Research On The Mechanism Of MYCN Amplified Small Cell Lung Cancer Based On The SKP2 Signaling Pathway Targeted Therapy

Background:Small Cell Lung Cancer(SCLC)is one of the most common malignant tumors in the world,accounting for about 15% of lung cancer,the sixth most common malignant tumor,its invasion and metastasis are very strong,and there is currently no effective treatment plan.Elucidating its occurrence and development mechanism is one of the key scientific issues that need to be solved urgently in the field of lung cancer research.Our previous studies have shown that SKP2 is an important component of ubiquitin ligase,mediating the degradation of various tumor suppressor proteins to promote cancer development and progression.It has been reported that the expansion of MYCN(encoding N-Myc)in tumor cells usually predicts a poor prognosis,but direct drug targeting of N-Myc is still difficult to achieve,so it is imperative to find alternative strategies to target N-Myc.Based on previous studies,we speculate that Skp2 may be the therapeutic target of MYCN amplified SCLC,and targeting Skp2 may selectively induce apoptosis in tumor cells,but the specific molecular mechanism has not been clarified.Objective:1.Study the molecular mechanism of downregulation of Skp2 to inhibit N-Myc;2.The therapeutic effect of targeted Skp2 on small cell lung cancer was confirmed at the animal level.Methods:1.Clinical samples were collected from 30 pairs of diseased tissues and adjacent tissues of small cell lung cancer,as well as 7 small cell lung cancer cell lines and 1normal tracheal epithelial cell line BEAS-2B,and the expression of N-Myc,p27 and Skp2 in SCLC tissues and cells was detected by Western-Blot,immunohistochemistry,RT-PCR and other methods.2.The plasmid was used to construct overexpressed Skp2 transfected cells and sh RNA low-expression Skp2 cells,and the apoptosis,migration and invasion changes of these two cells were observed by Tunel experiment,cell scratch experiment,Western-Blot and other methods.3.The relationship between N-Myc and Skp2 promoter region and p27 expression was detected by luciferase reporter analysis,Chip technology,RT-PCR and other technologies.4.In the animal model of nude mouse transplantation tumor,the nude mouse was injected with Skp2-expressing small cell cancer cells and sh RNA low-expression small cell cancer cells,and the survival and transplantation tumor of nude mice were observed one month later,and tumor tissues were taken for immunohistochemistry and Western-Blot detection.Results:1.Compared with normal tissues,N-Myc and Skp2 were upregulated in SCLC.Instead,P27 was down-regulated.2.Confirmed the successful silencing of Skp2 in H82 cells and overexpression in H196 cells.We believe that after overexpressing Skp2,H196 cell apoptosis decreases,migration and invasion increase significantly.3.It was confirmed that N-Myc was upregulated in SCLC and revealed its ability to transcriptionally regulate Skp2 to inhibit p27 and promote SCLC tumor growth.4.The influence of Skp2/p27 axis on N-Myc was verified by silencing experiment.SCLC cell apoptosis is reduced after N-Myc overexpression and can be reversed after Skp2 silencing.5.Animal experiments on transplanted tumors in nude mice: direct measurement of implanted tumors showed that the weight and volume of tumors decreased significantly after Skp2 silence,and the contrast was quite obvious,and the number of metastatic nodules in the lungs decreased after Skp2 knockdown.Conclusion:1.It was confirmed that N-Myc positively regulated Skp2 expression to negatively regulate the level of p27 in SCLC tumors.2.Through the signal axis of N-Myc/Skp2/p27,blocking the expression of Skp2 may bring new opportunities for the treatment of SCLC.