The Mechanism Of TRIB2 Inhibiting NF-?B Classical Pathway Through RFWD2 In Lung Adenocarcinoma Cells

Backgrounds:Lung cancer is one of the most malignant tumors with high morbidity and mortality rates in China,of which Adenocarcinoma is the most prevalent type and 85%of it is non-small-cell lung cancer.Over the past 30 years,lung cancers are usually diagnosed late.What's worse,the prognosis is poor and the survival rate is about 16%in 5 years.Therefore,new molecularly targeted therapy is urgently needed.It was reported before that TRIB2 is involved in the development of various cancers like leukemia,ovarian cancer and liver cancer.And our group find in those reports that TRIB2 is related to lung cancers,probably to the classical pathway of NF-?B.The NF-?B family is composed of p50,p65,which play a role in the form of heterodimeric and homodimeric complexes.However,the specific mechanism has not been reported.Objective:To investigate whether the mechanism of action of TRIB2 in lung adenocarcinoma is related to the NF-?B classical signaling pathway and to explore the gene targets to prevent the development of lung adenocarcinoma.Methods:1.MTT assay,clone formation assay,flow cytometry and trans-well assay were used to detect the effect of proliferation,apoptosis and migration in lung adenocarcinoma cells transfected pcDNA-TRIB2 and siR-TRIB2.In terms of molecular mechanism,the regulation of TRIB2 on NF-?B classical signaling pathway were detected by Western blot and Luciferase Reporter Assay.2.293T cells were transfected with Flag-TRIB2,and the interaction between TRIB2,I?B-?and ubiquitin-related molecules was detected by protein co-immunoprecipitation.A549 cells were transfected with Flag-TRIB2,GFP-TRIB2,HA-I?B-?,and the interaction between TRIB2,I?B-?and RFWD2 was detected by immunofluorescence colocalization.3.To further detect the specific domain interacted between TRIB2,I?B-? and RFWD2,it was divided according to the structural characteristics of TRIB2 and transfected into 293T cells.Then,the specific domain of TRIB2 interacting with I?B-?,RFWD2 was detected by co-immunoprecipitation.4.The lung adenocarcinoma cells was inhibit RFWD2 expression,then measured the effect of TRIB2 on cell proliferation,apoptosis and migration with MTT assay,clone formation assay,flow cytometry,real time cellular analysis(RTCA).Western blot assay used to dectect the variable of NF-?B classical signaling pathway-related molecule.5.Detecting the effects of TRIB2 and RFWD2 in lung adenocarcinoma cells in vivo using nude mice xenografts experiments.Results:1.Over-expression of TRIB2 could promote the proliferation and migration of lung adenocarcinoma cells and inhibit the apoptosis of lung adenocarcinoma cells.While the result of knockdown of TRIB2 was opposite.TRIB2 inhibits NF-?B classical signaling pathway.2.The results of co-immunoprecipitation assay showed TRIB2 was interacted with I?B-?,RFWD2.The co-localization region was detected by immunofluorescence.3.The results of segmental co-immunoprecipitation assay showed the pseudokinase domain and C-terminal of TRIB2 domain interacted with RFWD2.The TRIB2 domain containing only the pseudokinase structure or the N-terminal region might interact with I?B-?.4.Inhibition of RFWD2 expression in lung adenocarcinoma cells reverses the effect of TRIB2 on cell proliferation,migration,apoptosis and its effect on NF-?B classical signaling pathway.5.The results of nude mice xenografts showed that the volume of transplanted tumors decreased,the weight decreased,and the growth rate slowed down after knocking down TRIB2 or RFWD2.The result was opposited after overexpression of TRIB2.Overexpression of TRIB2 in lung adenocarcinoma cells inhibits the expression of RFWD2,compared with the overexpressed TRIB2 group,the volume of the transplanted tumor was reduced,the weight was reduced,and the growth rate was slowed downConclusion:TRIB2 could significantly promote the proliferation and migration of lung adenocarcinoma cells.TRIB2 was able to down-regulate the expression of NF-?B.The mechanismwas might be due to the negtive regulation of NF-?B signaling pathway by TRIB2 via RFWD2.Thus,RFWD2 is expected to become a new target for clinical treatment of lung adenocarcinoma.