Establishment of breast cancer cells with stable expression of ST3Gal Ⅲ gene RNA interference via lentivirus vector
10.13699/j.cnki.1001-6821.2017.08.011
- VernacularTitle:慢病毒载体法建立乳腺癌ST3Gal Ⅲ基因RNAi细胞模型
- Author:
Juan SONG
1
;
Hong-Lan WANG
;
Yu-Chun WANG
;
Hua TIAN
;
Chun-Hui XIA
;
Ye-Tong SHEN
;
Hong-Xia CUI
Author Information
1. 齐齐哈尔医学院药学院
- Keywords:
ST3Gal Ⅲ gene;
RNA interference;
transfection;
expression
- From:
The Chinese Journal of Clinical Pharmacology
2017;33(8):710-713
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish of breast cancer cells with stable expression of α 2,3-sialyltransferase Ⅲ (ST3Gal Ⅲ) gene RNA interference via lentivirus vector.Methods Four short hairpin RNA (shRNA) sequences targeting ST3Gal Ⅲ gene were designated,connected the synthesizd the DNA chains with linear pGLV3-H1-GFP carriers,and then lentivirus plasmid were constructed.Lentiviral vectors were packaged after identifying the sequences and the virus titer were measured.The recombinant lentiviral vector pGLV3-H1-GFP-shST3 was transfected into human breast cancer MDA-MB-231 cells,and transfected cells were divided into three groups:blank group (mock cells,M),control group (parent cells,P),RNA interfere groups (shST3-1,2,3,4).Then the expression of ST3Gal Ⅲ in transfected MDA-MB-231 cells was detected by real time-PCR and Western blotting.The content of α 2,3-sialic acids was analyzed by flow cytometry.Results The recombinant vectors of RNAi lentiviral vector targeting the ST3Gal Ⅲ gene were successfully constructed and all the virus titers were more than 2 × 10s TU · mL-1.The transfection efficiency was detected over 90％ by observing green fluorescence protein after the recombinant vectors transfected into MDA-MB-231cells.The inhibitory rate of ST3Gal Ⅲ mRNA expression in shST3-1,2,3,4 were 42.1％,66.7％,30.1％,80.9％ respectively with significantly (P ＜0.05).The lower expression of ST3Gal Ⅲ protein in shST3-1,2,3,4 were 35.92％,75.64％,37.12％,81.75％ respectively,compared with control group,were significantly decreased (P ＜ 0.05).The contents of α 2,3-sialic acids was reduced in shST3-4 with significantly (P ＜ 0.05).Conclusion The shST3-4 cells model which could interfere ST3Gal Ⅲ gene expression in MDA-MB-231 cells was successfully constructed.
