关键词: 人木糖基转移酶- Ⅰ, 短发夹状shRNA, 唾液腺肿瘤, 肌上皮细胞, 蛋白多糖

Abstract:

Objective To design and construct the plasmids expressing short hairpin RNA（shRNA）targeting human xylosyltransferase- Ⅰ（XT- Ⅰ）which is the initiating enzyme in the biosynthesis of proteoglycans（PG）. Methods Short chain oligonucleotides were designed according to the sequence of XT- Ⅰ provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil- 1 vector. The recombinant XT- Ⅰ shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT- Ⅰ expression vectors were transfected into salivary adenoid cystic carcinoma cell line（ACC-M） by LipofectomineTM 2000. The expression of green fluorescent protein（GFP） was detected by inverted fluorescent microscope and the rates of  transfection were detected by flow cytometer. Semiquantitative RT- PCR was used to detect the expression of mRNA level of XT- Ⅰin transfected ACC-M cells and the  rotein expression of XT- Ⅰ was detected by Western blot. Results The plasmids expressing shRNA targeting XT- Ⅰgene are called WJ1, WJ2, WJ3, WJ4, WJ5 and  J6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1 -WJ6  xpressed GFP successfully. And by RT- PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of  RNA level and 70.18% of protein level respectively. Conclusion The XT- Ⅰ shRNA expression vectors were constructed successfully which lays the foundation for  NAi study on the biosynthesis of PG in salivary gland tumors.

Key words: xylosyltransferase- Ⅰ, short hairpin RNA, salivary gland tumor, myoepithelial cells, proteoglycans