[Construction of heparanase gene-targeted small interfering RNA and its gene silencing effect].
To construct heparanase gene-targeted small interfering RNA(siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant melanoma A375 cell.



Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strand was synthesized and inserted into pRNATU6.1 vector, which was then identified by PCR and sequencing. Human malignant melanoma A375 cells were transfected with the constructed vector using lipofectamine method. Semi-quantitative PCR was performed to evaluate the heparanase-mRNA expression levels, and Western blot was performed to evaluate the expression of heparanase protein.



The vector containing siRNA was identified by PCR and sequencing; the results of semi-quantitative PCR and Western blot showed that the expression levels of both heparanase RNA and protein in transfected A375 cells were decreased significantly(P<0.05).



The heparanase gene-targeted siRNA and its vector were successfully constructed, which can reduce the heparanase gene and protein expression in transfected cells.
Liu
Xiao-yan
XY
The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Fang
Hong
H
Yang
Zheng-gang
ZG
D001483:Base Sequence; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D000099175:Heparanase
D005966:Glucuronidase; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099175:Heparanase