Part Ⅰ:the Expression Levels And Clinical Significance Of Skp1 Protein In Non-small Cell Lung Cancer Tissues And Peripheral Blood Part Ⅱ:biological Function Of Small Ubiquitin-like Modifier Activating Enzyme Subunit 1 In Occurrence And Progress Of Lung C

Part I:The expression levels and clinical significance of Skp1 protein in non-small cell lung cancer tissues and peripheral bloodBackground and objectiveS-phase kinase-associated protein 1(Skp1)is an specific adaptor protein of the Skpl-Cull-F-box(SCF)ubiquitin ligase complex,which can stabilize F-box protein,enhance the substrate binding ability,participate in cell cycle regulation,and is closely related to occurrence and progress of tumor.Studies have shown that SCF complex plays an essential role in tumorigenesis,and some F-box protein has been shown to be potential target for the treatment of human cancer.However,the role Skp1 plays during non-small cancer lung cancer(NSCLC)remains unclear,and whether it can be used as a therapeutic target for NSCLC remains to be further studied.The aim of this study was to detect the protein expression of Skpl in NSCLC tissues and peripheral blood,and to explore its clinical significance.MethodsThe expression of Skpl protein in 34 NSCLC tissues and 34 adjacent tissues was detected by Western blot.The expression of Skpl in 86 NSCLC tissues and 86 adjacent tissues was detected by tissue microarray and immunohistochemical staining method,and the relationship of Skpl protein expression with clinicopathological parameters and prognosis of NSCLC patients was analyzed.The plasma expression level of Skpl protein in 39 NSCLC patients and 27 healthy persons was detected by ELISA(enzyme linked immunesorbent assay).And the relationship of Skpl protein level in plasma with clinicopathological parameters of NSCLC patients was analyzed.ResultsThe expression level of Skp1 protein in NSCLC tissues was significantly higher than that in adjacent tissues by Western Blot and immunohistochemical staining method(P<0.001).The plasma level of Skpl protein in NSCLC patients was significantly higher than that in healthy person by ELISA(P=0.003).The simple factors analysis showed that the over-expression of Skpl protein in NSCLC tissues was statistically significant with poor prognosis in the middle to late stage(Ⅱ+Ⅲ)NSCLC patients produced by Kaplan-Meier method(P=0.001),and the multiple factors Cox regression analysis showed that Skpl protein could be used as the independent risk factors for the middle to late stage(Ⅱ+Ⅲ)NSCLC patients.The expression of Skpl protein had no significant correlation with gender,age,pathological type,lymph node metastasis and other factors in NSCLC patients(P>0.05).ConclusionSkp1 protein was over-expressed in NSCLC patients,and the over-expression of Skp1 protein in NSCLC tissues was statistically significant with poor prognosis in the middle to late stage(Ⅱ+Ⅲ)NSCLC patients,which could be used as the independent risk factors for the middle to late stage NSCLC patients.Part Ⅱ:Biological function of small ubiquitin-like modifier activating enzyme subunit 1 in occurrence and progress of lung cancerBackground and objectiveThe SUMO pathway is an important post-translational modification pathway,and the SUMO family-mediated post-transcriptional modification drives different stages of tumorigenesis by regulating a variety of cellular processes,including transcription,replication,chromosome segregation,and DNA repair.The SUMO activating enzyme E1 subunit(SAE1)is the first enzyme in the SUMOylation pathway,which plays an important role in Myc-mediated tumorigenesis.However,there are few studies on SAE1 in lung cancer,and the function is not yet clear.In the early stage of the laboratory,the differential expression of airway exfoliated cells in sputum of lung cancer and COPD patients was compared by mass spectrometry.The SAE1 was highly expressed in the sputum cells of lung cancer patients.Immunohistochemistry results showed that SAE1 protein was overexpressed in non-small cell lung cancer tissues and correlated with the pathological type of non-small cell lung cancer and the overall survival of patients with lung squamous cell carcinoma,suggestting the importance of SAE1 in lung cancer.SAE1 may play an important role in the development of lung cancer.The aim of this study was to investigate the effect of SAE1 on the malignant phenotype of lung cancer cells by changing the expression level of SAE1 in lung cancer cells,and further explore the biological functions of SAE1 in the development of lung cancer,and add further theoretical guidance.MethodsFirst,shRNA technology and plasmid cloning technology were used to knock down and overexpress the endogenous SAE1 gene in lung cancer cell lines,and SAE1 knockdown and overexpression plasmids were constructed to transiently and stably transfect lung cancer cell lines.The expression level of SAE1 in lung cancer cell lines was detected by Western blot,and the corresponding expression changes of Myc gene were detected.The effects of SAE1 gene expression changes on the proliferation and migration of lung cancer cells were detected by CCK-8 assay and transwell assay.Finally,the tumorigenicity of nude mice was used to compare the tumorigenicity of nude mice in each group,and then the effect of SAE1 gene on the degree of malignancy was evaluated.ResultsWestern blot confirmed the knockdown and overexpression of SAE1 gene in lung cancer cell lines at the protein level,and the Myc gene also showed corresponding changes.The CCK-8 assay showed that the proliferation of A549 cells and H1299 cells was significantly inhibited by knockdown of SAE1 gene,P<0.01.The overexpression of SAE1 gene significantly promoted the proliferation of H1299 cells and H520 cells,P<0.01.Transwell results showed that down-regulation of SAE1 gene expression significantly inhibited the migration of H1299 cells and H520 cells,P<0.01.In the tumor formation experiment of H1299 cells in nude mice,compared with the control group,the tumor-forming ability of the SAE1 knockdown group was significantly weakened,and the degree of malignancy was decreased,P<0.01.ConclusionOur results further confirmed that the carcinogenic activity of Myc is dependent on SAE1,and the up-regulation of SAE1 gene enhances the proliferation of lung cancer cells;after the down-regulation of SAE1 gene expression,the proliferation,migration and tumorigenic ability of lung cancer cells are inhibited.