| Objective The aim of this study was to investigate the expression of MYCN in non-small cell lung cancer and analyze the correlation of abnormal MYCN expression the clinical pathological parameters of NSCLC patients.Research the effect of MYCN on the proliferation of NSCLC cells and its its further regulation mechanism to make clear the impact of MYCN on the proliferation of NSCLC.Methods 1.Organization level:(1)8 pairs of NSCLC tumorous and the adjacent non-tumorous fresh tissues were selected to detect the expression of MYCN and PCNA by Western blot analysis;(2)immunohistochemistry was used to investigate the expression of MYCN,SOX2,Ki-67 in 140 paraffin-embedded NSCLC clinical specimens;(3)the correlation between MYCN and the clinicopathological parameters,Ki-67,SOX2 expression was analysed;2.Cellular level:(1)Western Blot was performed to detect the expression of MYCN in NSCLC cell lines(A549,H1299,Spca-1);(2)flow cytometry was performed to evaluate the effect of MYCN on the cell cycle;(3)after down-regulated MYCN expression by transfected with siMYCN,the expressions of MYCN,Cyclin D1 and PCNA were detected by Western blot,and the effect of MYCN on NSCLC proliferation was detected by flow cytometer and CCK8 assays;(4)after down-regulated and up-re regulated SOX2 expression by transfection,Western blot was performed to examine the expression of PCNA and downstream MYCN,Cyclin D1 to investigate the mechanism of SOX2-MYCN axis.Results 1.Tissue level:(1)Western blot analysis showed MYCN and PCNA protein in NSCLC tissues was significantly higher than that of corresponding cancer adjacent tissues;(2)immunohistochemistry showed MYCN、SOX2、Ki-67 was upregulated in NSCLC tissues compared with cancer adjacent tissues.The clinical statistics showed that overexpressed MYCN was correlated significantly related with smoking status(p=0.012),tumor size(p=0.008),pathology grade(p=0.005),grade(p=0.001),tumor recurrence(p=0.032),Ki-67(p<0.001),as well as SOX2(p<0.001);(3)Spearman’s correlation test showed a remarkable correlation did exist between the expression status of MYCN and that of SOX2(p<0.001)and Ki-67(p<0.001);(4)Cox proportional survival analysis indicated that pathology grade(p=0.035)and MYCN expression(p<0.001)were independent prognostic indicators of overall survival;(5)the survival curves showed that high expression of MYCN was significantly associated with poor overall survival rate(p<0.001)and poor disease-free survival(p<0.001)of NSCLC patients.2.Cellular level:(1)Western blot results indicated that all NSCLC cells expressed high levels of MYCN and SOX2,especially A549,but SOX2 expression in was the lowest,so ws choosed A549 and Spca-1 as experimental cell lines;(2)the results of serum starvation and releasing experiment showed that the expression of MYCN increased with the progression of cell cycle,which indicated that MYCN was involved in the progression of NSCLC cell cycle;(3)CCK-8 and flow cytometer assays showed down-regulated MYCN expression inhibited the proliferation of A549 cells;(4)Western Blot showed that up-regulated or down-regulated SOX2 expression could enhance or inhibit MYCN and Cyclin D1.Conclusions 1.Tissue level:(1)MYCN was an oncpgene,which was overexpressed in NSCLC tissued;(2)MYCN expression was negatively correlated with the prognosis of patients with NSCLC;2.Cellular level:(1)MYCN might promote proliferation of cells by regulating cell cycle;(2)Depletion of MYCN can inhibit proliferation of NSCLC cells,which provids a novel therapeutic target forNSCLC;(3)SOX2,which was a well-known oncogene,might be a upstream of MYCN;(4)SOX2 might promote proliferation of cells to develop NSCLC by MYCN. |
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