West China Journal of Stomatology
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SHI Hong, WANG Jie, WANG Xu, GU Hong- tao, HOU Ya- li, YU Li- jie
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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
SHI Hong, WANG Jie, WANG Xu, GU Hong- tao, HOU Ya- li, YU Li- jie. Construction of eukaryotic expr ession vector of shor t hairpin RNA targeting human xylosyltr ansfer ase - I gene[J]. West China Journal of Stomatology.
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