Investigation Of Pontin In Gliomagenesis By Activating TGF?/SMAD Signaling

Objectives:Pontin is an ATPase with complex cellular regulatory functions,and is involved in regulating gene transcription,chromatin remodeling,DNA damage response,macromolecular complex assembly,cell cycle and motility,all of which contribute to its core roles in facilitating cell proliferation and survival.Pontin has been proved to be overexpressed in various malignant tumors and is an indispensable key factor for tumor cell growth.Glioblastoma(GBM)is characterized by rapid proliferation and highly invasion,leading to the difficulty in radical resection during surgery and poor prognosis of the patients.At present,the relationship between Pontin and gliomagenesis remains unclear.Whether Pontin can be used as a prognostic indicator or a candidate therapeutic targets for glioma needs to be explored systematically.The purposes of this study are,(1)to verify whether Pontin is overexpressed in glioma and analyze its correlation with the grades and the prognoses of the glioma patients;(2)to test whether the alteration of Pontin expression can affect the proliferation and invasion phenotypes of the glioma cells;(3)to investigate the molecular mechanism by which Pontin impacts on intracellular gene transcription and signal pathway disorders,thus to exert important regulatory roles in gliomagenesis.Methods:1.Using the online database resources,the expression of Pontin mRNA in different malignant tumors including gliomas(GBM and LGG)was preliminarily analyzed,and the relationship between Pontin expression and the survival of glioma patients was predicted.Expression of Pontin protein in the immortalized astrocyte cell line UC2 and 7 GBM cell lines were detected and compared by Western blot.2.The expressions of Pontin and Ki-67 were detected by tissue microarray and immunohistochemistry(IHC),and the expression alteration of Pontin in gliomas of different grades were analyzed.The correlation between the expressions of Pontin and Ki-67 was analyzed by Pearson test.According to the clinical follow-up data,the relationship between Pontin expression and patients' survival was analyzed by Kaplan-Meier method.Cox's proportional hazards regression model was establishedto screen independent prognostic factors for the glioma patients.3.Two GBM cell lines(U87MG,U251)were respectively infected with indicated lentivirus to construct stable Pontin knockdown and overexpression sub-cell lines and control sub-cell lines.The proliferation and survival activities of each sub-cell line(Pontin knockdown and overexpression)were compared with those of the control sub-cell line by using the EdU,flow cytometry and the plate colony formation experiments,and the migration and invasion abilities were detected by transwell experiments in vitro.The above results could determine whether different Pontin expression could affect the biological behaviors of glioma cells.4.The U87 MG sub-cell lines with stable Pontin knockdown and overexpression and control sub-cell lines constructed above were used to establish the model of intracranial orthotopic xenografts in nude mice.The growth of the xenografts was monitored and photographed by in vivo imaging technology,the size of the xenografts was observed by HE staining,and the expressions of Pontin and Ki-67 were detected by IHC.5.The transcriptome sequencing and the bio-informatics analysis revealed that,Pontin may regulate TGF?/SMAD pathway and TGF?R2 gene expression.Then Western blot and qRT-PCR were performed to verify whether the expression of the downstream genes in this pathway could be influenced by Pontin.The agonist and inhibitor of this pathway were used to further clarify the effect of Pontin on the activation of this pathway and the malignant cellular phenotypes.6.Literatures and databases were comprehensively reviewed,and the luciferase reporter containing the TGF?R2 promoter was constructed.The luciferase activity was detected to determine the influence of Pontin knockdown or overexpression on this reporter.Co-immunoprecipitation(Co-IP)and immunofluorescence(IF)assays were combined to explore whether LEF1 can interact with Pontin and collectively bind to the TGF?R2 promoter to synergistically regulate TGF?R2 gene transcription.7.TGF?R2 was overexpressed in Pontin-knockdown GBM sub-cell lines and to verify the rescue effects of restoring TGF?R2 expression on proliferation,survival and migration phenotypes of the sub-cell lines by using EdU,colony formation and transwell experiments in vitro.Results:1.The predicted results from the online databases showed that Pontin mRNA was higher expressed in various types of tumors than that in the corresponding control group,including GBM and LGG.The predicted results also showed that the glioma patients with higher Pontin expression have significantly reduced Disease-free survival(DFS)and overall survival(OS).Western blot results showed that the expression level of Pontin in all the 7 GBM cell lines tested was significantly higher than that in UC2.The top 200 Pontin-co-expressed genes screened from cBioPortal are functionally enriched in biological processes like DNA replication,cell cycle and RNA splicing,indicating that Pontin might be involved in these processes in GBM cells.2.Tissue microarray and IHC results showed that Pontin was significantly higher expressed in gliomas than that in the non-tumoral control tissues,and the positive Pontin labeling index(LI%)increased with the increase of the glioma grade.The pairwise comparison among the glioma groups and the control group all showed significant difference(P<0.001)Pearson correlation analysis showed a significant positive correlation between Pontin LI% and Ki67 LI%.Kaplan-Meier survival analysis showed that when patients were divided into several subgroups according to age,IDH gene status or KPS score,the DFS and OS were worse in the high Pontin expression group than in the low expression group in the above subgroups.All the differences were statistically significant(P<0.01~0.0001).Cox's regression analysis showed that Pontin LI% and IDH gene status were two independent prognostic factors for glioma patients.3.Western blot confirmed the successful construction of the indicated sub-cell lines in which Pontin was stably knockdown and overexpressed.EdU assay confirmed that knockdown and overexpression of Pontin could reduce and increase the proliferation activity of GBM cells,respectively.Flow cytometry detection revealed that Pontin knockdown induced G1 arrest in GBM cells.Colony formation assay indicated that knockdown and overexpression of Pontin could inhibit and promote the survival of GBM cells,respectively.Transwell assay confirmed that knockdown and overexpression of Pontin could suppress and facilitate the migrationand invasion of GBM cells,respectively.4.Nude mice were orthotopically implanted with the U87 MG sub-cell lines.Tumor growth and size were significantly decreased in the Pontin-knockdown group compared with the corresponding control group,while the Pontin-overexpressed group showed the opposite effects.HE staining and IHC were performed in the xenografts,and the results showed that the size of the transplanted tumor in Pontin-knockdown group was smaller than that in the control group.The expressions of Pontin and Ki-67 were also lower in Pontin-knockdown group than those of the control group,while the Pontin-overexpressed group showed the opposite effects.5.Transcriptome sequencing analysis showed that Pontin knockdown could significantly inhibit the expressions of the TGF?/SMAD downstream genes and TGF?R2 gene.Western blot verified that the expression of TGF?R2,p-SMAD2/3 and CTGF in this pathway were all decreased and increased when Pontin was knocked down and overexpressed,respectively.TGF?1 and LY2109761 are known agonist and inhibitor of the TGF?/SMAD pathway,respectively.Stimulation of TGF?1 in the Pontin-knockdown group failed to up-regulate the downstream gene expression of this pathway,while LY2109761 inhibited the downstream gene expression of each group.The effects of agonist and inhibitor on the proliferation and migration of glioma cells were also consistent with the changes in the downstream gene expression of the TGF?/SMAD pathway.6.Through combined screening of Pontin binding proteins and possible transcription factor binding sites in TGF?R2 promoter,we speculated that LEF1 may be an important cooperator of Pontin to promote TGF?R2 gene transcription.TGF?R2 luciferase reporter assay showed that Pontin overexpression could increase the transcriptional activity of TGF?R2,and co-overexpression of Pontin and LEF1 could effectively amplify the transcriptional activation effect,while Pontin knockdown could reduce the transcriptional activity of TGF?R2.Co-IP results showed that LEF1 appeared in the immunoprecipitation complex of Pontin,suggesting that Pontin may bind to LEF1 in vivo.The results of IF indicated that endogenous Pontin and LEF1 co-localized in the nucleus.7.TGF?R2 was overexpressed in the Pontin-knockdown sub-cell line,thenWestern blot confirmed that the expressions of p-SMAD2/3 and CTGF,two important downstream proteins in the TGF?/SMAD pathway were rescued to the original levels.The results of EdU,colony formation and transwell migration assays revealed that TGF?R2 restoration could effectively rescued the impaired proliferation,survival and migration phenotypes caused by Pontin knockdown.Conclusions:1.Pontin is overexpressed in gliomas,and its expression increases with the elevation of glioma grade,and is positively correlated with Ki-67 expression.Therefore,its positive labeling index can be used as an auxiliary biomarker to assess the malignant degree of gliomas.Besides,Pontin expression is closely related to the survival of glioma patients and can be used as an independent prognostic factor.2.The possible mechanism by which Pontin activates the TGF?/SMAD pathway might be,Pontin as a transcription co-activator,interacts with the transcription factor LEF1,and they collectively bind to the promoter of TGF?R2 gene,then promote the transcription of TGF?R2 gene,thereby activating this pathway.3.TGF?R2 is an important target of Pontin to regulate the proliferation,survival,migration and invasion in glioma.Pontin can activate the TGF?/SMAD signaling pathway and up-regulate the expressions of the key effector protein CTGF,thus enabling glioma cells to achieve higher abilities in proliferation,survival,migration and invasion.4.The abnormal increase of Pontin expression in glioma is an important event contributing to gliomagenesis,and also an important molecular mechanism for glioma cells to possess unlimited proliferation,migration and invasion abilities.Targeted silencing of Pontin expression can inhibit the proliferation,survival,migration and invasion of glioma cells and exert tumor suppressive effects,which is a promising strategy for effective glioma intervention.