Expression Of ZBTB20 In The Brain Of Aged Mice And Its Effect On Proliferation,Differentiation And Migration Of N2a Cell

The zinc finger protein family is a member of the Transcription factors(TF)family in eukaryotes,which contains multiple subfamilies.ZBTB(Zinc finger and BTB domain-containing protein)belongs to the POK(POZ and Kriippel)family of the zinc finger protein family.The researchers first confirmed the presence of ZBTB20 in dendritic cells,which belongs to a new member of the ZBTB family,also known as DPZF,HOF,or ZNF288.Previous studies have proved that ZBTB20 plays an important role in many physiological processes,such as growth and development,immune regulation,blood sugar regulation,and pain modulation,etc.ZBTB20 gene is regarded as a tumor associated gene while its role in tumorigenesis is paradoxical.In non-small cell lung cancer and liver cancer,the expression of ZBTB20 is significantly up-regulated,and the expression of ZBTB20 is significantly negatively correlated with the prognosis of patients,indicating that ZBTB20 has important pathophysiological functions.While it is also a PTEN collaborating factor working as a tumor inhibitor.In neuroglioma,ZBTB20 knockdown decreased cell proliferation indicating its role in tumor promotor.However,ZBTB20 knockin significantly increased the expression of glial fibrillary acidic protein(GFAP),a differentiated glial marker,suggesting that ZBTB20 promote the neural cells differentiation.Taken together,the role of ZBTB20 gene in neural tumor is unclear.This research studies the possible role of ZBTB20 based on animal model and cell experiment.The incidence and type of brain tumor is associated with patient age,for example that the glioblastoma is more likely to happen in older persons,so we analyzed the expression of ZBTB20 gene in old mice(11 months).We also detected the location of ZBTB20 expression in brain.And we overexpressed ZBTB20 gene in N2a cells through lentivirus infection,to study the effect of ZBTB20 on cell proliferation,differentiation and migration ability.Method1.Preparation of experimental animals:C57BL/6 mice(11 months old)were selected.2.Immunohistochemical detection of ZBTB20,GFAP,NSE(neuron-specific enolase),MAP2(microtubule associated protein 2)expression in mouse brain slices:Adult mice were taken and whole brains were frozen for sectioning Frozen sections of 11-month-old male mice were subjected to immunofluorescence to observe the expression of ZBTB20 and its co-localization with GFAP and MAP2,NSE,GFAP.3.Lentivirus-mediated expression and identification of ZBTB20 in N2a cells:Lentiviral packaging was completed in HEK293T,virus supernatant particles were collected 48 hours later,filtered and stored.N2a cells were divided into ZBTB20-N2a group and Ctr-N2a group.They were infected with ZBTB20 overexpression lentivirus and control lentivirus in logarithmic growth phase.After 2 days of infection,they were screened with 2 μg/ml puromycin for 7 days.The green fluorescent protein was observed under a microscope(Enhanced green fluorescence protein,EGFP)expression,Western blot(WB)was used to detect the expression of ZBTB20.4.Cell proliferation and differentiation detection:CCK8 method and Plate cloning experiments were used to detect the proliferation of N2a cells after ZBTB20 knock-in,and WB method was used to detect the expression of GFAP and microtubule associated protein tau.5.Detection of cell migration ability:Using scratch experiment to explore the effect of ZBTB20 on the migration ability of N2A cells.Results1.Immunofluorescence results showed that ZBTB20 was expressed in the hippocampus and cortex od 11 months old mice.ZBTB20 is co-located with MAP2,NSE and GFAP,both in nuclei and out-nuclei.2.After lentivirus infection of N2a cells,after 7 days of puromycin screening,EGFP positive cells were above 90%,and the expression of ZBTB20 in the ZBTB20-N2a group was significantly increased.3.ZBTB20 knock-in significantly increased the expression of GFAP protein and tau protein in N2a cells,but there was no significant difference in cell morphology between the ZBTB20-N2a group and the Ctr-N2a group.3.The results of CCK8 method and plate cloning experiment showed that there was no significant difference in cell proliferation ability between ZBTB20-N2a group and Ctr-N2a group.4.The scratch test showed that there was no significant difference in cell migration ability between the ZBTB20-N2a group and the Ctr-N2a group.Conclusion1.ZBTB20 is expressed in mouse cortex and hippocampus,and is expressed in neurons and astrocytes.2.ZBTB20 knockin promotes GFAP and Tau’s expression in N2a cells,but it has no effects on the morphology of N2a cell.3.ZBTB20 knockin has no obvious effect on the proliferation and migration ability of N2A cells.