作者】 曹慧秋；

【导师】 文继舫； 周建华；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 背景Notch受体信号通路具有调控细胞分化、存活、增殖的作用,参与了肺脏的发生、发育,也参与了非小细胞肺癌的发生、发展。人类的Notch受体共有4个,即Notch1～4,均为单次跨膜受体。成熟的Notch受体由细胞外亚基(NEC)和跨膜亚基(NTM)组成,其中NTM包含一截短的膜内结构域和一个较大的胞内结构域(ICD)。当Notch受体信号通路被激活时,ICD从NTM上被酶切下来,并易位入核,启动靶基因Hes与Hey的转录,从而调控细胞分化、增殖或凋亡。肺鳞癌中4个Notch受体的表达情况目前还不完全清楚；Notch受体信号通路对肺鳞癌细胞凋亡的影响及相关分子机制也尚不明了；而且,Notch受体及Notch受体信号通路在肺鳞癌中受何种机制调控也尚不明确。目的1.分析Notch1～4蛋白在肺鳞癌中的表达及其临床病理意义。2.探讨Notch受体信号通路对肺鳞癌细胞凋亡的影响及其分子机制。3.探究肺鳞癌中Notch受体的表达及Notch受体信号通路的激活是否受YB-1基因的调控,并进一步研究Notch受体信号通路是否介导YB-1的凋亡抑制作用。方法1.免疫组织化学SP法检测64例肺鳞癌及癌旁非肿瘤肺组织中Notch1～4蛋白的表达。Spearman等级相关检验分析肺鳞癌中Notch1～4的表达与肿瘤的大小、淋巴结转移、临床分期的相关性。2.γ分泌酶抑制剂DAPT抑制肺鳞癌细胞株SK-MES-1细胞中Notch受体信号通路的活化,Annexin V-PI双染法检测细胞凋亡；Western blot检测PARP蛋白的剪切情况；Western blot及qRT-PCR检测XIAP、Survivin的表达水平；分离胞浆蛋白、线粒体蛋白、胞核蛋白,Western blot分别检测Smac蛋白,Endo G蛋白和AIF蛋白在胞浆、线粒体的含量以及EndoG蛋白和AIF蛋白在胞核中的含量；细胞免疫荧光检测Endo G口AIF蛋白的亚细胞定位。3.构建4个YB-1短发夹RNA(shRNA)干扰质粒pYr-1.1YB-1.shRNA,将其瞬时转染293细胞,RT-PCR检测YB-1mRNA,筛选出干扰效率最高的质粒。针对干扰效率最高的靶序列,应用点突变PCR在YB-1基因的表达质粒pYr-ads-1-YB-1上构建包含靶序列沉默点突变的质粒pYr-ads-1-YB-1M。将干扰效率最高的质粒pYr-1.1-YB-1-shRNA稳定转染SK-MES-1细胞,western blot检测YB-1蛋白的表达及PARP蛋白的剪切。将pYr-ads-1-YB-1或pYr-ads-1-YB-1M分别与pYr-1.1-YB-1-shRNA瞬时共转染SK-MES-1细胞,western blot检测YB-1的表达及PARP蛋白的剪切。4.一方面,在稳定转染pYr-1.1-YB-1-shRNA的SK-MES-1细胞中,应用RT-PCR及Western blot检测Notch1～4受体的表达及活化,RT-PCR检测Notch受体信号通路靶基因Hes1、Hey1的表达；另一方面,将YB-1基因的表达质粒pYr-ads-1-YB-1瞬时转染SK-MES-1细胞,Western blot检测YB-1蛋白的表达及Notch1-4受体的表达和活化,RT-PCR检测Hes1、Hey1的表达；最后,在瞬时转染pYr-ads-1-YB-1的SK-MES一1细胞中,应用DAPT抑制Notch受体信号通路,通过Annexin V-PI双染法检测凋亡,以验证YB-1是否通过激活Notch受体信号通路来抑制顺铂诱导的凋亡结果1.肺鳞癌组织Notch1～4蛋白的表达较癌旁支气管黏膜显著增强(P<0.05),其中Notch1、3的表达与肿瘤的大小呈正相关(r=0.334、0.421,P<0.05),Notch1.2的表达与淋巴结转移呈正相关(r=0.356、0.417,P<0.05)；4个Notch受体的表达与肺鳞癌的临床分期均无明显相关性(P<0.05)。2.Y分泌酶抑制剂DAPT可有效抑制SK-MES-1细胞中Notch受体信号通路的活化,并显著诱导细胞凋亡；不同浓度的DAPT作用SK-MES-1细胞能在翻译水平降低XIAP及Survivin的表达,诱导Smac蛋白从线粒体释放入胞浆,同时促进Endo G和AIF蛋白从线粒体释放入胞浆及胞核。3.4个pYr-1.1-YB-1-shRNA质粒中,2个干扰效率在60%以上,最高抑制率为70.3%；pYr-1.1-YB-1-shRNA质粒稳定转染SK-MES-1细胞能有效抑制YB-1蛋白的表达,并促进PARP蛋白的剪切；pYr-ads-1-YB-1M能回复pYr-1.1-YB-1-shRNA质粒转染所致的YB-1蛋白表达抑制及RARP蛋白剪切。4.YB-1可在转录和翻译两个水平调控Notch1的表达,并促进Notch1受体的活化及Hes1基因的转录,Notch2和Notch3表达及活化不受YB-1的调控,Notch4未能在各组SK-MES-1细胞中检测到。YB-1过表达可显著抑制顺铂诱导的凋亡,但Notch受体信号通路被DAPT抑制后,YB-1的凋亡抑制作用被解除。结论1. Notch受体在肺鳞癌组织中的表达显著上调,可能导致Notch受体信号通路异常激活。2.抑制Notch受体信号通路可通过caspase依赖及非caspase依赖的通路诱导肺鳞癌细胞凋亡。3. pYr-1.1-YB-1-shRNA转染SK-MES-1细胞可诱导细胞凋亡,凋亡效应是由于YB-1的表达被抑制而引起的,而非脱靶沉默效应所致。4. YB-1可调控Notch1受体的表达、活化及Notch受体信号通路,并通过Notch受体信号通路抑制肺鳞癌细胞凋亡。更多还原

【Abstract】 BackgroundNotch receptor signaling pathway (NRSP) regulates cell differentiation, survival and proliferation, which plays a key role in regulating lung development and is increasingly linked to carcinogenesis of non-small cell lung cancer. Notch is a transmembrane heterodimeric receptor family with four distinct members (Notch1～4) in humans. A mature Notch receptor contains an extracellular subunit (NEC) and a transmembrane subunit (NTM). The latter includes a short extracellular domain and a large intracellular domain (ICD). Upon interaction with transmembrane ligands expressed on the surface of neighboring cells, Notch receptors are activated by proteolytically cleaved, resulting in release of the ICD. Then the released ICD translocates into nucleus, where it initiates transcription of the hairy/enhancer-of-split (Hes) and hairy/enhancer-of-split related with YRPW motif (Hey) families. Hes and Hey family members in turn affect numerous pathways involved in differentiation, survival and proliferation. Up to now, no lung cancer trials are involved in investigating the differential expression of all the four Notch receptors, and whether NRSP could inhibit apoptosis and the related molecular mechanisms are still not fully understood. Moreover, the underlying reasons for the expression of Notch receptors and the activation of NRSP in lung squamous cell carcinoma (LSCC) have not been elucidated.Objective1. To determine the expression of Notch1～4in LSCC tissues and evaluate their clinical significances.2. To investigate the effect of NRSP on apoptosis and the relating molecular mechanism in LSCC. 3. To explore whether the expression of Notch receptors and the activation of NRSP are regulated by YB-1, and whether the activation of NRSP mediates the apoptosis-suppressive effects of YB-1.Methods1. Immunohistochemistry SP method was used to examine the expression of Notchl--4in64LSCC tissue samples and adjacent non-tumor lung tissue samples, Spearman rank correlation analysis was used to measure the correlations between immunohistochemistry staining of Notch receptors and tumor size, lymph node metastasis or clinical stage of LSCC.2. DAPT, a potent y-secretase inhibitor, was used to repress NRSP in LSCC cell line SK-MES-1cells. Apoptosis was determined by Annexin V and PI staining. Cleaved PARP was messured by western blot. XIAP and Survivin were assessed by qRT-PCR and western blot while the release of Smac from mitochondria to cytoplasm was evaluated by western blot. The subcellular locations of Endo G and AIF were observed by western blot and indirect immunofluorescence analysis.3. Four plasmids pYr-1.1-YB-1-shRNA carrying short hairpin RNA (shRNA) targeting YB-1were constructed. pYr-1.1-YB-1-shRNA were transiently transfected to293cells and RT-PCR was used to determine YB-1mRNA in293cells. Then the most effective plasmid was stably transfected to SK-MES-1cells. YB-1and PARP cleavage were examined by western blot in SK-MES-1cells. A mutant-type YB-1plasmid pYr-ads-1-YB-1M contains silent mutations in the region that is targeted by pYr-1.1-YB-1-shRNA was created by using point mutation PCR and pYr-ads-1-YB-1. pYr-1.1-YB-1-shRNA were co-transfected with pYr-ads-1-YB-1or pYr-ads-1-YB-1M to SK-MES-1cells. Both YB-1and PARP cleavage were examined by western blot.4. After stably transfected SK-MES-1cells with pYr-1.1-YB-1-shRNA, the expression and activation of Notch1～4were determined by RT-PCR and western blot, and the target genes of NRSP including Hes1and Heyl were examined by RT-PCR. Meanwhile, pYr-ads-1-YB-1was transiently transfected to SK-MES-1cells, western blot was used to dectected the expression of YB-1and activation of Notchl～4and RT-PCR was used to examine the expression of Hesl and Heyl. At last, DAPT was used to treat SK-MES-1cells that overexpressed YB-1by transfecting pYr-ads-1-YB-1transiently, and Annexin V and PI staining was used to examined the apoptosis induced by cisplatin.Results1. Expression of Notch1-4in LSCC were significantly up-regulated compared with bronchial epithelia of adjacent non-tumor lung tissues (P<0.05). Moreover, Notchl and Notch3were positively correlated with tumor size (r=0.334,0.421respectively, P<0.05) and both Notchl and Notch2were positively associated with lymph node metastasis of LSCC (r=0.356,0.417respectively, P<0.05). There was no significant correlation between Notch receptors expression and clinical stages.2. DAPT treatment could inhibit NRSP and induce apoptosis with marked increase in cleaved PARP, decrease in XIAP and Survivin proteins as well as concomitant release of Smac, Endo G and AIF from mitochondria.3. Of the four shRNA plasmids, two cut the amount of YB-1by60%in293cells with the maximal suppression rate arriving at70.3%. pYr-1.1-YB-1-shRNA could also significantly reduce YB-1protein and induce PARP cleavage in SK-MES-1cells. Importantly both the knock down of YB-1and the cleavage of PARP seen with pYr-1.1-YB-1-shRNA were rescued by the expression of the mutant type YB-1.4. Notchl was regulated by YB-1through both transcriptional and translational mechanism. Moreover, Notchl ICD protein as well as Hesl mRNA was modulated by YB-1. However, neither Notch2nor Notch3was influenced by YB-1. Notch4could not been examined in SK-MES-1cells. DAPT treatment could revert the apoptosis inducing by cisplatin in the SK-MES-1cells which overexpressed YB-1by transiently transfecteing wild-type YB-1plasmid.Conclusion1. Notch receptors are overexpressed in LSCC tissues, indicating that NRSP may be activated in LSCC.2. Suppressing NRSP could induce apoptosis through caspase- dependent and caspase-independent pathway.3. pYr-1.1-YB-1-shRNA could induce apoptosis in SK-MES-1cells through specifically knocking down YB-1rather than off-target effects.4. YB-1could regulate the expression and activation of Notch1, and thus activating NRSP. NRSP plays a vital role in apoptosis inhibition caused by YB-1.Figures32, Tables3, References100更多还原