| 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... |
PDF Full Text Request