CPEB4is Highly Expressed, And Regulates Cancer Progression In Human Glioma By Promoting Cell Proliferation And Migration

Gliomas are the most common malignant tumors of the central nervoussystem, account for about35.26%-60.96%(on an average44.69%) ofintracranial tumors in adults. Due to its invasive growth, there was often noobvious boundaries between the tumor and the normal brain tissue, and it wasdifficult to completely remove the tumor.Despite variety of the efforts wereperformed, this tumor remained incurable. This required us to find a newstrategy of gliomas treatment. In recent years，with the development ofgenetic engineering，the pathogenesis of glioma was explored from theperspective of molecular biology, the gene therapy for glioma graduallyattracted neurosurgery scholars’attention. CPEB4was recently discovered amember of the cytoplasmic polyadenylation element binding protein family(CPEBs), a sequence-specific RNA binding protein, CPEBs containing anRNA-recognition motif (RRM) and a zinc-finger， located on humanchromosome5q21region, located in the cytoplasmic, the length was7769bp.CPEBs was first discovered during oocyte maturation nearly two decades ago.The CPEBs play essential regulatory roles in the translation of definedmRNAs. Much of the works on CPEBs controlling of cytoplasmicpolyadenylation and translation were did in the early stage, it was found thatCPEBs mediated many processes including germ-cell development, celldivision and cellular senescence, and synaptic plasticity and learning andmemory. More recent work, however, has revealed that these processes aremore complex than previously thought; an explosion of studies showing thatCPEBs are not only expressed in a variety of somatic tissues, but haveessential functions controlling gene expression in the adult organism. These“new” functions of the CPEBs include regulating the balance betweensenescence and proliferation and its pathological manifestation, tumor development. CPEBs are widely expressed in a variety of tissues and tumors,with partially overlapping patterns. A latest study demonstrated that CPEB4was found to be upregulated in pancreatic ductal cancer and glioblastoma. Inthis case, CPEB4over-expression, correlates with increased malignancy,tumor growth and vascularization. However, the expression of CPEB4ingliomas have not been described in detail.Due to the above background, We carried out experiments includingimmunohistochemistry, Western blot and Real-time PCR to examin theprotein and mRNA levels of CPEB4in normal brain tissues and differentgrades of gliomas. Furthermore, RNA interference technology was performedto knockdown the expression of CPEB4in glioma cell line for exploringCPEB4’s role in the glioma cells proliferation, apoptosis and invasion.This study contains the following three parts:Part one: Expression of CPEB4in human glioma tissues and its clinicalsignificance.Objective: To explore the expression of CPEB4in normal brain tissueand glioma tissues of different grades.Methods:1Sample collection: Sixty-three clinical glioma samples and nine normalbrain tissues were collected randomly from the patients immediately aftersurgical resection at the Second Hospital of Hebei Medical UniversityNeurosurgery department from2011October to2012November. Thetumor specimens were all diagnosed by the neural pathologist, normalbrain tissue samples were obtained from the patients with cerebralhemorrhage or trauma who urgently needed the decompression surgery.Specimens were collected by two parts in each patient, one part was fixedin10%formalin, another part was stored in2ml sterile vials andimmediately frozen in liquid nitrogen.2Immunohistochemistry was performed to detect the expression of CPEB4protein in nine cases of normal brain tissue and63cases of glioma tissue,and the expression difference was compared, correlation with pathological grade was analyzed.3Real-time qPCR was performed to examin CPEB4mRNA expressionlevels.4Western blot was conducted to measure CPEB4protein expression innormal brain tissue and glioma tissues.Results:1Immunohistochemical results: the total positive rate of CPEB4in gliomatissues was88.89%(56/63), CPEB4expression was negative or weak innine cases of normal brain tissue, CPEB4positive expression rate inglioma tissues was significantly higher than that in normal brain tissues,with significant statistical significance（P＜0.01）；Positive rate forlow-grade (I, II grade) and high-grade (III, IV grade) glioma tissues was75%(15/20) and95.4%(41/43) respectively, the difference wasstatistically significant (P <0.05).2Western blot results: CPEB4protein expression in glioma tissues wassignificantly higher than that in normal brain tissues. The relativeexpression of CPEB4protein in normal brain tissue and I, II, III, IV gradegliomas were respectively0.070±0.012,0.417±0.044,0.495±0.043,0.709±0.080,0.713±0.027. CPEB4protein expression levels in the low-grade (I, II grade) gliomas lower than the high-grade (III, IV grade)(P<0.05).3Real-time qPCR results: CPEB4mRNA relative expression was notstatistically significant in normal brain tissue, low-grade and high-grade(P>0.05).Conclusions:1Immunohistochemistry and Western blot analysis both concluded CPEB4protein was highly expressed in human glioma tissue, and the expressionlevels increased with glioma grade, CPEB4protein expression level in IIIand IV grade gliomas higher than I and II grade gliomas, with significantstatistical significance; However, there was no significant difference inthe CPEB4expression at mRNA level. For this inconsistency between protein and RNA expression level, we analyzed this may related with thepoor stability of the RNA.2The high expression of CPEB4was significantly positively correlated withlevel of gliomas, it can be used as an important indicator of clinicalgrading of gliomas.Part two: The expression of CPEB4in human glioma cell lines U87andU251.Objective: To detect the expression of CPEB4in human glioma cell linesU87and U251,and select the one which CPEB4expressed higher for furtherbiological function tests.Methods:1Western blot was performed to examin CPEB4protein expression inU87and U251cells.2Real-time qPCR was conducted to examin CPEB4mRNA expressionlevels in U87and U251cells.Results:Both protein and mRNA expression of CPEB4in human gliomacell line U87were higher than that in U251(P <0.05).Conclusions: U87cell line can be selected as CPEB4advantageexpressing cell strain, and further study of the biological function tests targetsCPEB4will be done.Part three: CPEB4lentiviral vector infected human glioma cell line U87and its effect on cell proliferation, apoptosis and invasion.Objective: To construct the interference lentiviral expression vectortargeting CPEB4, and infect cell line U87with constructed CPEB4interference lentiviral vector, to investigate inhibition of RNA interference onCPEB4expression in U87cell and its impact on the U87cell proliferation,apoptosis and invasion.Methods:1The interference lentiviral expression vector targeting CPEB4wasconstructed: Accorded strictly with CPEB4gene sequence information andthe RNA interference sequence design principles, designed three RNA interference target sequences, then synthesized SiRNA and transfectedwith three kinds siRNA into U87cells, screened the best interfere effectsiRNA by western blot, s1ynthesized double-stranded DNA Oligo, withcohesive ends including BamHI and the ECORI. Double-stranded productobtained after annealing can be directly connected to the BamHI andECORI linearized pSRL-SIH1-H1-GFP vector without cutting. Connectedthe product into competent cells of bacteria, then extracted plasmid fromthe bacterial culture and enzyme digested, sequenced for comparing.Identification of positive clones indicated that a successful CPEB4RNAinterference lentiviral vectors constructed.2CPEB4SiRNA was used to infect glioma cell line U87with RNAinterference in vitro, divided into transfected group, negative control groupand blank control group;3CPEB4protein and mRNA expression changes were detected on first day,second day, third day, fourth day, fifth day after CPEB4-siRNA lentivirusinfected U87cells by Western blot and Real time qPCR.4Cell cycle and apoptosis level changes were detected in U87cells infectedwith CPEB4SiRNA lentivirus by flow cytometry (FCM).5Cell proliferation was observed on first day, second day, third day,fourth day, fifth day, sixth day and seventh day after U87cells infectedwith CPEB4-SiRNA lentiviral by MTT analysis.6Invasiveness in vitro was analyzed in U87cells transfected with lentiviralby Transwell invasion assay.Results:1We successfully constructed and packed the lentiviral expression vectortargeting CPEB4, and DNA sequencing confirmed the insertion sequencewas correct.2Western blot and Real time qPCR results: CPEB4protein and mRNArelative expression in lentiviral infection group was significantly lowerthan blank control group and negative control group, the difference wasstatistically significant (P <0.05); No significant difference between the blank control group and negative control group (P>0.05);3Cell cycle results: Relative to the negative control group and blank controlgroup cells, U87cells of lentiviral infection group were arrested in G1phase, cells in S phase reduced; Cell state and cell cycle of U87cells innegative control group and blank control group unchanged; apoptosislevels in three groups analyzed by flow cytometry: Cells relative to theblank control group (0.13±0.08)%and negative control group (0.47±0.12)%, apoptosis rate in U87cells of lentiviral infection group increased,up to (19.16±3.85)%, with statistical difference (P <0.01);4MTT assay result: from the third day onwards, compared with thenegative control group (0.476±0.048) and non-transfected group (0.495±0.031), cell growth rate in lentiviral infection group (0.42±0.05) began toslow down, the difference was statistically significant (P <0.05),and thedegree of inhibition was increasingly apparent with infection timeextending. There was no significant difference between thenon-transfected group and the negative control group (P>0.05). Thisdemonstrated that CPEB4siRNA had significantly inhibition on theproliferation of U87cells;5Transwell invasion assay results: After CPEB4siRNA lentiviralinfection, the number of U87cell-penetrating significantly reduced, lowerthan the negative control group and blank control group, the differencewas statistically significant. In other words, CPEB4could promoteinvasion of glioma cells in vitro.Conclusions:CPEB4targeting transfection can effectively knock down the expressionof this gene in human glioma tumor cell line U87, tumor cell proliferation andinvasion was significantly inhibited, cell cycle was arrested, and promoteapoptosis increased, provides a new target for glioma gene therapy.