Preliminary Study On The Feasibility Of YAP As A Therapeutic Target For NF1-related Neurofibroma

Objective:To investigate the expression and location of YAP in neurofibromas of Neurofibromatosis Type I and assess the feasibility of using YAP as a target to treat neurofibroma.To provide new ideas for the targeted treatment of neurofibromas.Methods:1.Based on the atabase of the Department of Oral Pathology,the Ninth People`s Hospital Affiliated to Shanghai Jiaotong University School of Medicine,we selected the pathological samples of neurofibromas of Neurofibromatosis Type I that were surgically removed from January 2009to December 2019.Immunohistochemical staining was used to campare the expression level and location of YAP in normal nerve and tumor tissue.2.For further study on the corrlation between the NF1 geng and YAP,a schwann cell modal NF1kd RSC96 with NF1 gene knockdown by lentiviral was established.Real-time PCR,Western blot and immunofluorescence were used to identified the expression of NF1 geng.Meanwhile,Western blot and cell confocal were used to campare the changes of YAP expression and nuclear aggregetion between NF1 geng knockdown group and control group.3.CCK-8 assay was used to assess the effect of YAP inhibitor CA3 on the proliferation of NF1 knockdown group.4.A tumor-bearing mouse model of NF1^kdRSC96 was established to study the effect of YAP inhibitor on tumor growth in vivo.Results:1.YAP is highly expressed in the tumor tissue,and the phenomenon of intranuclear aggreation is more obvious.2.Successfully constructed NF1^kdRSC96 cell line with reduced NF1geng by lentiviral enterference vector.The m RNA expression of NF1 gene was down-regulated by 70%,and the expression of NF1 protein was down-regelated by 65%.The total protein level of RAS was increased as well as p-ERK which is a downstream protein of the RAS/MAPK pathway.Compared with the control group,the expression of YAP protein in NF1^kdRSC96 group was significantly up-regulated.The result of cell confocal showed that intranuclear aggreation of YAP protein was enhanced.Western blot results confirmed the increase of YAP protein level in nucleus.3.The YAP inhibitor CA3 and the MEK 1/2 inhibitor Selumeitnib can both inhibit proliferative activity of NF1^kdRSC96 cell line.The semi-inhibitory concentrations of CA3 in the NF1^kdRSC96 group and control group were 0.7063±0.0233μM and 0.9583±0.0354μM,with statistical significance（P<0.05）.The semi-inhibitory concentrations of Selumeitnib in the NF1^kdRSC96 group and control group were 57.55±2.43μM and 53.98±3.55μM,with no statistical significance.4.The drug experiment results of tumor-bearing mice show that both CA3 and Selumeitnib can effectively delay the growth of the tumor,and the drug toxicity can be tolerated,but there is no significant difference in tumor size between the two groups.Conclusions:1.The expression of YAP and ability of nucleas entry are increased in the neurofibroma;2.Targeted inhibition of YAP in NF1-related Schwann cells can inhibit tumor cell growth and cell proliferation;3.The results provide new ideas for further study on pathogenesis and targered therpy of NF1...