The Mechiasm Study Of E3 Ligase RFWD3 In The Development And Progression Of Lung Cancer

Background: Lung cancer is the leading cause of cancer death worldwide,with short5-year survival rate.Ubiquitination is playing an important role in regμl ating the life activities of organisms and is closely related to the development of tumors,cardiovascμl ar and other diseases.Ubiquitination is achieved throμgh a series of reactions formed by E1 ubiquitin-activating enzyme,E2 ubiquitin-coupled enzyme and E3 ubiquitin ligase.E3 ligase RFWD3 plays an important role in the development of tumorigenesis.To date,its function and mechanism in NSCLC are unclear.Methods: Immunohistochemistry(IHC)was firstly conducted to detect the expression level of RFWD3 in the cancer tissues and its adjacent normal tissues of NSCLC patients,and to explore the relationship between the expression level of RFWD3 and the survival time of patients.Then we constructed two stable RFWD3 overexpression or knockdown NSCLC cell lines,A549 and H1299,and performed Incucyte and Transwell experiments to study the RFWD3 effects on cell proliferation,invasion and migration.In addition,flow cytometry experiment was conducted to detect the effect of RFWD3 on the cell cycle of NSCLC cell lines.By using RNA-seq analysis,we explored the downstream pathways of RFWD3 that may have effects on NSCLC.We used coimmunoprecipitation combined with mass spectrometry to identify the proteins that may bind to RFWD3.Afterwards,co-immunoprecipitation,immunofluorescence,and Biacore experiments were performed to verify the candidate protein that interact with RFWD3.Finally,we established a subcutaneous xenograft lung tumor mouse model by subcutaneous injection of RFWD3 overexpression or knockdown NSCLC cells,and measured the tumor size regμl arly as to explore the RFWD3 effect on NSCLC tumor growth in vivo.Results: In NSCLC patient,the expression of RFWD3 in tumor tissues was significantly higher than that in adjacent normal tissues.The survival of patients with high expression of RFWD3 was significantly shorter than that of patients with low expression(p<0.05).Stably overexpression of RFWD3 in NSCLC cells coμl d significantly increase the cell proliferation rate when compared with that of the control cells,while stably knocking down of RFWD3 coμl d significantly inhibit NSCLC cell proliferation(P<0.05).The invasion and migration ability of NSCLC cells that overexpress RFWD3 is significantly enhanced,while knockdown of RFWD3 coμl d significantly inhibit cell invasion and migration.Both overexpression or knockdown of RFWD3 can affect the DNA synthesis phase(S phase)of NSCLC cells.Analysis of mass spectrometry resμl ts found that the differentiated proteins with significant changes are mainly enriched in the cell cycle pathway.After mass spectrometry analysis,it was shown that CDK4 is one protein that can bind to RFWD3.The immunofluorescence staining confirmed that RFWD3 coμl d co-localize with CDK4.The in vivo resμl ts showed that the tumor overexpressing RFWD3 had a significantly higher growth rate than that of control tumor,while the RFWD3-knockdown tumor had a significantly lower growth rate than that of control tumor.Conclusion: RFWD3 can promote the NSCLC cell proliferation by mediating cell cycle pathway,sμggesting that RFWD3 may be used as a potential target for molecμl ar targeted therapy of lung cancer.This study might provide the new insights for treatment of lung cancer.