The Role And Mechanism Studies Of Proteolipid Protein 2(PLP2) In The Progression Of Glioblastoma

BackgroundGliomas constitute approximately 75%of the malignant primary brain tumors in adults.The life expectancy of patients with glioblastoma(GBM,WHO grade IV)is on average 14 months after diagnosis.Currently,the main therapies for glioma include surgical resection,oral alkylating agents and radiation.Despite great advances in therapeutic interventions against glioma which are the results of many scientists and clinicians' efforts,current treatment strategies have shown only limited survival benefits.Consequently,a deeper understanding of the mechanisms underlying glioma growth and progression is important in order to develop new therapeutic strategies.The endoplasmic reticulum(ER)is characterized by a special structure whose shape and architecture controls the biological functions which occurs within this organelle.It is composed of a relative complex system of membranes that gives rise to the nuclear envelope and peripheral ER,which is composed of sheets and tubules.In fact,the ER structure is dynamic and can adapt to meet cellular demands in response to physiological or pathological stimuli.During cellular stress,as misfolded proteins are accumulated in the ER,ER dysfunction leads to the activation of unfolded protein response(UPR),and this is referred as a condition of ER stress.Several endogenous and exogenous factors may influence the alteration of cellular conditions,inducing variations in ER homeostasis and its appropriate functioning.A cascade of signaling events to re-establish ER homeostasis can be caused by this.Among these events,activation of transcription factor 6(ATF6),protein kinase RNA-like ER kinase(PERK),and inositol-requiring enzyme 1(IRE1)are the three main transmembrane proteins initiating UPR signaling in ER stress response.They are maintained in an inactive state in normal conditions and they are activated through dissociation from GRP78(binding protein,BIP),the master regulator of UPR when ER stress is triggered.It has been reported that enhancing ER stress could exert cytotoxicity in GBM cells,which suggests that endoplasmic reticulum stress may play an important role in glioma.The PLP2 gene,also known as A4/A4LSB,was first discovered in colon epithelial cells.The resulting protein PLP2 represents an integral membrane protein that localizes to the endoplasmic reticulum(ER).Recently,PLP2 has also been reported to be involved in cell growth,proliferation and migration in several cancers such as melanoma,breast cancer and osteogenic sarcoma.The exact function of PLP2 under normal conditions is not clear.It has been shown that PLP2-knockout mice display increased ER stress in neurons under hypoxia,that leads to apoptotic cell death.Also,PLP2 seems to be important during normal gastrulation,but currently its exact function especially the mechanisms in glioma is unclear.Based on the study summary of the above research basis,this study analyzed the bioinformatics of public databases and collected specimens from clinical glioma surgery patients for immunohistochemical(IHC)staining,meanwhile,related molecular biological experimental methods such as RNA interference,transmission electron microscopy,EdU staining,CCK-8 cell viability assay,flowcytometry,Western blot,lentiviral transfection,and orthotopic intracranial models in nude mice were used to investigate the role and possible underlying mechanisms of PLP2 in gliomas.The study found that compared with low-grade gliomas,the expression of PLP2 in high-grade gliomas was significantly increased,and the high expression of PLP2 was related to the poor prognosis of the disease.Mechanistically,knockdown of PLP2 in glioma cell lines can activate endoplasmic reticulum stress,which in turn leads to increased apoptosis and autophagy.In PLP2-knockdown U87 and U251 glioma cell lines,the application of the autophagy inhibitor chloroquine(CQ)would further increase apoptotic cell death.At the same time,orthotopic U87-shPLP2 and U251-shPLP2 intracranial xenograft models in vivo revealed that PLP2 inhibition reduced glioma growth.Therefore,our results indicate that the high expression level of PLP2 plays an important role in the progression of glioblastoma,and the combination of PLP2 molecular targeted therapy and autophagy inhibitors can be one of the research directions for future glioma treatment strategies.PART ? The expression of PLP2 in gliomas and its relationship with tumor proliferation and prognosisObjectives1.To identify the expression of PLP2 in gliomas.2.To investigate the relationship between PLP2 expression and tumor proliferation and prognosis.Methods1.Detect the expression of mRNA levels of PLP2 in the three major public databases Rembrandt,The Cancer Genome Atlas(TCGA),and The Chinese Glioma Genome Atlas(CGGA)and identify the relationship between high PLP2 expression and survival prognosis by Kaplan-Meier analysis.2.Immunohistochemical staining was used to detect the expression level of PLP2 in sample sections from clinical glioma patients with different pathological grades.3.Western blot was used to identify the expression abundance of PLP2 in human normal astrocyte cell line NHA and glioblastoma cell lines U87,A172,U251,and T98.4.Small RNA interference(siRNA)was used to knock down PLP2 and the knockout efficiency was confirmed by Western blot.5.Cell counting kit-8(CCK-8)and EdU staining were used to detect the effects of PLP2 knockdown on the proliferation of glioblastoma cell lines.Results1.High expression of PLP2 is associated with high tumor grade of glioma and poor prognosis of glioma patientsBy analyzing publicly available databases from Rembrandt,Cancer Genome Atlas(TCGA),and The Chinese Glioma Genome Atlas(CGGA),the expression levels of PLP2 in human glioma specimens in three databases were examined.Compared with normal brain tissue,the expression levels of PLP2 in gliomas,especially malignant glioblastoma,were significantly up-regulated(P<0.001).PLP2 immunohistochemical staining was performed on 72 paraffin-embedded glioma tissue samples(16 with WHO grade ?,21 with grade ?,and 35 with grade ?)and 5 normal brain tissue samples collected from our department.Quantitative analysis of average staining scores showed that PLP2 levels increased with increasing WHO grades of gliomas.In addition,Western blot analysis of normal human astrocyte cell lines NHA and human glioblastoma cell lines U87,A172,U251,and T98 showed that all glioma cell lines showed higher PLP2 protein expression levels compared to NHA.According to Kaplan-Meier survival analysis results from three public databases,patients with high PLP2 expression have a shorter overall survival compared with groups with low PLP2 expression,and the differences are statistically significant(Rembrandt P<0.0001,TCGA P<0.05,CGGA P<0.0001).2.Down-regulation of PLP2 expression inhibits cell proliferation of U87 and U251 glioma cell linesSmall interfering RNA(siPLP2)was used to inhibit the expression of PLP2 in U87 and U251 glioma cell lines.Western blot results revealed that significant knockdown of PLP2 expression level was obtained.Furthermore,CCK-8 results showed that treatment of U87 and U251 glioma cell lines with siPLP2,significantly inhibited cell viability in both cell lines after 24 h,48 h,and 72 h respectively.EdU fluorescence staining analysis further confirmed that the percentage of cells with proliferative activity which were stained with red color were significantly reduced after treatment of U87 and U251 with siPLP2 for 48 h(P<0.001).ConclusionsThe expression level of PLP2 in gliomas is correlated with tumor grade,and glioma patients with high expression levels of PLP2 have poor prognosis.In biological experiments,down-regulation of PLP2 by small interfering RNA siPLP2 inhibits the proliferation of glioma cells.PART ? Knockdown of PLP2 activates endoplasmic reticulum stress(ER stress)response and inhibits glioblastoma growth in vivoObjectives1.To explore the regulation and mechanism of PLP2 knockdown on ER stress response in glioma cells.2.To determine the effect of PLP2 knockdown on the growth of glioblastoma in vivo.Methods1.Transmission electron microscope(TEM)was used to observe the changes of endoplasmic reticulum structures in glioma cells after knockdown treating by two small interfering RNA sequences.2.The expression of ER stress-associated pathway proteins after PLP2 knockdown were detected by Western blot.3.Construction of PLP2 stable knockout cell lines U87-shPLP2 and U251-shPLP2 were carried out by lentiviral infection.4.Intracranial orthotopic tumor model in nude mice was used to detect the effect of PLP2 knockdown on the growth of glioblastoma in animal experiments.5.HE staining and immunohistochemical staining were used to detect the expression of related proteins in tumor samples from mice.6.Kaplan-Meier analysis was used to obtain the prognosis survival curve of different groups of mice.Results1.Down-regulation of PLP2 activates ER stress response in glioma cellsTEM images showed that the cells of the siPLP2 knockdown treatment group had significantly changed endoplasmic reticulum structures,the lumen of the ER was markedly dilated and even fragmented compared with the cells of the siNC treatment group after 48 hours of siRNA treatment in U87 and U251 cell lines.Western blot results showed that,compared with siNC-treated cells,the representative proteins of the ER stress in the siPLP2 knockdown-treated U87 and U251 cells,including CCAAT enhancer binding protein homologous protein(CHOP)and glucose regulatory protein 78(GRP78)were significantly increased,while related proteins in the signaling pathway such as phosphorylated protein kinase RNA-like ER kinase(p-PERK),phosphorylated inositol requiring enzyme 1(p-IRE1?),phosphorylated eukaryotic initiation factor 2(p-eIF2?)were upregulated as well.These results indicate that PLP2 knockdown activates the endoplasmic reticulum stress response in glioma cells.2.Down-regulation of PLP2 inhibits glioblastoma growth in vivoLentiviral shRNAs were designed for stable PLP2 knockdown in glioma cell lines,and satisfactory knockout efficiency was verified by Western blot experiments.Orthotopic tumor models were established by implanting U87-shNC cells,U87-shPLP2 cells,U251-shNC cells and U251-shPLP2 cells intracranially into nude mice.The HE staining results showed that for both cell lines U87 and U251,the average tumor volume after PLP2 knockout was smaller than that of the shNC control group.Kaplan-Meier survival analysis showed that the overall survival of shPLP2 groups were significantly prolonged(P<0.01,P<0.001).IHC staining for PLP2,and markers for proliferation(Ki-67),and apoptosis(cleaved-caspase 3)was performed on sections from the U87-shNC and U87-shPLP2 xenografts.Decreased levels of PLP2 and Ki-67 expression and increased level of cleaved-caspase3 were observed in xenografts from U87-shPLP2 mice,compared to shNC controls.ConclusionsPLP2 knockdown activates the ER stress response in glioma cells,and inhibits the growth of glioblastoma in vivo.PART ? Regulation of down-regulation of PLP2 expression on ER stress-induced apoptosis and autophagy in glioblastomaObjectives1.To determine whether PLP2 knockdown mediates ER stress-induced apoptosis.2.To determine whether PLP2 knockdown mediates ER stress-induced autophagy.3.To investigate the crosstalk between apoptosis and autophagy induced by PLP2 knockdown mediated ER stress.Methods1.Two small interfering RNA with different sequences siPLP2-1 and siPLP2-2 were applied to knockdown PLP2 in U87 and U251 cell lines.2.Flow cytometry after Annexin V/PI staining under different treatment conditions was carried out to detect the level of apoptosis occurrence.3.Transmission electron microscopy(TEM)was used to observe the number of autophagosomes with typical bilayer membrane structure in glioblastoma cell lines after PLP2 knockdown.4.Western blot experiments were used to detect the expression of some proteins related to autophagy and apoptosis under different treatment conditions.5.The autophagy inhibitor Chloroquine(CQ)was used to pretreat the glioblastoma cell lines before PLP2 knockdown,and the effect of autophagy inhibition on the occurrence of apoptosis was examined afterwards.Results1.Down-regulation of PLP2 increases endoplasmic reticulum stress-mediated apoptosis via CHOP inductionSiPLP2 down-regulation in both cell lines induced apoptosis in both early(Annexin V+/PI-)and late(Annexin V+/PI+)stages.This was further confirmed by western blot analyses of apoptosis related markers.The levels of cleaved-caspase 9,cleaved-caspase 3 and cleaved-PARP were up-regulated after treatment with siPLP2 for 48 h in both cell lines.SiRNAs were used to suppress the expression of CHOP.U87-shNC and U87-shPLP2 cells that were infected by the negative control shRNA and PLP2-knockdown shRNA,were transfected with siNC or siCHOP for 48 h.A reduction was observed in shPLP2-induced cleaved-caspase 3 and cleaved-PARP expression in cells treated with siCHOP,which indicates that down-regulation of PLP2 expression promotes ER stress induced apoptosis via CHOP in glioma cells.2.Down-regulation of PLP2 increases endoplasmic reticulum stress-mediated autophagy via CHOP inductionTEM is commonly used to identify the formation of autophagosomes,which is characterized by their double-membrane structures.The TEM analysis clearly demonstrated increased production of autophagosomes after siPLP2 treatment,compared to siNC treatment.The autophagosome numbers in each cell was then quantified,and a significant increase was observed for both cell lines.U87 and U251 cells treated with siPLP2 were also assessed for classical markers associated with autophagy.PLP2 knockdown led to increased levels of ATG5 and LC3B-?/LC3B-?ratio and a decreased level of P62 expression.This indicates an enhanced autophagic flux in the siPLP2 treated cells.Western blot analysis of U87-shNC and U87-shPLP2 cells treated with siNC or siCHOP was conducted afterwards,and the increased level of LC3B-?/LC3B-?,induced by shPLP2,was reduced in the absence of CHOP whereas the decreased level of p62,induced by shPLP2,was augmented in the absence of CHOP,which indicates that down-regulation of PLP2 also induces autophagy via ER stress-induced CHOP in glioma cells.3.Autophagy inhibition by chloroquine(CQ)augments apoptotic cell death induced by PLP2 knockdownCells were pretreated with or without 10 ?M CQ for 1 h prior to treatment with siPLP2 for 48 h.Treatment with CQ increased apoptosis induced by PLP2 knockdown in U87 and U251 cells as detected by flow cytometry.Statistical analysis revealed that the percentages of apoptotic cells were significantly increased in the siPLP2+CQ group,compared to the siPLP2 group.Consistent with these data,the protein levels of cleaved-PARP were also increased in siPLP2+CQ-treated cells,compared to siPLP2-treated cells.Taken together,these results show that autophagy inhibition augments apoptotic cell death induced by PLP2 knockdown.ConclusionsPLP2 knockdown regulates ER stress-mediated apoptosis and autophagy in glioblastoma cell lines.Pretreatment with autophagy inhibitors CQ can increase the apoptotic effect of PLP2 knockdown,combined treatment with autophagy inhibitors may enhance the inhibitory effect of PLP2 knockdown on glioblastoma.