| Objective: Salivary adenoid cystic carcinoma (SACC) is one of the mostcommon malignancies of salivary gland, which is composed of neoplasticmyoepithelial cells (NMCs) and duct epithelial cells. NMCs hold the functionof producing and secreting proteoglycans (PGs) which provide nutrition andmicroenvironment for the biological behavior of SACC.PGs are macromolecular glycoprotein and generally exist in mammaliancells. There are much more proteoglycans in the tumor matrix than in thenormal tissue. The synthesis of PGs is starting from xylosyltransferase (XT)catalyzes transfer of xylose to selected serine residuces in the PGs core protein,froming O-glycosidic bond. Two kinds of XT were found now, XT-Ⅰ and XT-Ⅱ.XT-Ⅰ plays a key role in rate-limiting step in the biosynthesis ofGAG-congtaining PGs. XT-Ⅱ represents an isoform of XT-Ⅰ with less than55%amino acid sequences similar to XT-Ⅰ, and also has its function forbiosynthesis of proteoglycans.RNA interference (RNAi) is one of the most widely used methods ingene research. By this way, double-stranded RNA (dsRNA) composed ofsense strand RNA and anti-sense strand RNA was inserted to cells, which willlead to the specific degradation of the corresponding mRNA and the silence ofthe complementary gene.In this study, the inhibited effects of proteoglycans biosynthesis after thesilence of XT-Ⅱ and XT-Ⅰ&XT-Ⅱ by RNAi was compared. The morphologicalchange and apoptosis of the gene-silenced SACC-83cells were observed.Methods:1The expression vectors of short hairpin RNA (shRNA-WJ7,shRNA-WJ8, shRNA-WJ9) targeting XT-Ⅱ gene were transfected intoSACC-83cells, and the effective plasmid was selected and the cells transfected were shRNA-WJ7. shRNA-WJ4used as the effective plasmidtargeting XT-Ⅰ was selected by Jie Wang group. The shRNA-WJ7wastransfected into SACC-83cells named SACC-83-WJ7(group SACC-83-WJ7).shRNA-WJ4targeting XT-Ⅰ and shRNA-WJ7targeting XT-Ⅱ were mixed andtransfected into SACC-83cells (group SACC-83-WJ4&WJ7). TheshRNA-HK was transfected into SACC-83cells used as blank-transfectedcontrol (group SACC-83-HK). SACC-83cells were used as non-transfectedcontrol (group SACC-83).2The XT-Ⅰ and XT-Ⅱ gene expression was measured by Real-time PCR.3The content of proteoglycans was detected by Blyscan Assay Kit.4The cells of four groups were selected to detect the apoptosis bymicroscope and flow cytometry.Results:1Successfully transfection of shRNA-WJ7, shRNA-WJ8andshRNA-WJ9was performed into the SACC-83cells and showed the stableexpression of green flourescent protein (GFP). The transfection efficiency was31.71%,22.05%,33.11%respectively. The transfection efficiency of groupSACC-83-WJ4&WJ7was34.81%. The transfection efficiency of groupSACC-83-HK was51.10%.2The expression of XT-Ⅱ decreased by39.11%with shRNA-WJ7anddecreased by14.88%with shRNA-WJ8. It is no decreased with shRNA-WJ9.The efficient inhibition was group SACC-83-WJ7. The expression of XT-Ⅰdecreased by36.87%and XT-Ⅱ by10.23%in group SACC-83-WJ4&WJ7.There is no change in group SACC-83-HK.3The proteoglycans content was129.63μg,82.38μg,193.50μg and202.25μg in group SACC-83-WJ7, SACC-83-WJ4&WJ7, SACC-83-HK andSACC-83respectively. The rate of inhibition compared group SACC-83-WJ7,SACC-83-WJ4&WJ7, SACC-83-HK with group SACC-83was35.91%,59.36%,4.33%respectively. The proteoglycans content of groupSACC-83-WJ7was lower than that of group SACC-83-HK and groupSACC-83respectively (P＜0.05). The proteoglycans content of group SACC-83-WJ4&WJ7was lower than that of group SACC-83-HK and groupSACC-83respectively (P＜0.05). There was significant difference ofproteoglycans content between group SACC-83-WJ7and groupSACC-83-WJ4&WJ7(P＜0.05), while no difference of proteoglycans contentbetween group SACC-83-HK and group SACC-83was found (P＞0.05).4No morphologic change of apoptosis was observed by electronmicroscope.5The rate of apoptosis in group SACC-83-WJ7, SACC-83-WJ4&WJ7,SACC-83-HK and SACC-83was3.17%±0.76,2.40%±0.13,2.49%±0.09and1.40%±0.22respectively. There was no obvious apoptosis peak in each group.Conclusion:1The single silence of XT-Ⅱ gene in SACC-83cells could suppress thebiosynthesis and secretion of proteoglycans significantly.2The silence of both XT-Ⅰ gene and XT-Ⅱ gene in SACC-83cells wasstronger than that of single silence of XT-Ⅱ gene in the biosynthesis andsecretion of proteoglycans. |
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