Effects Of Notch-1Gene Silencing On Perineural Invasion Of Salivary Adenoid Cystic Carcinoma In Vitro

Background Salivary adenoid cystic carcinoma(SACC) is one more common salivary gland cancer, accounting for7.5%-10%of malignant tumors of the salivary gland, can occur at any age,40-60years old accounted for the vast most, but women slightly more than men, occur in the parotid, submandibular and sublingual glands and minor salivary glands, such as the palate. SACC has the characteristic of perineural invasion (PNI), Often invades the nervous and towards distantal metastasis along nerve growth,which is one of the important biological characteristics different from other head and neck cancer and closely related to its recurrence, metastasis and prognosis. Although SACC develops relatively slowly, it can invade nerves and spread along them at it’s early stage, resulting in facial paralysis, numbness, pain and other symptoms of nerve damage, bringing great difficulties to the clinical treatment and poor prognosis, and thus exploring the SACC perineural invasion mechanism has important clinical significance.In addition to SACC, the neurotropic phenomenon is also can be seen in malignant melanoma, prostate cancer, pancreatic cancer and colorectal cancer. Therefore, the current domestic and foreign scholars tend to view perineural invasion as the fifth diffusion transfer mode except the traditional four ways including local tumor invasion,metastasis of blood, lymphatic metastasis and body cavity planted.Neurotropic characteristics of malignant tumors have been proposed as early as a century ago. Early studies suggest that PNI is mainly along the perineural lymphatic invasion of tumor cells,now,it’s believed that there is a potential loose gap around nerves in the anatomy, instead of lymphatic,tumor cells grow along the gap because it has the minimum resistance. This growth pattern suggest that tumor cells may appear distantal metastasis in the early stage. even part of the nerves connected to tumors are resected during surgery,the possibility of postoperative recurrence is high due to the distant never fibers have already been infiltrated by cancer cells. So far, the mechanism of perineural invasion of SACC is unclear, which is currently a hot topic at home and abroad.SACC’s perineural invasion behavior has a variety of related genes and signaling pathways involved in,which include the Notch signaling pathway. Notch signaling pathway is a minority signal transduction systems regulating cell proliferation and apoptosis repeatedly. Studies found that Notch signaling pathway is closely linked to cell differentiation, proliferation, apoptosis,adhesion and epidermal cells to mesenchymal transition and it is essential for the normal development of most organizations. In the process of cell differentiation, Notch signaling has four functions:(1) involved in embryonic development. Notch signaling up-regulate notch molecules of cell surface, while reduced expression of its ligand Delta;Conversely, the Delta express to the cells themselves can cut the notch molecules.(2) involved in T cell development.(3) maintain hematopoietic stem cell self-renewal.(4) regulate angiogenesis.Abnormal control of notch can cause tissue dysplasia,and may lead to tumorigenesis and it’s important role in the development of tumor as follows: maintaining an undifferentiated state; involved in cell fate decisions; induce terminal differentiation; regulating tumor angiogenesis. The mechanism is in the Notch receptor activation by ligand generated by neighboring cells, through the interaction among a series of molecules. according to the type of tissue or cell background of tumor cell, it play a different role in the regulation of proliferation, differentiation and apoptosis of tumor cells.Notch gene was found in Drosophila in1919, loss of partial function of the gene will cause the gap at the edge of the Drosophila wing (notch), Notch gene thus named.1991, Notch-1in a human T lymphoblast leukemia was first identified, indicating that the notch signaling pathway is related to the tumor. Studies suggest that notch family genes Notch-1, Notch-2, Notch-4are related to occurrence, development and metastasis of SACC. The research group in preliminary studies, combined with laser capture microdissection and the human genome chip technology, builded perineural invasion associated gene expression profile of SACC successfully the first time, validated by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), and used cluster analysis and gene functional analysis to study the function of Notch-1gene.Finally,the gene was found highly expressed in specific perineural invasion group.This result is consistent with two other gene expression profiling studies of the SACC published recently.ACC-M cell lines was a highly metastatic lung cell lines of SACC screened from ACC-2cell lines in1996, whose transfer capabilities increased significantly compared to ACC-2,the transfer rate increased from18%to96%,and Metastases weight from0.31g to0.88g. Establishment of ACC-M cell lines with high metastatic ability that provides an extremely important experimental model to discuss the mechanism of invasion, metastasis of SACC and helps select anti-invasion, metastasis drugs effectively. Although neurotropic researchs of SACC has gained a lot of progress in recent years, the mechanism have not yet been fully elucidated and is still in the exploratory stage. So far it show no reports about effects of Notch-1gene silencing on perineural invasion ability of high metastatic ACC-M cell lines in vitro home and abroad.Objective Using RNA interference (RNAi) technology inhibited Notch-1gene expression effectively and specifically, to understand the changes of neurotropic ability of ACC-M cells in vitro after Notch-1gene silencing.Methods1. Establishment of Notch-1gene RNA interference stably transfected cell lines ACC-M by designing shRNA sequences targeting Notch-1genes, constructing recombinant lentivirus, then transfecting ACC-M cells,we gained stably transfected cell lines ACC-M of Notch-1gene RNA interference.2. Impact of Notch-1gene silencing on proliferation, perineural invasion capability of high metastatic adenoid cystic carcinoma cell lines (ACC-M).By cell growth curve determined by MTT assay, flow cytometry instrument to detect the cell cycle change, improved Transwell cell perineural invasion assay,we tested effects of Notch-1gene silencing on proliferation, perineural invasion capability of ACC-M in vitro.Results1. We established Notch-1gene RNA interference stably transfected cell lines ACC-M Successfully. Building Notch-1RNAi lentiviral vector pLenR-GPH-Notch-1, and then identifying the vector by double digestion and DNA sequencing; when four plasmids co-transfected293T cells, a lot of green fluorescence could be seen; we measured virus titer of5.8×108TU/ml after concentrated; to infect293T cells after MOI (multiplicity of infection) was1, the infection efficiency was more than90%observed under a fluorescence microscope. The ACC-M cells were infected by the three groups of concentrated lentiviral vector each. Combined with qRT-PCR and Western Blot test results, and the third group lentiviral vector showed best interference effect, Notch-1gene mRNA and protein levels were inhibited significantly.2. MTT and flow cytometry results showed that Notch-1gene silencing may not infect the cell proliferation of ACC-M cells. Modified transwell cell invasion assay demonstrated that Notch-1gene silencing could inhibit perineural invasion capability of ACC-M cells in vitro.Conclusion1. Notch-1RNAi lentiviral expression vector and stably transfected cell lines can block Notch-1gene expression effectively and specifically,thus it is a good tool to study the gene’s function.2. Notch-1gene silencing may not infect the cell proliferation of ACC-M cells, but it can significantly inhibit the perineural invasion capacity of ACC-M cells in vitro, suggesting that Notch-1gene may become perineural invasion-related biomarkers and therapeutic target genes of SACC.