The Role Of Fstl1/DIP2A/FOS Signaling Pathway In Glioma Proliferation And Drug Resistance

Happening on neuroderm and originating fromneurogliocyte,giloma is primary intracranial tumor with height heterogeneity.It is characterized as high morbidity,recurrence and mortality.Uncontrolled proliferation and insensitivity to chemotherapeutic agents are the main reasons for the rapid progression of glioma,short survival time and difficult to be cured.At present,the treatment of glioma is mainly based on surgery,postoperative radiotherapy and chemotherapy.However,current treatment regimens do not significantly inhibit tumor recurrence and improve patients’ survival.Tumor proliferation and drug resistance are mainly caused by inactivation of tumor suppressor genes and abnormal activation of oncogenes.Therefore,a more comprehensive understanding of the molecular mechanism of tumor proliferation and drug resistance may provide a novel therapeutic strategy for treatment of glioma.Follistatin-like protein 1(Fstll)is a secreted glycoprotein that was originally cloned from the mouse osteoblastic cell line MC3T3-E1.The structure of Fstll is similar to that of follistatin;therefore,the protein encoded by Fstll is also named FRP(follistatin-related polypeptide).Fstll protein belongs to the SPARC(secreted protein,acidic,rich in cysteine)family,and comprises a calcium binding motif,a von Willebrand factor type C domain,Kazal type serine protease inhibitors,and follistatin-like domains.However,a study by Hambrock et al.revealed the EF-hand calcium-binding domains of Fstll are non-functional,suggesting that,despite its sequence homology to other members of the SPARC family,Fstl1 has unique features.Sundaram GM et al.reported that miR-198 derived from the 3’-untranslated region of Fstll messenger RNA,and KSRP(The KH-type splicing regulatory protein,also known as KHSRP)a trans-acting RNA-binding protein is involved in regulating the processing of exonic miR-198.Many studies have shown that Fstll plays an important role in the development of ovarian cancer,cervical cancer,lung cancer,pancreatic cancer and prostate cancer.Previous studies show that increased Fstll expression was correlated with glioma grade and overall survival in different grade gliomas.However,the function and mechanism of action of Fstll in GBM(glioblastoma multiforme)development have not yet been investigated.In the first part of this project,we employ the database of CGGA(Chinese Glioma Genome Atlas)and TCGA(The Cancer Genome Atlas)to analyze the expression of Fstll in glioma tissues and normal brain tissues respectively,gradually explore the relevance between the level of Fstll and patient’s outcome.Then we adopt shRNA(short hairpin RNA)and Fstll overexpression plasmid to downregulate and upregulate Fstll of glioma cells respectively,and estimate the degree to which Fstll exerts influence the proliferative capacity of glioma cell via CCK-8 and colony formation assays.And the apoptosis was measured by flow cytometry.Ectopic expression of Fstll in GBM cells promoted cells growth,whereas depletion of Fstll inhibited GBM cells proliferation in vitro and in patientspecific orthotopic GBM xenograft models.C-fos which has oncogenic activity is frequently overexpressed in many cancers,including glioma,and enhances the invasion,proliferation and drug resistance of glioma.Tanaka et al.reported that overexpression of Fstll suppressed c-fos expression.In contrast,Kim et al.reported the opposite result,where the mRNA and protein levels of c-fos were significantly increased in Fstll-transduced osteoclasts compared with control cells.In order to verify the relationship between Fstll and c-fos in glioma,we downregulated and upregulated Fstll in difference glioma cells respectively.And we revealed that the downregulation of Fstll significantly decreased the RNA and protein levels of c-fos,while forced expression of Fstll promoted c-fos transcriptional activation.The ectopic expression of Fstll in GBM cells promoted acquired resistance to temozolomide,whereas depleting Fstll sensitized GBM cells to temozolomide in vitro and in vivo.Co-immunoprecipitation assays verified the interaction between DIP2A(disco interacting protein 2 homolog A)and Fstll in GBM cells.Moreover,DIP2A overexpression decreased and DIP2A knockdown increased the expression of c-fos.Ectopic expression of DIP2A reversed Fstll overexpression-promoted c-fos transcriptional activation.And downregulating DIP2A abrogates the effects of Fstl1 on c-fos expression.DIP2A is a member of the disconnected(disco)-interacting protein 2(DIP2)family that contains DMAP,CaiC,and AMP-binding domains.Immunoprecipitation experiments revealed that DIP2A was involved in the DMAP1-HDAC2 repressive transcription complex by forming a complex with DMAP1(DNA methyltransferase 1 associated protein 1)in the nucleus.The overexpression of DIP2A enhanced the transcriptional inhibition of c-fos by recruiting the HDAC2(histone deacetylase 2)repressive transcription complex.In addition,Fstll overexpression impaired the interaction between DIP2A and the DMAP1-HDAC2 repressive transcription complex and weakened the RNA transcriptional silencing function of HDAC2 at the c-fos gene.Therefore,Fstll is involved in the regulation of glioma proliferation and drug resistance through DIP2A/HDAC2/c-fos signaling pathway.MicroRNAs(miRNAs)has been attracting increasing attention in the field of tumor research for the past decade.It receives transcription regulation of molecular upstream,and passed the signals through targeting specific mRNA 3’-untranslated region(3’-UTR),which was regarded as post-transcriptional regulation of gene expression.With the development of systematic biology,the research on mi RNA evolves as well,upgraded from single-functional to a holistic level.A large number of reports demonstrate that unique miRNA expression profile is relevant to the diagnosis,staging,progressing,apoptosis and therapeutic efficacy of tumor,functioning as a significant hub molecular in the network of tumor regulation.In the second part of this project,we employ the database of CGGA to analyze the expression of miR-198 in glioma tissues and normal brain tissues respectively,gradually explore the relevance between the level of miR-198 and patients’ outcome.To understand the potential role and mechanism of miR-198 in GBM,we adopted the bioinformatic algorithm TargetScan to identify potential target genes of miR-198.Among the candidates,we found that seed sequence of miR-198 matched 3’-UTRs of MGMT(06-methylguanine-DNA methyltransferase).Combining the microRNA target prediction by bioinformatics,we construct luciferase reporter gene plasmid.Dual luciferse reporter assay system gave the verification that MGMT is direct target of miR-198.MGMT is the most popular mechanism of temozolomide resistance in glioblastoma.The DNA repair protein O6-methylguanine-DNA methyltransferase restores the structural integrity of O6-meG bases by transferring the methyl group to a cysteine residue(Cysl45)within its own active site.Overexpression of MGMT restored miR-198-induced chemosensitivity to temozolomide.Therefore,overexpression of miR-198 enhanced chemosensitivity of GBM cells to temozolomide through targeting MGMT in vitro and in vivo.Fstll mRNA alternative splicing generates anti-proliferation miR-198 or pro-proliferation FRP and the mechanism that regulates splice shifting is incompletely understood.In the third part of this project,our study recovered that H19 upregulation impaired KSRP association with the Fstll 3’-UTR and favored KSRP binding to H19.In the absence of H19,KSRP promoted miR-198 maturation by binding with high affinity to the terminal loop of Fstll 3’-UTR in the nucleus through recruiting both Drosha and Dicer complexes.And upregulation of H19 clearly prevented KSRP from locating in the nucleus and arrested it in the cytoplasm.Altogether,H19 prohibited the maturation of miR-198 through affecting nuclear translocation of KSRP.