[Inhibition of invasive and chemotactic abilities of SKOV3 cells by human epithelial growth receptor-2 small interfering RNA].
OBJECTIVE
To investigate the effects of RNA interference (RNAi) targeting human epithelial growth receptor-2 (HER-2) gene on the invasive and chemotactic ability of SKOV3 cells.


METHODS
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the positive control. Lipofectamine 2000 mediated transient transfection was conducted to transmit the siRNA into SKOV3 cells. Three pairs of specifically targeted (HER-2 siRNAI, HER-2 siRNAII, HER-2 siRNAIII) sequence were selected in the coding region of HER-2 mRNA. Transfection of HER-2 siRNA was conducted with lipofectamine 2000 in ovarian carcinoma cell line SKOV3. The HER-2 gene expression was assessed by real-time PCR and western blot assays. Changes of invasive and chemotactic capacity of SKOV3 cells were measured by polycarbonates coated with or without matrigal.


RESULTS
Western blot results showed that the expression of GAPDH protein was decreased in specifically transfected cells and with the increase of siRNA dose, the expression of GAPDH protein was decreased. GAPDH protein gray value in control group, different doses (0.5, 1.0, 1.5, 2.0 microg) GAPDH siRNA interference groups were 0.6855 +/- 0.0259, 0.5698 +/- 0.0275, 0.4542 +/- 0.0296, 0.3341 +/- 0.0178 and 0.1816 +/- 0.0180, respectively. There was a significant difference in each group (F = 198.126, P < 0.01). Both HER-2 siRNAII and siRNAIII could inhibit the expression of HER-2 protein. There was a significant difference in inhibition of HER-2 expression between the siRNAIII (0.1562 +/- 0.0067), siRNAII (0.2162 +/- 0.1589) groups and the other groups (F = 69.461, P < 0.01). In comparison of the relative expression levels in each dose (0.5, 1.0, 1.5, 2.0 microg) group, the difference was significant (F = 174.53, P < 0.01). The relative expression of HER-2 mRNA in HER-2 siRNA group after the 1(st), 3(rd), 6(th) interference were 0.0506 +/- 0.0017, 0.0266 +/- 0.0011 and 0.0154 +/- 0.0020, respectively. There was a decreasing trend in expression levels at different times (P < 0.01). The invasion and chemotactic capacity of SKOV3 cells were decreased after transfection of HER-2 siRNA into the cell line (F = 53.707, P < 0.01 vs F = 11.361, P < 0.01).


CONCLUSIONS
HER-2 siRNAIII can silence the mRNA expression in a time and dose dependent manner. HER-2 siRNA can inhibit cell invasion and chemotactic capacity of SKOV3 cells. The application of HER-2 siRNA extends the list of available therapeutic modalities in the treatment of human ovarian cancers.
Zhang
Shu-lan
SL
Department of Gynaecology and Obstetrics, the Second Affiliated Hospital, China Medical University, Shenyang, China.
Lu
Yan-ming
YM
Meng
Li-rong
LR
Zhao
Yan-yan
YY
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005260:Female; D018734:Genes, erbB-2 / Q000187:drug effects*; D006801:Humans; D009361:Neoplasm Invasiveness; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases