The Inhibited Effect Of Proteoglycans On The Neurotropic Growth Of Salivary Adenoid Cystic Carcinoma

Objectives:Salivary adenoid cystic carcinoma (SACC) is one of most common malignancy in salivary gland tumors, accounting for approximately 22% of human malignancy of salivary gland tumors. The tumor grows slowly with strong invision ability. The neural invasion in early stage would lead to patients with paresthesia, numbnesss and pain. The facial paralysis caused in parotid gland by SACC gives not only anguish to patients, but also greatly difficulty to clinical treatment.SACC is composed of neoplastic myoepithelial cells (NMCs) and duct epithelial cells. Because of the bilateral differentiation, NMCs hold the function of secreting proteoglycans (PGs) which is different from the normal myoepithelial cells. The abundant proteoglycans produced by NMCs make SACC typical cribriform structures full with PGs which provide nutrition for the biological behaviour of SACC.PGs are macromolecules composed of core protein and GAG chains. As the major components of the extracellular matrix, PGs generally exist in mammalian cells and almost body tissues, and participate in and regulate many biological processes, such as extracellular matrix (ECM) deposition, cell membrane signal transference, moisture retention and substance exchange between the orgnainzations. Studies found that there much more PGs and GAGs in the tumor matrix than in the normal or surrounding tissues, and PGs play a key role in such processes as tumor cell growth, adhesion and migration, cell differentiation and morphogenesis.The initial step of PGs biosynthesis is that xylosyltransferase (XT) catalyzes transfer of xylose to selected serine residues in the PGs core protein, forming O-glycosidic bond. And then, initiate the biosynthesis of GAGs side chains. Now there are two kinds of xylosyltransferase: xylosyltransferase-I (XT-I) and xylosyltransferase-II (XT-II), XT-I known as the chain-initiating enzyme is involved in rate-limiting step in the biosynthesis of GAG-containing PGs. XT-II represents an isoform of XT-I with 55% amino acid squences similar to XT-I,and also has its function for biosynthesis of PGs.RNA interference (RNAi) is a technology of post-transcriptional gene silence (PTGS) caused by the introduction of a homologous and complementary double-stranded RNA (dsRNA). Result in special degration of mRNA and loss of the relative functional phenotype.In this study, the technology of RNAi was used to silence the expression of XT-I at gene level, and the biosynthesis of PGs was inhibited. The inhibited effect of PGs biosynthesis was observed in vivo. The results provided new theory for understand of the neurotropic mechanism of SACC, and provided new idea and method for clinical treatment of SACC.Methods:1. The XT-I gene expression of SACC-83 was silenced by transfection of constructed XT-I expression vectors shRNA-WJ4 and shRNA-HK (negative control) into SACC-83 cells by LipofectameneTM 2000.2. The expression of the mRNA level of XT-I in SACC-83 was detected by the Real-Time PCR technology.3. The assay of PGs contents of the SACC-83 cells by Blyscan Assay Kit .4. The effect of down-regulated PGs on the ability of neural invasion of SACC-83 cells by silencing the XT-I geneEighteen three-week-old, 14~15g, female BALB/C nude mice were divided into 3 groups of 6 mice each randomly. Forty-eight hours after transfection, SACC-83-WJ4 cells, SACC-83-HK cells and SACC-83 cells were collected repectively, 0.2 ml cell suspension containing 1×107 cells per milliliter was injected into the muscle at the midpoint of line between tail root and right knee joint. The mice were anesthetized and killed at the 15th day after injection. All the right legs were fixed in 10% formaldehyde, decalcification with 10% formic acid for 48 hours, and samples were embedded in paraffin, continuous sections at 2.5μm, H&E stain for histological observation by light microscope was done to evaluate the neurotropic ability of SACC.Statistical analysis: All the data were analyzed by the software SPSS 13.0, analyze for statistical significance by using one way ANOVA. P<0.05 was considered statistically significant.Results:1. Successfully transfection of shRNA-WJ4 and shRNA-HK into the SACC-83 cells with stable expression of green fluorescent protein (GFP). The transfection efficiency was 43.3%.2. The expression of XT-I was inhibited by 43.0% after 48h transfection of shRNA-WJ4 by Real-Time PCR assay.3. The content of PGs was down-regulated by 30.25 % after 48h transfection.4. In the experimental neural invasion models, the rate of neural invasion of group SACC-83-WJ4 was 33.33%, significantly lower than that of group SACC-83-HK(100%)and group SACC-83(100%), (P<0.05). The ability of neural invasion of the SACC-83 cells was cut down after down-regulation of PGs in SACC-83 cells by silencing the XT-I gene.Conclusion:XT-I gene of SACC cells was silenced by RNAi, PGs secretion was reduced and neurotropic invasion behavior of SACC was inhibited obviously.