Effects of small interfering RNA targeting heparanase-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cell lines.
OBJECTIVE
Heparanase-1 (HPA-1) can promote angiogenesis and metastasis of malignant tumors and plays an important role in the genesis and development of tumors. This study was to explore the effects of specific small interfering RNA (siRNA) targeting HPA-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cells.


METHODS
The expression of HPA-1 in Hca-F, Hca-P, and Hepa1-6 cells, which have high, low, and no metastatic potential, respectively, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). After transfection with two specific siRNAs targeting HPA-1, siRNA-1 and siRNA-2, and treatment with heparin, invasiveness of Hca-F cells was observed by Matrigel invasion assay.


RESULTS
HPA-1 was negative in Hepa1-6 cells while positive in both Hca-F and Hca-P cells. The expression levels of both HPA-1 mRNA and protein were obviously higher in Hca-F cells than in Hca-P cells. HPA-1 proteins could be secreted into culture supernatant of Hca-F and Hca-P cells, and the amount of secreted HPA-1 detected by Western blot analysis was larger in Hca-F cells than in Hca-P cells (1.34 ± 0.02 vs. 0.60 ± 0.01, P < 0.001), which was consistent with the results of ELISA. Both siRNA-1 and siRNA-2 downregulated the expression of HPA-1 and the siRNA-2 did more efficiently. The number of invasive Hca-F cells treated with siRNA-2 or heparin alone was larger than that of Hca-F cells treated with combination of them (9 ± 1 vs. 4 ± 1, P = 0.013; 15 ± 2 vs. 4 ± 1, P = 0.008), but smaller than that of untreated Hca-F cells (9 ± 1 vs. 22 ± 2, P = 0.006; 15 ± 2 vs. 22 ± 2, P = 0.026).


CONCLUSIONS
The combined application of specific siRNA targeting HPA-1 and heparin is more effective in inhibiting the invasiveness of mouse hepatoma cells.
Yu
Sheng-Jin
SJ
Department of Basic Medical Sciences, Medical College of Eastern Liaoning University, Dandong, Liaoning 118000, PR China.
Kang
Xiao-Hui
XH
Zhang
Jia-Ning
JN
Wang
Hong-Mei
HM
Xie
Tao
T
Wang
Wei
W
Wang
Shu-Jing
SJ
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D015536:Down-Regulation; D005966:Glucuronidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection; D000099175:Heparanase
D005966:Glucuronidase; D006493:Heparin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099175:Heparanase