Study On The Expression Of Hevin In Glioma And Its Effect On Neuron-glioma Synapse

Objective:Glioma-related epilepsy is one of the most common clinical complications in patients with glioma.Seizures are the first symptom of about 30%-50%of patients with brain tumors,es-pecially gliomas.According to cortical EEG,about 75%of glioma cases have long-term ab-normal epileptiform discharges in the peritumoral cortical area after tumor resection.This phenomenon is especially common in patients with low-grade gliomas.However,conven-tional antiepileptic drugs are not effective for this treatment,and the control of its recurrence is still not ideal.The recurrent seizures of epilepsy promote the recurrence and progression of gliomas as feedback,making patients with poor prognosis and seriously affecting their quality of life.Clinical data suggest that there is a close correlation between glioma-related epilepsy and glioma biological progression which may have a common pathological mechanism.Therefore,exploring the mechanism of glioma-related epilepsy may have the dual significance of controlling epileptic seizures and tumor biological progression.The latest research shows that neurons and glioma cells in glioma can form a special‘cellular network’structure neuron-glioma synapses（NGS）.This type of structure affects both the biological progression of glio-mas and cortical excitability.This study aimed to elucidate the role of Hevin in the structural remodeling of the‘cellular network’of glioma-associated epilepsy,especially focusing on its effects on NGS.Methods:1.Through bioinformatics analysis,the difference between the low-grade glioma（LGG）and high-grade glioma（GBM）data in the TCGA（The Cancer Genome Atlas）database was analyzed,and then the differential genes were used to conduct KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway enrichment analysis and GO（Gene Ontology）functional enrichment analysis.Identified synapse-related genes and target Hevin as the research object.2.Collected surgical specimens from clinical patients,studied the expression of Hevin in mRNA and protein levels in different grades of glioma tissues（WHO Ⅱ,WHO Ⅲ and WHO Ⅳ）and normal control cortex（Control）,and verified the results with bio-information analysis data.3.Primary culture of E19 SD fetal mouse cortical neurons,and establishment of a co-culture cell system of SD fetal mouse cortical neurons（RN）and human glioma cell lines U251and U87（i.e.:RN+U251,RN+U87）.Built NGS model in vitro,and further used morphology and other methods to analyze the numbers and types of synapses.4.U251 and U87-luc2（U87 with luciferase gene）cells were transfected with recombi-nant Hevin lentiviral activation vector and Hevin lentiviral interference vector,respectively.U251 and U87-luc2 transfected with control lentiviral activation vector and control lentiviral interference vector were used as controls(U251^Lv-NC and U251^sh-NC,U87-luc2^Lv-NC and U87-luc2^sh-NC),respectively.SPARCL1-overexpressed glioma cells(U251^Lv-SPARCL1,U87MG-luc2^Lv-SPARCL1)and SPARCL1-knockdown glioma cells(U251^sh-SPARCL1,U87MG-luc2^sh-SPARCL1)were screened.Coculture systems with RN were further established one by one,namely:RN+U251^Lv-SPARCL1,RN+U251^sh-SPARCL1,RN+U87MG-luc2^Lv-SPARCL1,RN+U87MG-luc2^sh-SPARCL1.The effect of glioma cells-derived Hevin on NGS was explored by in vitro ex-periments combined with morphological methods.5.3-4w NCG（NOD/Shilt JGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt）immunodeficient mice and four types of U87 cells(U87-luc2^Lv-NC,U87MG-luc2^Lv-SPARCL1,U87-luc2^sh-NCand U87MG-luc2^sh-SPARCL1)were used to establish glioma xenograft models.Intravital fluores-cence imaging was used to assess glioma size.Combined with immunoelectron microscopy and immunofluorescence,the effect of glioma cell-derived Hevin on NGS was further ex-plored in vivo.6.Used four types of U87 cells(U87-luc2^Lv-NC,U87MG-luc2^Lv-SPARCL1,U87-luc2^sh-NCand U87MG-luc2^sh-SPARCL1)to explore the effect of tumor-derived Hevin on the proliferation and migration of glioma in vitro.Combined with the results of in Intravital fluorescence imaging,the effect of Hevin on the proliferation and migration of glioma in vivo was further explored.Results:1.Compared with LGG and GBM,there were 1490 genes up-regulated and 965 genes down-regulated.More up-regulated differential genes were related to synapses.Among them,124 genes represented by Hevin were recognized as synaptic organizers;2.Hevin had different expression changes in 26 cancer types compared with normal corresponding tissues,especially in LGG and GBM.Hevin can be used as an independent factor to influence the prognosis of glioma and played a protective role in the progression of glioma;3.In the core of gliomas,there were mainly more glial cells.However,there were more neurons in the tumor infiltration area.Correspondingly,Hevin was more expressed in GFAP-positive glial cells in the core of the tumor,while in the peripheral infiltration area of the tumor was more co-labeled with Neu N-positive neurons;4.Compared Control with different WHO grade of gliomas,it was found that the distri-bution of Hevin around the tumor in gliomas was positively correlated with excitatory synap-ses,which was similar to the expression of Hevin in gliomas.5.NGS model was successfully constructed in vitro and passed the test.In addition,Hevin from glioma can affect the number of NGS on distal tumoral microtubules（TMs）.Conclusions:Hevin derived from glioma may affect the progress of the remodeling of glioma perimeter cortex by regulating neuron-glioma cell synapses,and affect the prognosis of patients with glioma.