Transcription Factor PHF19 Regulates The Proliferation And Metastasis Of Glioblastoma

PHD Finger Protein 19(PHF19),a critical component of Polycomb repressive complex 2(PRC2),is crucial in maintaining the repressive transcriptional activity of some developmental regulatory genes and plays essential roles in biological processes.Abnormal expression of PHF19 causes dysplasia or serious diseases including chronic myeloid disorders and tumors.However,the biological functions and molecular mechanisms of PHF19 on glioblastoma(GBM)remain unclear.Here,we discover that PHF19 expression is positively associated with GBM progression,including cell proliferation,migration,invasion and tumorigenesis.Using XAV-939,a Wnt/β-catenin inhibitor,we find that the effects of PHF19 on GBM cells were β-catenin dependent.We also demonstrate that PHF19 expression is positively correlated with the expression of cytoplasmic β-catenin.PHF19 stabilized β-catenin through inhibiting the transcription of SIAH1,an E3 ubiquitin ligase of β-catenin,by directly binding to SIAH1 promoter region.Taken together,our results revealed a novel PHF19-SIAH1-β-catenin axis as a potential and promising therapeutic target.1.High PHF19 expression is associated with poor prognosis in patients withglioblastomaTo clarify whether the PHF19 is a prognostic factor for poor survival,we performed immunohistochemical staining(IHC)on the primary tissue microarray samples from glioma patients.The results showed that PHF19 expression levels are significantly increased in the advanced stages of glioma compare to adjacent normal tissues.Then,we examined PHF19 expression in four GBM cell lines(U-118 MG,U-87 MG,A172,LN-229)and normal astrocytomas(SGVP12).We found that PHF19 is commonly expressed in the four GBM cells,but not the SGVP12.The A172 cell line,which has no tumor-formation ability,has a relatively low PHF19 expression.To further confirm whether PHF19 is a prognostic marker for glioblastoma,we conduct the survival data from 2 databases(Tumor Glioma-French-284 and Tumor Glioma-Kawaguchi-50)which are available from the online R2 genomics analysis and visualization platform.The 2 databases all showed that high PHF19 expression is strongly associated with a poor overall survival.Moreover,Kaplan–Meier analysis showed that PHF19 expression levels is positively associated with the grades of glioma.Taken together,Our data suggest that PHF19 is highly expressed in GBM,high level of PHF19 is associated with poor prognosis for patients with glioblastoma.2.PHF19 is necessary for GBM cell proliferationTo investigate the role of PHF19 in GBM cell proliferation,we knocked down PHF19 by using two independent shRNA sequences against PHF19 in GBM cell lines(U-87 MG and LN-229),named as shPHF19 #1and shPHF19 #2.Western blot analysis show that the shPHF19 #1 exhibited the most significant reduction of PHF19.MTT assays also showed that the shPHF19 #1 exhibited the significant decline in growth curves.Hence,the following experiments were all performed using the highly effective shPHF19#1,which used as a representative shPHF19,shGFP was used as a negative control.BrdU assays were performed to show that PHF19 knockdown led to significant reduce in DNA synthesis compared with control cells.Then,we examined the cell cycle distribution of PHF19 knockdown cells and control cells by flow cytometry and found that knockdown of PHF19 induce cell cycle arrest at the G1/S phase.In order to explore the molecular mechanism underlying PHF19-induced cell cycle arrest,we detected some G1/S cell cycle phase-related proteins.At last,we found that the expression CDK4,CDK6,and Cyclin D were reduced but the p21 was increased when knocked down PHF19.To further confirm the effect of PHF19 on cell proliferation,we then overexpressed PHF19 in PHF19-knockdown cells,So that the expression of PHF19 in U-87 MG and LN-229 to were rescued.After overexpression of PHF19,the PHF19-knockdown cells rescue the retarded proliferation,suggesting that the reduced cell proliferation caused by knocking down PHF19 is because of the decrease in PHF19 expression in GBM cells.In general,these results indicate that PHF19 was necessary for GBM cell growth and proliferation.3.PHF19 is essential for GBM cell migration and invasionAs one of the most aggressive brain tumor cells,GBM cells have a strong ability to migrate and invade.In order to verify whether PHF19 is related to cell migration and invasion of GBM,migration assays and invasion assays were performed.Stable knockdown of PHF19 resulted in a significant reduction in the ability of GBM cells migration and invasion.To further explore the role of PHF19 on cell migration and invasion,Western blot analysis were performed to detect the mesenchymal markers(Snail,Slug,ZEB1 and N-cadherin)epithelial-to-mesenchymal transition biomarker(β-catenin)and a epithelial marker(E-cadherin).The cells stable knockdown of PHF19,decreased the levels of Snail,Slug,ZEB1,N-cadherin,MMP9 and β-catenin,but the E-cadherin was increased.When PHF19 expression was restored in PHF19-knockdown LN-229 cells,the ability of tumor formation and colony formation have been restored.Corresponding,PHF19 and Ki-67 expression were also restored in the tumor samples.These results suggested that PHF19 play an important role in the tumorigenesis of GBM cells.4.PHF19 is closely related to ubiquitination degradation of β-cateninThe previous date has shown that β-catenin was downregulated in PHF19 knockdown cells.And the downstream of β-catenin was detected subsequently.Not surprisingly,expression of C-myc,CyclinD1,and MMP7 were decreased as well,while it was restored after PHF19 expression was recovered.We hypothesized that PHF19 plays the regulatory role through β-catenin.To test this hypothesis,XAV-939,a Wnt/β-catenin signaling inhibitor was used to treat PHF19 overexpression cells.The promotion of cell proliferation,migration and invasion was successfully blocked after XAV-939 treatment,suggesting that the proliferative and metastatic effects of PHF19 on GBM cells were β-catenin dependent.As is verified that β-catenin was downregulated after PHF19 knock-down,the mechanism by which the expression of β-catenin was decreased need further exploration.Therefore,the mRNA level of β-catenin was first analyzed by RT-PCR.Relative to WB results,there is no significant change on mRNA level,suggesting that PHF19 may regulate β-catenin in a post-transcription way.The cellular distribution of β-cateninTo assess the relationship between PHF19 expression and the activation of β-catenin,we performed nuclear and cytoplasmic extraction assay.β-catenin was remarkable lowered in cytoplasmic but not in nuclear.In view of the fact that β-catenin was mostly degraded through ubiquitin-proteasome pathway in cytoplasmic.We consider whether the reduction of β-catenin protein level was due to PHF-19 associated proteasomal degradation.And then the proteasome inhibitor MG132 was used in our next assays.The decrease of β-catenin caused by PHF19 knockdown was abolished after MG132 treatment.To be convinced,we examined the turnover rate of PHF19 protein in the presence of cycloheximide(CHX),an inhibitor of protein synthesis.The turnover rate of PHF19 was enhanced in PHF19 expressing LN-229 cells compared with control plasmid expressing cells Taken together,these results demonstrated that PHF19 stabilized β-catenin by suppressing proteasomal degradation.On account of the fact that ubiquitination is a pivotal step for proteasomal degradation pathway,we test whether PHF19 can regulate β-catenin ubiquitination.In vitro ubiquitination assays were carried out using Flag-PHF19.The results showed that the ubiquitination of β-catenin was significantly decreased after PHF19 overexpressed.Taken together,these findings demonstrated that PHF19 increased β-catenin stability by reducing the ubiquitination of β-catenin.5.PHF19 regulates the transcription of SIAH1 which is the ubiquitination ligaseof β-cateninPHF19 is unable to mediate the ubiquitination of β-catenin directly for it is a transcriptional regulator.Thus,we examined mRNA levels of ctnnb1 regulators,such as BTRC,SIAH1,GSK3,and so on.Only SIAH1 was significantly upregulated when knocked down PHF19.Next,a dual-luciferase reporter assay was designed to study the mechanism by which PHF19 regulates SIAH1 expression in PHF19-knockdown LN-229 cells.The fragment of SIAH1 promoter regions were designed and inserted into the pGL3 basic vector.We co-transfected the shPHF9/Flag-PHF19 plasmid and the SIAH1 Promoter into 293 FT cells.The empty pGL3 basic vector was used as the control.The results demonstrated that SIAH1 promoter activity was significantly increased by knocking down the PHF19.Meanwhile,SIAH1 promoter activity was reduced when co-transfected with Flag-PHF19.To determine whether the suppression of SIAH1 promoter activity was caused by PHF19 binding,a ChIP assay was performed to map the PHF19-bindinglocus on the SIAH1 promoter.As shown in,PHF19-binding sites were enriched in the promoter proximal region D(-358 bp to +115bp).This finding confirmed our previous results,showing that PHF19 inhibits SIAH1 transcription by directly binding to its promoter.