Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells |
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Authors: | Lei?Fan Qiang?Wu Xiaojuan?Xing Yudong?Liu Email author" target="_blank">Zengwu?ShaoEmail author |
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Institution: | 1. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2. Southeast Forensic Science Service, Hanjing 210000, China |
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Abstract: | The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma
cells (MG63 cells) were investigated in the present study. Two complementary oligonucleotide strands were synthesized and
inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene. The expression vector containing short hairpin
RNA (pGenesil-shRNA) was constructed successfully. MG63 cells were randomly allocated into 3 groups: blank group, empty vector
(pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group. Under the induction of Lipofectamine
2000, the recombinants were transfected into MG63 cells. Heparanase gene expression level was detected by RT-PCR and Western
blotting. Cell proliferation was measured by MTT assay. Cell invasiveness and metastasis were examined by cell adhesion and
Transwell-ECM assays. HUVECs migration assay was applied for the detection of angiogenesis. As compared with negative controls,
the mRNA and protein expression levels of heparanase were down-regulated by 76.1% (P<0.01) and 75.3% (P<0.01) respectively in the pGenesil-shRNA transfected group. Meanwhile, the proliferation, adhesiveness, invasiveness and
angiogenesis properties of MG63 cells were all significantly inhibited. It was suggested that targeted silencing of heparanase
gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells. |
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Keywords: | siRNA shRNA heparanase gene silencing osteosarcoma gene expression invasion metastasis |
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