OBJECTIVETo study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I .

METHODSASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. 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 and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively.

RESULTSThe expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05).

CONCLUSIONSpecific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.

Cell Adhesion ; genetics ; physiology ; Cell Movement ; genetics ; physiology ; Flow Cytometry ; Gene Silencing ; HeLa Cells ; Humans ; Neoplasm Invasiveness ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialyltransferases ; biosynthesis ; genetics ; Transfection

In this study, we have shown that gene expression of human GD3 synthase (hST8Sia I) is suppressed by triptolide (TPL) in human melanoma SK-MEL-2 cells. To elucidate the mechanism underlying the downregulation of hST8Sia I gene expression in TPL-treated SK-MEL-2 cells, we characterized the TPL-inducible promoter region within the hST8Sia I gene using luciferase constructs carrying 5'-deletions of the hST8Sia I promoter. Functional analysis of the 5'-flanking region of the hST8Sia I gene demonstrated that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB, functions as the TPL-inducible promoter of hST8Sia I in SK-MEL-2 cells. Site-directed mutagenesis and ChIP analysis indicated that the NF-kappaB binding site at -731 to -722 is crucial for TPL-induced suppression of hST8Sia I in SK-MEL-2 cells. This suggests that TPL induces down-regulation of hST8Sia I gene expression through NF-kappaB activation in human melanoma cells.
Cell Proliferation/drug effects ; Diterpenes/*pharmacology ; Down-Regulation ; Epoxy Compounds/pharmacology ; Genes, Reporter ; Humans ; NF-kappa B/metabolism ; Phenanthrenes/*pharmacology ; Promoter Regions, Genetic ; Sialyltransferases/*biosynthesis/genetics ; Tumor Cells, Cultured