Study On The Effect Of ARID1B Gene On The Signal Pathway Of Human Neuroblastoma Cells Based On Bioinformatics

Objective:To construct ARID1 B lentivirus interference vector and establish ARID1 B gene silencing human neuroblastoma SK-N-SH cell line,then study the role of ARID1 B gene in abnormal intellectual development by clarifying the potential transcriptional regulation and binding sites of ARID1 B gene’s downstream genes.Subjects and Methods:1.The virus was packaged and transfected into SK-N-SH cell line by the construction of lentivirus interference plasmid vector carrying ARID1 B sh RNA and then establish a stable SK-N-SH/ARID1B-cell line which ARID1 B gene is silenced.Firstly,the target gene plasmid YSH-LV001-sh RNA,was constructed with host cells 293 T and co-transfected with virus packaging the helper plasmids p CMV-VSVG and ps PAX2.Secondly,synthesize the recombinant lentivirus efficiently.And then detect the titer of the virus.Finally,the recombinant lentivirus was transfected into SK-N-SH cell line,and the transfection efficiency of lentivirus was detected under fluorescence microscope.2.The lentiviral vector containing sh RNA was transfected into the experimental group and the lentiviral vector without sh RNA was transfected into the negative control group.RNA and protein were extracted from the two groups and analyzed by RNA-seq as well as i TRAQ proteomics.Through the bioinformatics analysis,we can get the different results of m RNA and protein between the two groups.GO and KEGG analysis were carried out in transcriptome and proteome,and the cluster heat map was drawn.At the gene level,the differentially expressed genes were clustered according to the multiple of differential expression,and the differentially expressed genes which were closely related to neural development were selected.3.According to the results of bioinformatics analysis,the differentially expressed genes screened by bioinformatics analysis were verified by cell experiments.The differentially expressed gene in which the expression of m RNA and protein decreased after ARID1 B gene knockout was verified and screened by QRT-PCR and Western blot experiments.Results:1.The target gene plasmid vector YSH-LV001-sh RNA,was constructed by lentivirus interference vector medium,and the negative control group SK-N-SH-ctrl,was set up to package the virus and infect SK-N-SH cell line.The SK-N-SH/ARID1B-cell model with stable ARID1 B gene silencing was successfully established,and the transfection efficiency was more than 90%.2.Through the proteomic analysis of RNA-seq and i TRAQ in the experimental group as well as the control group,we screened out the differentially expressed genes closely related to nerve development: ARID1 B.3.Through the detection of QRT-PCR and Western blot,the results showed that compared with the control group,the ARID1 B gene successfully interfered with the stable transgenic strain in the experimental group,and the expression decreased by 60.1%compared with the control group.Among the other three indicators,FOS gene expression decreased most significantly,down-regulated by about 97%;STAG2 gene expression decreased by about 73.8%,and ARC gene expression decreased by about 20%.Compared with the protein level,the expression level of STAG2 protein in the experimental group decreased obviously,and the stable transgenic strain interfered with ARID1 B gene significantly interfered with the protein expression of STAG2 gene.Conclusions:In this study,the SK-N-SH/ARID1B-cell model of ARID1 B gene silencing was successfully constructed by lentivirus interference vector,which provides a solid foundation for functional detection of the cell model established in the next step.Through cell experiments for exploring the molecular mechanism of the effect of ARID1 B gene on neural development,ARID1 B gene may regulate the expression of STAG2 gene by influencing its’ transcription,and then affect the transmission of BMP pathway,and finally affect the expression of key genes in central neuron differentiation.