Systematic Analysis And Functional Research Of Hippo Pathway Regulated By Deubiquitinase And Tetraspanin Family Members

The Hippo signaling pathway was first identified by genetic screening in Drosophila melanogaster.It is mainly composed of two kinase cascades and downstream transcription complex.The known core components of Hippo signaling pathway in mammals include two kinase complexes of MST1/2(mammalian STE20-like protein kinase1/2)/SAV1(Salvador)and downstream LATS1/2(Large tumor suppressor kinase1/2)/MOB1(MOB kinase activator 1A/B)and YAP(Yes-associated protein)-TEADs(TEA domain)transcription complex.When Hippo pathway is activated,MST1/2 phosphorylates downstream LATS1/2 and MOB1.LATS is phosphorylated and activated,and then phosphorylates downstream YAP and TAZ(transcriptional co-activator with PDZ-binding motif).On the one hand,phosphorylated YAP and TAZ binds to 14-3-3 protein,resulting in their retention in the cytoplasm.And on the other hand,it is degraded through the ubiquitin proteasome pathway,thereby inhibiting the transcription of the downstream target genes.When Hippo pathway is inactivated,the activities of the MST1/2 and LATS1/2 kinases are inhibited.And YAP could not be phosphorylated.Then the non-phosphorylated YAP enters the nucleus,and binds to transcription factor TEADs to activate transcription of downstream target genes.Previous studies have shown that Hippo signaling pathway plays an important role in regulating organ size,tissue regeneration and tumorigenesis.Hippo pathway isregulated by a variety of extracellular signals,and its effector YAP/TAZ plays a key role in signal regulation.Abnormal regulation of Hippo signaling pathway can lead to tumorigenesis,and YAP shows high expression in various tumors.However,the molecular mechanism of Hippo pathway on tumorigenesis and progression is still unclear.We observed that many members of the deubiquitinating enzymes and Tetraspanin families showed high expression in tumors through bioinformatics analysis,and whether deubiquitinating enzymes and Tetraspanin families might regulate the initiation and progression of cancers by affecting the Hippo signaling pathway.Therefore,it is important to explore systematically the relationship between these two family members and the Hippo signaling pathway in order to understand the functions in the initiation and progression of cancers.Meanwhile,it provides new clues for further understanding of Hippo pathway.The dual luciferase reporting system was used to carry out the screening of Hippo pathway.The activity of Hippo signaling pathway was detected by the Firefly luciferase reporter plasmid TBS-luciferase,and the Renilla luciferase plasmid pRL-TK was used as control for normalization.Firstly,in order to validate the feasibility of this reporting system,we performed a series of positive control experiments: we constructed overexpression plasmids(EGFP-N1-YAP,EGFP-N1-LATS2,EGFP-N1-MST2)and shRNA plasmids(shYAP,shLATS2)for the core members of this pathway respectively.The HEK-293 T cells were then seeded in96-well plate.Subsequently,TBS-luciferase reporter plasmid,pRL-TK and target plasmids were co-transfected into HEK-293 T cells.After 72 hours,Dual-Luciferase?reporter assay kit was used to detect the luciferase activity of the cell and then calculates the ratio(Firefly/Renilla).The results showed that the activity of the TBS-luciferase significantly increased after transfection of plasmids overexpression YAP and shRNA knocking down LATS2.On the contrary,the activity of the TBS-luciferase significantly decreased after transfection of plasmids overexpression LATS2,MST2 and knocking down YAP.This indicates that the reporter system can sensitively reflect the activity of Hippo pathway and can be used to screen genes thatregulate Hippo pathway.Next,we constructed the shRNA plasmid and the overexpression plasmid(Flag-DUBs)of deubiquitinating enzyme family.The dual luciferase reporting system was used to screen the deubiquitinating genes.The results showed that fifteen genes(USP2,USP18,USP20,USP22,USP29,USP36,USP42,USP43,USP49,USP52,USP54,PARP11,OTUD5,OTUB1,OTUD7B)of the deubiquitinating enzyme family had up-regulated the activity of Hippo pathway in different degrees.Similarly,we screened Tetraspanin family using the same system and observed that thirteen genes(TSPAN2,TSPAN8,TSPAN9,TSPAN10,TSPAN13,TSPAN14,TSPAN15,TSPAN17,TSPAN21,TSPAN23,CD82,CD63,TSPAN33)of the family had up-regulated the activity of Hippo pathway.Subsequently,we further detected the mRNA levels of the downstream target genes(CTGF,CYR61,ANKRD1)regulated by Hippo pathway with RT-qPCR,so as to verified the effect of candidate genes screened by dual luciferase reporting system on Hippo pathway.The results showed that the mRNA levels of CTGF,CYR61 and ANKRD1 were increased after the overexpression of USP2,USP29,USP52,PARP11 and OTUD7B of deubiquitinating enzyme family.Tetraspanin family genes TSPAN13,TSPAN14,CD82,TSPAN8,TSPAN9 and TSPAN23 have up-regulated the mRNA levels of target genes.We further detected the protein levels of the core members(YAP,p-YAP,LATS1,MST1 and p-MST)of Hippo signaling pathway by Western blot,in order to confirm the regulation of the candidate genes on Hippo pathway.The results showed that shRNA knocking down of USP2 significantly increased the protein levels of p-YAP and LATS1.The Hippo signaling pathway plays an important role in regulating cell proliferation and differentiation.And previous experiments have shown that the deubiquitinating enzyme gene USP2 could regulate the Hippo pathway.Therefore,in order to determine whether the USP2 gene affects cell proliferation,we first construct a cell line that stably knocks down USP2,and then measure the viability of the cellswith MTS.The results showed that shRNA knocking down of the USP2 gene inhibits cell viability.In summary,combined the screening results of Hippo pathway by dual luciferase reporting system with the mRNA changes results of the target genes detected by RT-qPCR,it was concluded that deubiquitinating enzymes USP2?USP29?PARP11and OTUD7 B could activate YAP.Western blot results showed that USP2 may affect Hippo pathway by regulating the protein levels of p-YAP and LATS1.Cell proliferation assay results showed that knocking down of USP2 gene inhibited cell viability.In the screening of Tetraspanin family genes,we observed that after shRNA knocking down of TSPAN13,TSPAN14,CD82,TSPAN8,TSPAN9 and TSPAN23,the activity of TBS-luciferase was significantly inhibited,indicating that these Tetraspanin candidate genes could regulate Hippo signaling pathway in some degree.The aim of this project is to find new genes regulating Hippo signaling pathway,systematically screening two protein families of deubiquitinating enzymes and Tetraspanin family.It laies a theoretical foundation for the discovery of a new mechanism regulating Hippo pathway,and provides a basis for the functional testing and molecular mechanism research in vivo,and provides us with new targets for clinical cancer therapy.