The Construction And Significance Of SUZ12 Overexpression And Knockout Stable Cell Line In MPNST

ObjectiveMalignant peripheral nerve sheath tumor,which originated from Schwann cells or Schwann cell precursor cells,is a kind of invasive soft tissue sarcoma with highly malignant,rapid development,easy to metastasize and recurrence,and poor prognosis.Approximately 5-10% of all soft tissue sarcomas.Most previous genetic studies have focused on the NF1,TP53,CDKN2 A and p16INK4 A genes.However,some researchers have recently sequenced MPNST and found that mutational inactivation of PRC2 core components SUZ12 and EED in MPNST is also a frequent event.SUZ12 is a gene encoding Zeste homolog 12 protein,which is an important core component of PRC2.It can ensure the function of H3K27me3 in epigenetic modification by stabilizing the PRC2 complex.In order to further explore the specific role of SUZ12 in MPNST,we used the lentiviral vector system to construct MPNST stable cell lines which overexpression and knockout SUZ12 gene separately,and initially explored the effect of SUZ12 on MPNST through cell phenotype,immunohistochemistry and other related assays,and lay the foundation for further mechanism research and in vivo experiments.MethodWe detected the abundance of SUZ12 m RNA expression in two MPNST cell lines by RT-qPCR,and determined the overexpression group and the knockout group.The full-length SUZ12 gene was synthesized by PCR,the specific sg RNA interference target sequence was designed and synthesized,and the recombinant expression plasmid was constructed,which was verified by enzyme digestion and sequencing.These recombinant plasmids was transfected into 293 T cell to package lentivirus,and the titer was determined by fluorescence method.The optimal MOI for lentiviral infection and the dose for puromycin selection were determined,and ST88-14 and STS26 T cells were transfected to construct overexpression and knockout MPNST stable cell lines.Fluorescence microscopy was used to observe the expression of green fluorescent protein in stable transfected cell lines.RT-qPCR and Western blotting verified the role of overexpression group,and sequencing,mutation kit and Western blotting verified the role of knockout group.We used CCK8 and Colony formation assay to determine whether SUZ12 affected MPNST proliferation ability,and Wound-Healing assay and Transwell assays to determinewhether SUZ12 affected MPNST invasion and migration ability.Western blotting confirmed the correlation between the expression level of SUZ12 and the expression level of H3K27me3.IHC analyzed the expression and prognosis of H3K27me3 in clinical specimens of MPNSTs.Results1.RT-qPCR results showed that the expression level of SUZ12 m RNA in STS26 T cells was 2.5 times higher then that in ST88-14 cells.Therefore,we performed SUZ12 overexpression in ST88-14 cells and performed SUZ12 knockout in STS26 T cells.The results of restriction digestion and sequencing showed that the overexpression and knockout plasmid vector was successfully constructed.MOI=100 and MOI=10 are the optimal MOI values for overexpressing lentivirus and CRISPR/Cas9 lentivirus,respectively,and the optimal dose for puromycin selection was 2 μg/m L.Fluorescent microscope was used to observe the stable expression of green fluorescent protein in the stably cell lines.RT-qPCR and Western blotting showed that SUZ12 m RNA and protein in LV-SUZ12 were significantly higher than that of control group.Sequencing of PCR products showed that there were no SNP sites in sg RNA-1 and sg RNA-2 sequences,which can be used in subsequent assays;the mutation kit test results showed that compared with the negative control group,a band of the expected size appeared in the sg RNA-1group,that is,a mutation was successfully introduced at the target site;therefore,sg RNA-1 was used for subsequent experiments.Western blotting results also showed that the protein content of SUZ12 in the sg-SUZ12 group was significantly lower than that in the control cells.2.CCK8 assay showed that cell growth after SUZ12 overexpression was significantly reduced on the fifth day compared with control cells,and Colony formation assay showed that cells with SUZ12 overexpression formed smaller and fewer colonies compared with control cells.The overexpression of SUZ12 significantly reduced the migration ability of MPNST cells in the Wound-Healing assay and the Transwell migration assay.The overexpression of SUZ12 significantly reduced the invasion ability of MPNST cells in the Transwell invasion assay,but the cell proliferation,invasion,and migration ability in the SUZ12 knockout group was no statistical difference.Western blotting results showed that the expression of H3K27me3 in the LV-SUZ12 group was significantly increased after SUZ12 overexpression,but there was no significant difference in the SUZ12 knockout group.IHC results showed that 90.5%(38/42)of the tissue samples showed acomplete loss of SUZ12 expression,9.5%(4/42)were weakly positive,and SUZ12 was under-expressed in all MPNST specimens.73.8%(31/42)of H3K27me3 showed different degrees of deletion in MPNST.More importantly,the H3K27me3 low expression group(negative + weak positive)has significantly worse overall survival time than the high expression group(medium positive + strong positive)(OS,P = 0.042),but there is no significant difference in disease-free survival(DFS,P = 0.106).Conclusion1.We successfully constructed a recombinant lentiviral vector for the overexpression and knockout SUZ12 gene,and established ST88-14 stable cell lines stably expressing SUZ12 and STS26 T stable cell lines knocking out SUZ12.2.SUZ12 overexpression in part of MPNST reduces the proliferation,invasion and migration ability of MPNST and increases the expression level of H3K27me3.3.H3K27me3 can not only be used as a diagnostic marker for MPNST,its expression status can also provide a reference for disease prognosis.