Hypoxia-induced SPOP Attenuates Growth And Metastasis Of Choriocarcinoma Cells By Targeting PTEN/PI3K/AKT Pathway And Inhibiting EMT

Background/Aims:Hypoxia microenvironment and hypoxia inducible factor（HIF-1）play an important role in many tumors,and choriocarcinoma（CC）is in a high hypoxia response state compared to normal chorionic tissues or other tumors,so it is a good model to investigate the adaption of tumors to hypoxia microenvironment.Speckle?type POZ protein（SPOP）,a novel cancer-associated protein,was previously reported to play promoting or inhibiting roles in different malignant tumors.This research aims to explore the expression and biological functions of SPOP in CC,the relationship between SPOP and HIF-1αunder hypoxic conditions and the molecular mechanisms underlying of SPOP in CC.Methods:（1）The expression of SPOP protein in CC tissues and normal chorionic tissues was detected by immunohistochemical staining.Western blotting and qRT-PCR methods were used to detect the expression of SPOP in three trophoblast cells including HTR8-SVneo,JAR and JEG3.（2）The lentiviral vectors of SPOP over-expression or knockdown were constructed and transferred into JAR cells.We observed the multiplicity of infection using q RT-PCR and western blotting.（3）Transwell chambers method and wound healing assay were used to detect the invasion and migration of JAR cells after the transfection with lentivirus.The effect of SPOP on cell proliferation activity was detected by CCK8 method.In vivo,the effect of SPOP on the growth and proliferation of JAR cells in nude mice was tested.（4）Flow cytometry was used to detect the effect of SPOP on cell apoptosis and cell cycle.（5）Western blotting method was used to detect the expression of PTEN/PI3K/AKT signaling pathway associated proteins and EMT related proteins,including PTEN,PI3K,p-AKT,p-GSK3β,E-cadherin,N-cadherin and vimentin.（6）Western blotting and qRT-PCR were used to detect the expression of SPOP protein after hypoxia treatment by chemical reagent COCL₂.The model of low HIF-1αwas constructed using mall interfering RNA（siRNA）transfection,and the expression of HIF-1αand SPOP were tested by western blotting.（7）Western blotting method was used to detect the expression of EMT associated protein in hypoxic condition.The localization of SPOP in normoxic and hypoxic conditions was detected by cellular immunochemistry and cell fractionation assay.（8）Western blotting and Transwell chamber assay were used to explore whether SPOP participates in hypoxia-induced EMT.（9）We investigated the entirely alterations of gene expression at transcript level following SPOP over-expression in JAR cells using microarray technology,and further verified the possible mechanisms of SPOP in regulating the occurrence of CC.Results:（1）Compared with normal chorionic tissues and trophoblast cells（HTR8）,we demonstrated that SPOP expression was lower in CC tissues and CC cell lines（JAR and JEG3）.（2）Compared with the empty control group,induction or depletion of endogenous SPOP correspondingly suppressed or promoted CC cells growth,invasion and proliferation activity both in vitro and in vivo.（3）Flow cytometry was used to detect cell apoptosis and cell cycle.Apoptosis results showed that over-expression of SPOP could increase the number of apoptotic cells,while the knockout group had no significant effect on cell apoptosis.The results of the cell cycle suggested that alterations of SPOP levels could affect nearly all period of the cell cycle（including G1,S,and G2/M）.（4）SPOP can regulate the expression of PTEN/PI3K/AKT signaling pathway and EMT related protein.The results showed that the expression of PTEN and E-cadherin was up-regulated after over-expression of SPOP,and the levels of PI3K,p-AKT,p-GSK3β,N-cadherin and vimentin were down-regulated.However,SPOP knockdown had the opposite results.（5）CC cells received hypoxia treatment resulted in significantly improved expression of HIF-1α,and a gradual increased of SPOP protein level,while hypoxia had no significant effect on SPOP mRNA level.（6）Immunocytochemical experiments and cell fractionation assay showed that SPOP predominately distributed in the cytoplasm of JAR and JEG3 cells but mainly accumulated within the nucleus of HTR8 cells.However,after hypoxia treatment for 48h,the expression of SPOP was markedly enhanced both in the nucleus and cytoplasm,especially in the nucleus.（7）Hypoxia could promote EMT process of CC cells,and further experiments showed that hypoxia-induced SPOP was involved in the EMT transformation.（8）Microarray technology showed that forced SPOP expression resulted in robust effects on gene expression:31 genes were up-regulated and 940 genes were down-regulated.GO analysis results confirmed that SPOP is indeed involved in regulating the growth and proliferation of CC.Conclusion:These data indicate that hypoxia-induced SPOP could suppress growth and metastatic potentials via inactivating the PI3K/AKT signaling pathway and inhibiting EMT,which serves as a potential and novel target for the treatment of CC.However,many questions still need to be explored.