A Study On The Role Of HHEX In The Migration Of Non-small Cell Lung Cancer Cells

Lung cancer is the carcinoma with the highest rates of incidence and fatality in the world.As other malignant tumors,lung cancer can cause much death dues to its strong metastatic capability.The metastasis of lung cancer is an extremely complex process.And the enhanced motility of lung tumor cells is the basis of their metastasis.Therefore,to find critical target regulating movement of tumor cells is very important for lung cancer therapy.HHEX(Human haematopoietically expressed Homebox)is located at 10q23.33 of human chromosome and the length is 1776bp.The protein has 270 amino acids that can be divided into three parts:proline riched N-terminal domain(1-136aa),homebox domain(137-197aa),and acidic amino riched C-terminal domain(198-270aa).The homebox domain can bind to DNA,so HHEX can act as a transcription factor to regulate some genes expression.Its N-and C-terminal domain are the effective domains,which positively and negatively regulates gene expression respectively.Apart from regulating gene expression,HHEX also plays an important role in embryonic development,angiogenesis,stemness maintenance,lymphocyte differentiation,the occurrence of type-2 diabetes and leukemia.In addition,the study found that HHEX can regulate the movement of prostate cancer and breast cancer cells.However,whether HHEX can regulate the movement of lung cancer cells and the molecular mechanism has not been reportedRHOA,CDC42 and RAC1 are three important members of RHO-GTPases and play significant roles in regulation of cell motility.For those RHO-GTPases,their activation is premise of their functions.They became inactive by binding to GDP through the roles of RHOGAP and RHOGDI or active by binding to GDP through RHOGEF.Activated RHO-GTPases promotes assembly of microfilaments by activating downstream signaling pathways,which ultimately acts on some microfilament-binding proteins such as ARP2/3,mDia,MLC,CFL1,etc.,to regulate cell movement.CFL1 is a member of the actin dissociation factor ADF/COFILIN family.It binds to G-actin and F-actin to promote depolymerization of actin filaments.The sever activity of CFL1 is regulated by several mechanisms including competitive inhibition of PIP2,pH and phosphorylation.For example,CFL1 phosphorylation at Ser3 inhibits its binding to microfilaments,thus repress its functions.Therefore,phosphorylation and dephosphorylation of CFL1 is important to regulate cell movement.In this study,NSLCCs were used as material.And some experimental methods like:overexpression,small RNA interference.Western blot,co-IP,wound-healing scratch and confocal were also applied.We focused on the mechanism of migration regulated by HHEX.Our results showed:1.HHEX expression was controled by in Calu-1 cells and A549,the wound-healing scratch assay suggested cell migration was inhibited by HHEX.2.Down-regulation of HHEX in Calu-1 cells,A549 cells and H1792cells,F-actin was stained with TRITC-phalloidin,cell protrusions formation was visualized by confocal laser scanning microscope.Images indicatedup-regulation of HHEX decreased protrusion formation.3.Lamellipodium and filopodia formation decreased when HHEX expression was upregulated.4.CFL1 phosphorylation level was promoted by HHEX knock down in Calu-1 cells and A549 cells.And HHEX overexpression decreased CFL1 phosphorylation in H1299 cells and H157 cells.5.The activation of RHO-GTPase was detected with RHO activation assay kit.Results showed RHOA and CDC42 activation was repressed by HHEX overexpression in A549 cells and H1299cells.6.Plasmids of HHEX and RHOGDIA were co-transfected into A549 cells,H1299 cells and H157 cells,the WB results suggested RHOGDIA promoted CFL1 phosphorylation decrease induced by HHEX.7.HHEX interacted with RHOA/CDC42.And this interaction would enhanceinteraction of RHOGDIA and RHOA/CDC42.Conclusion:HHEX could promote interaction of RHOGDIA with RHOA and CDC42 thus repressed activation of RHOA and CDC42,which resulted in significant inhibition of RHOA/CDC42-p-CFL1 pathway.This effect led to less protrusions formation in cell edges,thus repressed cell migration of of non-small cell lung cancer cells...