The inhibited effect of proteoglycans by XT-Ⅰ gene silenced on the invasive activity of salivary pleomorphic adenoma
Objective To inhibit the biosynthesis of proteoglycans(PGs)in salivary pleomorphic adenoma(PA),and to observe the inhibition of invasion activity of PA cells.Methods ①Xylosyltransferase-1(XT-Ⅰ)expression vector shRNA-WJ4 and shRNA-HK(negative control)was constructed.②The sample was obtained from a patient with a parotid pleomorphic adenoma resection in Department of Oral Maxillofacial Surgery,Hospital and College of Stomatology,Hebei Medical University.Histologic stain and immunohistochemical stain against Hyaluronan synthase 2(HAS2)and Heparan Sulfate Proteoglycan were performed.③Primary culture of salivary PA was adopted,and PA cells were cultured in RPMI 1640 medium containing 20％fetal bovine serum.The second generation of PA cells was identified by immunocytochemical stain against calponin,and S-100 protein as well as CK7.④The XT-Ⅰgene expression of PA was silenced by transfection of constructed XT-Ⅰ expression vector shRNA-WJ4 and shRNA-HK(empty-vector control)into PA cells by LipofectameneTM2000.Three groups were included,group PA(blank control),group PA-HK(empty-vector control),group PA-WJ4(XT-Ⅰ expression vector),and MTT was used to detect the growth of 3 groups.⑤The expression of the mRNA level of XT-Ⅰ in 3 groups was detected by the Real-Time PCR technology.⑥The content of PGs from 3 groups was detected by Blyscan Assay Kit.(7)Matrigel invasion assay was used to observe the inhibited effect of PGs on the invasive activity of PA cells by silencing the XT-Ⅰ gene.⑧The statistical analysis was performed by SPSS 13.0 software.And data were exported to generate the column chart by Excel software.Results ①Silenced XT-Ⅰexpression vector shRNA-WJ4 and shRNA-HK(empty-vector control)was constructed successfully.②Hyaluronic acid and heparan sulfate were observed richly in the mucoid-,myxoid-and chondroid-type tissues of salivary PA.③After 5～7 days of primary culture,short spindle-shaped or polygonal PA cells emigrated from the tissue.Immunocytochemical stain showed anti-calponin,S-100 positive in neoplastic myoepithelial cells and CK7 positive in neoplastic epithelial cells.④Successful transfection of shRNA-WJ4 and shRNA-HK was performed into PA cells with stable expression of green fluorescent protein(GFP).The transfection efficiency was 44.2％.The results of MTT showed that the cells of group PA-WJ4(XT-Ⅰ expression vector)grew slowly.⑤The expression of XT-Ⅰwas inhibited by 28.0％after 48h transfection of shRNA-WJ4,detected by Real-time PCR assay.⑥The content of PGs was down-regulated by 27.2％after 48 h transfection by Blyscan Assay Kit.⑦Matrigel invasion assay showed that the cell number of group PA-WJ4(XT-Ⅰ expression vector)migrating to the lower compartment through the microporous membrane was 23.83±2.93,much lower than that of group PA-HK(empty-vector control)(64.50±3.94)and group PA(blank control)(67.50±2.35),(P＜0.0l).The inhibitory rate was 64.70％.Conclusion XT-Ⅰ gene of salivary PA cells was efficiently silenced by RNAi,PGs secretion was reduced and invasion activity of PA cells was inhibited obviously.
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