The Inhibited Effect Of Proteoglycans On The Invasive Activity Of Salivary Pleomorphic Adenoma

Objectives:Salivary pleomorphic adenoma (SPA) is the commonest benign neo-plastic lesion of salivary gland, accounting for approximately over50%ofsalivary epithelial tumors. The tumor is easily recurrent and in implantinggrowth clinically. The facial paralysis caused in parotid land by SPA givesnot only nguish to patients, but also greatly difficulty to clinical treatment.Histopathologically, SPA is composed of neoplastic myoepithelial cells(NMCs) and duct epithelial cells. Unlike the normal myoepithelial cells,NMCs obtain the functionality of producing abundant extracellular matrixwith chondroid or mucoid stroma which is full of proteoglycans (PGs).PGs are macromolecules made up of core protein and GAG chainsattached to the core protein. As the major components of the extracellularmatrix, PGs regulate many important cell functions, such as differentiation,proliferation, adhesion, migration, protein synthesis or degradation.Xylosyltransferase is the initial enzyme which plays a key role in thebiosynthesis of proteoglycans, which initiates GAG biosynthesis onto PGsmolecules. There are two kinds of xylosyltransferase currently known:xylosyltransferase-I (XT-I) and xylosyltransferase-II (XT-II), while XT-I iskey to the biosynthesis of proteoglycans, which initiates the rate-limited stepin the proteoglycans biosynthesis.RNA interference (RNAi) is a biological process in which RNA mole-cules inhibit gene expression, typically by causing the destruction of specificmRNA molecules. As a valuable research tool, RNAi has been used inexperimental studies in which abnormal proliferation and metastasis of tumorcells can be inhibited efficiently.In this study, the expression of XT-I is to be silenced at gene level by RNAi, to inhibit the biosynthesis of proteoglycans in SPA cells. The effect ofdown-regulated proteoglycans will be observed in vitro. This research willprovide new idea and method for clinical treatment of SPA.Methods:1The cell culture of SPA cellsThe sample was obtained from a patient with a tumor resection in Depar-tment of Oral Maxillofacial Surgery, Hospital and College of Stomatology,Hebei Medical University. Primary culture of SPA was adopted, and SPA cellswere cultured in RPMI1640medium containing20%fetal bovine serum.2The identification of SPA cellsThe second generation of SPA cells was identified by immunocy-tochemical stain against calponin, CK8, and S-100protein.3Cell transfection and screeningThe XT-I gene expression of SPA was silenced by transfection ofconstructed XT-I expression vector shRNA-WJ4and shRNA-HK (negativecontrol) into SPA cells by LipofectameneTM2000.4Valuation of the silence efficiencyThe expression of the mRNA level of XT-I in SPA was detected by theReal-Time PCR technology.5Determination of PGs contentThe content of PGs from the SPA cells was determinated by BlyscanAssay Kit.6Cell invasion assayThe inhibited effect of proteoglycans on the invasive activity of salivarypleomorphic adenoma by silencing the XT-I gene.Results:1The cell culture of SPAAfter5~7days of primary culture, short spindle-shaped or polygonal SPAcells emigrated from the tissues.2The identification of SPA cellsImmunocytochemical stain showed calponin, S-100and CK8positive in the cytoplasms of SPA cells.3Cell transfection and screeningSuccessful transfection of shRNA-WJ4and shRNA-HK was performedinto SPA cells with stable expression of green fluorescent protein (GFP). Thetransfection efficiency was44.2%.4Detection of the silence efficiencyThe expression of XT-I was inhibited by28.0%after48h transfection ofshRNA-WJ4, detected by Real-Time PCR assay.5Determination of PGs contentThe content of PGs was down-regulated by27.2%after48h trans-fection.6Cell invasion assayMatrigel invasion assay showed that the cell number of group SPA-WJ4migrating to the lower compartment through the microporous membrane was23.83±2.93, much lower than that of group SPA-HK (64.50±3.94) and groupSPA (67.50±2.35),(P＜0.01). The inhibitory rate was64.70%.Conclusion:XT-I gene of SPA cells was efficiently silenced by RNAi, PGs secretionwas reduced and invasion activity of SPA was inhibited obviously.