NGF Positively Regulates N-RAS To Promote Perineural Invasion Of Salivary Adenoid Cystic Carcinoma

ObjectiveTo investigate the role of NGF and N-RAS in perineural invasion(PNI)of salivary adenoid cystic carcinoma(SACC),analyze the correlation between NGF and N-RAS and the effect of NGF on the malignant biological behavior of SACC cells,and further clarify the role of NGF-mediated N-RAS in regulating perineural invasion of SACC.Methods1.The relationship between NGF,N-RAS and PNI of SACC:seventy-five case of paraffin specimens of SACC in our hospital were collected and divided into the PNI group and the non-PNI group.The expressions of NGF and N-RAS in paraffin specimens of SACC in the two groups were detected by immunohistochemistry,and the correlation was analyzed in the same time.2.(1)The regulatory effect of NGF on N-RAS:Exogenous NGF and NGF inhibitor PD90780 were added to detect the changes in the expression level of N-RAS in SACC cells by Western blot.(2)The effect of NGF on the malignant biological behavior of SACC:Changes in cell proliferation ability were detected by CCK-8 and clone formation assay,changes in migration ability were detected by Wound healing and Migration experiments,and changes in invasion ability were detected by Invasion experiments.(3)NGF-mediated N-RAS regulates PNI of SACC:To construct a co-culture model of ACC-M and rat dorsal root ganglion(DRG),simulation of PNI in SACC in vitro.Using this model to observe the growth of DRG axons under different levels of NGF,the proliferation and migration of SACC cells.Results1.NGF and N-RAS were mainly expressed positively in the cytoplasm of SACC cells,and the positive rates were 89.1%and 80.4%in PNI group,and 44.8%and 51.7%in the non-PNI group,respectively.And the expression was strongly positive in the nerve invasion site and negative in the non-nerve invasion site.The positive expression rate of NGF in PNI group of SACC was significantly higher than that in the non-PNI group(χ~2=17.317,P=0.000),as same as N-RAS(χ~2=6.896,P=0.009).Spearman rank correlation analysis showed that there was a positive correlation relationship between the expression of N-RAS and NGF in SACC(r=0.294,P=0.011).2.(1)After culturing the exogenous NGF group,NGF inhibitor PD90780 group and control group for 24h and 48h,results of Western Blot showed that compared with the ACC-M NC group,the expression of N-RAS in the exogenous NGF group increased at 24h and 48h,decreased in the inhibitor PD90780 group.(2)Compared with the ACC-M NC group,the proliferation,migration and invasion abilities of cells in the exogenous NGF group were significantly enhanced,and inhibited significantly in the inhibitor PD90780 group.(3)The co-culture model of ACC-M/DRGs was successfully constructed.Compared with the single DRG culture group,in the co-culture system of ACC-M NC/DRGs,the proliferation rate of SACC cells was accelerated,and more axonal growth was observed around the DRGs.In exogenous NGF+ACC-M/DRG co-culture system,compared with the ACC-M NC/DRG,it could be seen that the SACC cells proliferate faster and DRG nerve axons grow specificity towards SACC cell colony.When axons after contacted with SACC cells,the SACC cells migrated along the axon to the main body of the dorsal root ganglion and surrounded the axon.Conclusions1.NGF and N-RAS are closely related to the PNI of SACC.And there is a positive correlation between NGF and N-RAS in the PNI of SACC.2.NGF positively regulate N-RAS in SACC to enhance the abilities of proliferation,invasion and metastasis of SACC cells.The PNI of SACC is the result of the interaction between nerve cells and SACC cells.NGF positively regulates N-RAS to promote the perineural invasion of SACC.