FAM212B Activates Rac1/ERK Signaling Pathway And Promotes The Molecular Mechanism Of Malignant Phenotype Of Lung Cancer Through Binding With DVLs

Objective:FAM212B is the Family of protein sequence similarity 212 member B,also known as INKA2（Inka box actin regulator 2）and C1orf183（Chromosome 1 open reading frame 183）.FAM212B has been identified as a downstream target of FRA-2/AP-1 gene in melanoma studies up to now^[1].However,the downstream signaling pathway of FAM212B is still unclear,and the expression and molecular mechanism of FAM212B in NSCLC are also unclear.Methods:1.First,immunohistochemistry was used to analyze the expression and subcellular localization of FAM212b in NSCLC specimens,and statistical analysis was used to analyze the clinicopathological factors of FAM212b and NSCLC specimens.Western blotting was used to detect the expression of FAM212b in HBE and six commonly used NSCLC systems,and laser confocal experiment was used to detect the localization of FAM212b in NSCLC.2.After that,G418 screening technology was used to construct cell lines stably expressing FAM212B,and si RNA technology was used to bidirectional regulate the expression level of FAM212B,and the proliferation and invasion ability of FAM212B in NSCLC was detected by cell viability test and cell invasion and migration test in vitro.3.We used luciferase reporter gene assay and western blot assay to study whether FAM212b exerted its molecular mechanism through activation of classical Wnt signaling pathway,and further screened and confirmed that FAM212b exerted its role through ERK signaling pathway.4.At the same time,we used immunoprecipitation method and immunofluorescence assay to explore the combination of FAM212b and DVLS.In addition,according to the structural domain of FAM212b and the research characteristics that C-terminal cleavers and C-terminal cleavers contained VWV sequence,C-terminal cleavers and C-terminal cleavers were constructed,and Western blotting and in vitro proliferation and invasion experiments were used to further determine whether the downstream changes of FAM212b were dependent on the combination with DVLs.5.At this point,we considered how the combination of FAM212B with DVLS affected the activity of ERK signaling pathway.Therefore,we speculated that the combination of FAM212B with DVLS could activate the small GTPase Rac1,and the three could form a complex to activate the ERK signaling pathway after mutual binding.At the same time,we by building Rac1 mandatory inactivation mutants,observe FAM212B make Rho GTPase activity-after the change of the downstream impact factor of change and biological function,at the same time,application of Rac1 E Hop-016 specific inhibitors to suppress the activation of Rac1/ERK signaling pathways,both more confirmed FAM212B through combination with DVLs activating Rac1/ERK signaling pathway this hypothesis.Results:1.First,we found that FAM212B was negatively expressed in normal bronchial epithelial tissues and alveolar epithelial tissues,and was significantly overexpressed in the cytoplasm of squamous cell carcinoma and adenocarcinoma tissues.Its expression was closely correlated with TNM staging,lymph node metastasis and poor prognosis of NSCLC.At the same time,we found that the expression of FAM212B in non-small cell lung cancer cell lines was also significantly higher than that in normal bronchial epithelial cell lines,and was also localized in the cytoplasm.2.After the overexpression of FAM212B,the proliferation and invasion ability of NSCLC were significantly increased;At the same time,the results were obviously opposite after the interference of FAM212B expression.These results suggest that FAM212B plays a carcinogenic role in NSCLC.3.There was no significant change in the activity of classical Wnt signaling pathway after bidirectional regulation of FAM212B expression;The expression of classical Wnt target genes MMP-7,c-MYC and Cyclin D1 did not significantly change after biaxial expression of FAM212B,indicating that FAM212B did not play a molecular role by affecting Wnt signaling pathway.Later,we screened in detail the signaling pathways related to proliferation:ERK/JNK/SPAK/P38,etc.,and we found that FAM212B could activate the ERK signaling pathway.4.Immunoprecipitation assay indicated that FAM212B could bind to DVLS.Simultaneously,the laser confocal assay showed that FAM212B and DVLS were co-localized in the cytoplasm.We called plasmids containing over FAM212B–Wild Type,knock out the c-terminal VWV sequence structure are called FAM212B-ΔC,only knock out INKA2 structure are called FAM212B-ΔF,found the c-terminal VWV knockout after transfection expression sequence of p-ERK expression,show that the c-terminal VWV FAM212B protein molecules sequence for regulating the ERK signaling pathways play a key role.In addition,we found that P-ERK expression was significantly increased after co-transfection of FAM212B wild-type&DVLS.Common transfection FAM212B-ΔC&DVLs p-ERK expression quantity compared to fall;Functional studies also proved that the proliferation and invasion abilities of FAM212B&DVLs were significantly increased after co-transfection,which indicated that FAM212B was dependent on the combination with DVLs to activate the ERK signaling pathway.5.Through the small GTPases experiment,we detected whether FAM=212B affected Rac1 activity.Rac1-GTPRs were significantly up-regulated after overexpression of FAM212B,which proved that FAM212B could activate RAC1 activity.Therefore,we speculated that the combination of FAM212B and DVLS could activate the ERK signaling pathway through enhanced RHO-GTPase activation.At the same time,we found that Fam212B,DVLS and Rac1were inter bound by immunoprecipitation assay.Laser confocal analysis indicated that FAM212B and RAC1 were co-localized to the cytoplasm,and DVLS and RAC1 were mainly co-localized to the cytoplasm.Finally,we used RAC1-specific inhibitor EHOP-016 and transfected RAC1 mutants to suppress the activity of RAC1 signaling pathway,and found that p-ERK expression was significantly down-regulated,and proliferation and invasion abilities were significantly decreased.Conclusions:Fam212b protein can activate RAC1 through binding with DVLS,and then activate ERK signaling pathway,promoting the occurrence and development of NSCLC.