The Role Of MARCH7 In Non-small Cell Lung Cancer And Its Mechanism

Objective:The aim of the study was to explore the role of E3 ubiquitin-ligase MARCH7 in the development of non-small cell lung cancer(NSCLC),and to explore the underlying molecular mechanism,so as to provide a new idea for the experimental basis for the treatment of NSCLC in the future.Methods:1、The clinical significance of MARCH7 in NSCLC was investigated by statistical analysis:Western blot and immunohistochemistry were applied to examine the expression of MARCH7 in NSCLC cancer tissue samples.The correlation between MARCH7 expression and clinicopathological features of NSCLC was satistically analysed,and the influence of MARCH7 on the prognosis of NSCLC patients were studied.2、The effect of MARCH7 on the proliferation of NSCLC cells was studied by in vitro approaches:The siRNA was constructed and stably transfected into A549 cells to inhibit the expression of MARCH7.The effects on cell cycle and proliferation were detected by CCK-8,clone formation and flow cytometry.The activation of the downstream NF-κB and Wnt/β-catenin signaling pathways was assessed by luciferase reporter gene,and the expression of the pathway molecules was measured by Western blot.3.The effect of MARCH7 on NSCLC tumorigenesis was studied by in vivo experiments:Study animals were randomly divided into three groups,three animals per group,to establish nude mouse transplantation tumor model.Spirit,diet and stool of nude mice was observed daily,the weight of mice and the size of tumor were measured every two days.After record relevant measurement data,the tumor growth curve was established respectively by approximate volume and tumor growth days for vertical and horizontal coordinates.The mice were euthanized at the twenty-first day and tumors were collected.The expression of PCNA and Ki-67 was valued by immunohistochemistry to verify the proliferation of the tumor.Meanwhile,the expression changes of NF-κB and Wnt/β-catenin signaling pathway molecules were detected by Western Blot.Results:1、To analyze the clinical significance of MARCH7 in NSCLC:Western blot and immunohistochemistry results showed that the expression of MARCH7 in NSCLC cancer tissues was significantly higher than that in paracancerous tissues.Statistical analysis of tissue microarray staining results and clinicopathological parameters of NSCLC patients revealed that MARCH7 expression was closely related to TNM stage,degree of tumor differentiation and lymph node metastasis of NSCLC patients.Furthermore,univariate and multivariate analysis and survival curve analysis displayed that MARCH7 might be a prognostic factor in NSCLC patients,and its high expression was closely associated with poor prognosis.2、MARCH7 regulates cell proliferation by mediating the activation of NF-κB and Wnt/β-catenin signaling pathways:MARCH7 siRNA was constructed and stably transfected into cells.CCK-8 assay indicated that the growth rate of tumor cells in the interference group was considerably reduced.The number of colonies in the interference group decreased and the number of cells in the colony was also decreased in the plate clone formation experiment.Flow cytomography presented that G0/G1 phase cells were predominantly increased after blocking endogenous MARCH7 expression,and G0/G1 phase arrest occurred in A549 cells.After A549 cell line was transfected with MARCH7-siRNA,the reporter gene activity of NF-κB signaling pathway and Wnt/β-catenin signaling pathway was significantly reduced validated by double luciferase reporter gene assay.Western Blot analysis showed that the expression of NF-κB P50,NF-κB P65 and β-catenin were decreased,while the expression of E-cadherin was elevated.3、MARCH7 promotes the growth of transplanted tumor:MARCH7 overexpressed virus was constructed and transfected into A549 cells,then subcutaneously injected into nude mice.At the same time,blank control group was set up for comparative study.It was demonstrated that tumors were formed in all nude mice at the injection site,but the tumor volume was significantly larger in the overexpressed MARCH7 group than control nude mice at the same period.Elevated expression of PCNA and Ki-67 was observed in tumor mass of the overexpressed MARCH7 group measured by immunohistochemical analysis,accompanied by enhanced level of NF-κB P50,NF-κB P65 and P-catenin detected by Western Blot.Conclusion:1、MARCH7 is highly expressed in NSCLC cancer tissues,and its high expression level is closely related to TNM stage,degree of tumor differentiation as well as lymph node metastasis of NSCLC patients.High expression of MARCH7 predicts poor prognosis in patients with NSCLC.2、The data of in vitro experiment presented that MARCH7 can promote the proliferation of NSCLC cells by activating NF-κB and Wnt/β-catenin signaling pathways.3、By establishing the xenograft tumor model in nude mice,we demonstrated that MARCH7 can promote the proliferation of tumor cells and tumor growth in vivo,and may become an independent therapeutic target of NSCLC.