[Combinatorial effects of ST6Gal I siRNA and antisense oligonucleotide-mediated gene silence on metastasis ability of cervical carcinoma cells].
OBJECTIVE
To study the combinatorial effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting alpha 2,6 sialyltransferase (ST6Gal I) on the cell adhesion and invasiveness of human cervical carcinoma cell line, HeLa with over-expressing ST6Gal I.


METHODS
The siRNA and ASOs targeting to ST6Gal I were designed and synthesized chemically, and then with lipofectamine 2000, transferred into HeLa cells, which were cultured and divided into 7 groups: blank control group, liposome group, siRNA group transfected with ST6Gal I siRNA, ASO1 group transfected with ST6Gal I ASO whose target site is same as siRNA, ASO2 group transfected with ST6Gal I ASO whose target site is different with siRNA, siRNA+ASO1 group transfected with siRNA and its homologous ASO1, siRNA+ASO2 group transfected with siRNA and its nonhomologous ASO2. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion or invasiveness to extracellular matrix (ECM) was analyzed by using CytoMatrix kit or cell invasion assay kit, respectively.


RESULTS
The expression of ST6Gal I mRNA, the amount of a2,6-sialylation of cell surface, the cell adhesion and invasion to ECM were remarkably decreased in siRNA group, ASO1 group, ASO2 group, siRNA+ASO, group and siRNA+ASO2 group, and all significantly lower than those in the blank control group and liposome group (all P < 0.05), especially in siRNA+ASO2 group. The difference was significant between siRNA+ASO2 group and siRNA group (all P < 0.05). No significant difference was observed between siRNA+ASO1 group and siRNA group, neither between blank control group and liposome group (all P > 0.05).


CONCLUSIONS
The synthesized chemically specific siRNA targeting to ST6Gal I can effectively downregulate the ST6Gal I expression in HeLa cell and further lead to the decline of cell adhesion and invasiveness to ECM, and use of associating siRNA with ASO targeting to same gene but different oligonucleotide location possesses the synergy and additive effect on targeted gene expression knocked down.
Yuan
Tian-Hong
TH
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Li
Ming-Yuan
MY
Li
Wan-Yi
WY
Li
Hong
H
Jiang
Zhong-Hua
ZH
D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D009361:Neoplasm Invasiveness / Q000235:genetics; D009362:Neoplasm Metastasis / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012799:Sialyltransferases / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase