Investigation Of The Role Of MINA In Glioblastoma Cell Proliferation And Tumorigenesis And The Underlying Mechanisms

Glioblastoma is infamous as a most common and deadly brain tumor for its high proliferation ability and its aggressiveness and infiltration capacity in metastasis.Although the surgical and pharmacological therapies has got some fruits,glioblastoma remains hardly to cure and has a low survival rate.Therefore,identifying alternative therapeutic approaches is urgent and critical,which attaches more importance to the exploration of the mechanisms underlying glioblastoma initiation and progression.MINA was first discovered as a nuclear protein transcriptionally regulated by MYC in human beings.High expression of MINA was reported in many cancers and is an indicator of poor prognosis in several cancers.Although some studies speculated that MINA could act as a potential oncogene based on its involvement in cell proliferation and tumorigenesis,no molecular characterizations were described in these reports.Epigenetic modification on core histones has been shown of great importance for the regulation of chromatin dynamics and gene transcription,and usually causes tumorigenesis.Lysine demethylation on H3K9 trends to activate gene transcription.And MINA is involved in reducing the tri-methylation of histone H3K9(H3K9me3).So we aimed to investigate the role of MINA in glioblastoma and reveal the underlying mechanisms,considering the H3K9me3 demethylating activity of MINA.The main results are as follows:1.MINA is universally expressed in glioblastoma cell lines and is a prognostic indicator in patients with glioblastomaElevated MINA expression has been reported in numerous cancers,and indicates poor prognosis in several cancer cases.However,the role MINA plays in glioblastoma has not been well defined yet.First,we detected the expression level of MINA in several glioblastoma cell lines: LN-229,U-251 MG,U-87 MG,U-118 MG and A-172.And the results showed that MINA is commonly expressed in those cell lines.MINA expression detection and the pathology analysis in five primary glioblastoma specimens and their corresponding peritumoral tissues showed that primary tumor specimens processed a much higher MINA level than their corresponding peritumoral tissues where MINA was barely expressed.Database analysis further confirmed the indicative meaning of MINA in glioblastoma even glioma prognosis.Progression-free survival Kaplan-Meier analysis of two glioblastoma and two glioma databases showed that high MINA level was strongly correlated with a poor outcome,whereas low MINA level was associated with good overall survival.Moreover,MINA expression was significantly and gradiently increased along with the tumor stage advancing.These results demonstrated that MINA could be a meaningful prognostic marker and might play an oncogenic role in tumor development.2.MINA promotes glioblastoma cell proliferation and clonogenicity Next,we knocked down MINA in two glioblastoma cell lines: LN-229 and U-87 MG,and then investigated the cell viability after knocking down MINA.Compared with the control groups,sh MINA groups showed sharp declines in the growth curves and a dose-dependent influence by MINA silence.Brd U assays reached the consistent results that the Brd U-positive rates in sh MINA#2 groups were much lower than the corresponding control groups.Soft agar assays showed that the sh MINA groups formed much smaller and fewer colonies than the corresponding control groups.These results together indicated that MINA is indispensable for glioblastoma cell proliferation and clonogenicity.To further confirm the significance of MINA in glioblastoma cell proliferation and clonogenicity,we recovered MINA expression after the sh MINA#2 interference by overexpressing a full-length MINA that resistant to the #2 sh RNA targeting(MINA-R).The overexpression efficiency was examined and the results showed that MINA was successfully overexpressed after knocked down by sh MINA#2.Accordingly,we investigated the cell growth,cell proliferation and clonogenic ability of sh MINA#2/MINA-R cells using MTT,Brd U and soft agar assays respectively.Data showed that recovery of MINA after knocking it down rescued the cell growth,cell proliferation and colony formation,suggesting that MINA could reliably accelerate glioblastoma cell proliferation and clonogenicity.3.MINA regulates glioblastoma cell cycle progression To investigate how MINA accelerated glioblastoma cell proliferation,we performed microarray profiling of LN-229 cells with or without MINA knockdown.Gene Ontology(GO)analysis showed that genes down-regulated by MINA silence were significantly enriched for GO terms correlated with the cell cycle progression.In addition,pathway analysis revealed that among the down-regulated genes,those within the ‘CDK Regulation of DNA Replication' pathway were highly enriched.These results inspired us to explore the effect of MINA on the cell cycle course of glioblastoma cells.Flow cytometry analysis was conducted and showed that MINA knockdown in LN-229 and U-87 MG cells led to a G1 and G2/M arrest,together with a remarkable decline of the cell population in S phase;and recovery of MINA after knocking it down significantly facilitated the cell cycle transition.These results suggested that MINA is crucial for glioblastoma cell cycle progression.4.MINA transcriptionally activates Cyclins and CDKs To gain more insight into the molecular mechanism how MINA regulated cell cycle progression,we analyzed our microarray data and found that several Cyclins and CDKs genes which,collectively,are required for the progression from the interphase to the mitotic phase were down-regulated significantly.The validating results of q TR-PCR assays showed that deficient MINA expression led to a remarkable decrease of CCNB,CCND,CCNE,CDK1,CDK2 and CDK4 m RNA expression.Western blot analysis of several Cyclins and CDKs which are closely linked with cell cycle progression further confirmed the results.Moreover,we found that MINA knockdown resulted in a notable down-regulation of Cyclin B1,D1,E2,CDK1,CDK2 and CDK4,meanwhile,recovery of MINA reversed this inhibition phenotype and dramatically up-regulated the above genes expression.Cyclin D/CDK4 and Cyclin E/CDK2 complexes are required for the cell cycle progression from G1 to S phase,and Cyclin B/CDK1 complex is essential for cells to transit through the G2/M checkpoint.Our findings suggested that MINA controls cell cycle progression probably through transcriptionally regulating the gene expression of these complexes.To verify this hypothesis,we performed chromatin immunoprecipitation(Ch IP)assays to examine whether MINA could bind to those genepromoters.As predicted,significant enrichment of MINA was found at the promoters of CCND1,CDK4,CCNE2,CDK2 and CDK1.Furthermore,to provide more relevant data,dual-luciferase reporter assays were applied and the results showed that CDK2 and CDK4 promoter activity were significantly reduced after knockdown of MINA.Together,our results provide evidence that MINA modulates glioblastoma cell cycle by directly and transcriptionally activating the Cyclins and CDKs.5.MINA accelerates cell cycle progression through H3K9 demethylation There are increasing evidence that epigenetic modifications on histones play important roles in gene transcriptional activation or silence.And it has been reported that MINA has a demethylase activity on H3K9me3 whose demethylation usually activates genes expression.Hence,we designed experiments to determine whether the regulation of glioblastoma cell cycle progression by MINA is related with the H3K9 methylation alteration.First,we examined the H3K9 methylation status after down-regulation of MINA in LN-229 cells by western blot analysis.The results showed that MINA knockdown led to a marked increase in H3K9me3 and a notable decrease in H3K9me1,with the level of H3K9me2 not varied apparently.The Jmj C domain in MINA is essential for MINA demethylating activity,therefore to further confirm our results,we constructed a MINA-mutant plasmid with the N-terminal(139–271aa)of wild type(WT)MINA deleted,which thus lost the Jmj C domain together with the H3K9me3-demethylating activity.This MINA mutant is conjunct with a Flag tag and is also resistant to the sh MINA#2 interference(MINA?Jmj C-R).We then analyzed the alteration of the H3K9 methylation after knocking down MINA and subsequently and respectively overexpressing MINA-R or MINA?Jmj C-R.We found that in the course of time,with the decrease in MINA caused by sh MINA#2 interference,the level of H3K9me3 exhibited a significant increase,meanwhile the level of H3K9me1 exhibited a gradual decrease.And after the recovery of the full-length MINA by overexpression of MINA-R,the level of H3K9me3 got an observable decline,whereas the H3K9me1 level restored as time went on.These results suggested that MINA knockdown blocked the transition from tri to mono methyl on H3K9,and the block was reversed by the recovery of functional MINA.Notably,overexpression of the mutant MINA with MINA?Jmj C-R after sh MINA#2 interference hardly had any effect on the H3K9 methylation status influenced by MINA knockdown: neither H3K9me3 nor H3K9me1 changed distinctly with the increase of mutant MINA.In addition,immunofluorescence staining of H3K9me1,me2 and me3 reached the consistent results,indicating that MINA functioned to demethylate H3K9 from tri to mono methyl and the deletion of its Jmj C domain disabled this ability.Accordingly,using the above lose-regain/defect methods,we studied the relationship between the demethylation function of MINA and the cell cycle progression of glioblastoma cells.By detecting three most effective cell cycle related proteins in response to MINA function,we found that the expression of Cyclin E2,CDK2 and CDK4 gradually decreased in response to the loss of MINA and restored along with the regain of functional MINA,in agreement with the block of H3K9me3 demethylation and the recovery of H3K9me3 demethylation,respectively.Overexpression of the Jmj C-deleted MINA which lost the demethylating activity in MINA-silenced LN-229 cells could not rescue the expression of Cyclin E2,CDK2 nor CDK4,indicating that deficiency or defects of MINA disabled cells to demethylate H3K9me3 and thus inhibited genes expression.Together,these findings suggested that MINA activates Cyclins and CDKs transcription through H3K9me3 demethylation.6.MINA promotes glioblastoma tumorigenesis in vivo We further evaluated the role of MINA in tumorigenesis of glioblastoma cells.Subcutaneous xenograft experiments on NOD/SCID mice were carried out and the results demonstrated that MINA knockdown in LN-229 cells resulted in much smaller values in both tumor volumes and weights than the control group;and after functional MINA expressed in the MINA-knockdown cells,the tumor formation was significantly rescued;while overexpression of mutant MINA with Jmj C domain deleted in MINA-knockdown cells could not reverse the tumorigenic inhibition,during the same time course.Further western blot analysis of the xenograft tumors exhibited a similar expression pattern to that of the glioblastoma cell lines: Cyclin B1,D1,E2,CDK1,CDK2,CDK4 and H3K9me1 expression was remarkably down-regulated in MINA-knockdown tumor,while by contrast,H3K9me3 was obviously up-regulated;and these were reversed significantly by overexpression of MINA-R but not MINA?Jmj C-R.H&E staining assay and IHC assay detecting MINA and Ki67 expression were also performed and further supported the western blot results.And the Ki67 expression exhibited a synchronous variation with MINA expression,suggesting that MINA is essential for glioblastoma.In summary,our study has expounded that MINA is a prognostic indicator in patients with glioblastoma,and is crucial in glioblastoma cell proliferation and tumorigenesis by controlling the cell cycle progression through transcriptionally regulating Cyclins and CDKs expression via the demethylation of H3K9me3.These findings,for the first time,reveal the role of MINA in glioblastoma initiation and progression,and provide insights into the molecular mechanism underlying MINA-controlled cell proliferation.As MINA is frequently overexpressed in different cancers and is positively correlated with Ki67,the molecular pathway described here could be of significance in other tumors,and MINA,as a novel and critical cell cycle regulator,could work as a molecular target for anticancer therapy.Moreover,the discovery that the control of cell cycle by MINA is closely linked with the demethylation on H3K9me3 provide meaningful support for the connection between the demethylation on H3K9me3 and cell cycle regulation.