Silencing of Heparanase Gene-Targeted by Small Interfering RNA_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LIU Qizhi ¹ , WANG Lie ²

1.Fuzong Medical College of Fujian Medical University, Fuzhou 350025, China;;
2.Research Institute of General Surgery, Fuzhou General Hospital of Nanjing Military Region, Fuzhou 350025, China;

Corresponding author：

Keywords：

RNA interference; Heparanase; Plasmid; MDA-MB-231 cell line; Transfection; Real-time polymerase chain reaction

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Objective According to heparanase’s gene sequence of GenBank, 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 breast cancer MDA-MB-231 cell.
Methods Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector, which was identified by sequence identify. Human malignant breast cancer MDA-MB-231 cell was transfected with the constructed vector with lipofectamine method. Fluorescence photograph was taken. Real-time PCR (RT-PCR) was performed to evaluate the level of heparanase mRNA expression.
Results Four kinds of heparanase gene-targeted hairpin siRNA were designed, then were inserted into pGPU6/GFP/Neo vector after annealing. Sequencing indicated the construction was successful. Fluorescence photographs showed MDA-MB-231 cells were transfected successfully. RT-PCR showed that heparanase mRNA expression levels were inhibited significantly （P lt;0.05）.
Conclusion The heparanase gene-targeted siRNA and its vector are successfully constructed and MDA-MB-231 cells are transfected successfully. Heparanase mRNA expression levels are significantly inhibited by siRNA vector, which provide a new method for the treatment of cancer.

Citation： LIU Qizhi ,WANG Lie. Silencing of Heparanase Gene-Targeted by Small Interfering RNA. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(2): 142-146. doi: Copy

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23.	Gtte M， Yip GW. Heparanase, hyaluronan, and CD44 in cancers: a breast carcinoma perspective [J]. Cancer Res, 2006； 66(21): 1023310237.
24.	童强，舒晓刚，卢晓明，等. STAT3基因短发夹RNA表达质粒的构建及其对胃癌细胞生长和侵袭的抑制作用 [J]. 中国普外基础与临床杂志， 2009； 16（1）： 611.
25.	刘利平，陈孝平，杨盛力，等. 沉默缺氧诱导因子1 对肝癌细胞化疗敏感性影响及相关机理的研究 [J]. 中国普外基础与临床杂志， 2008； 15（4）： 234239.

1. BenZaken O, Shafat I, GingisVelitski S, et al. Low and high affinity receptors mediate cellular uptake of heparanase [J]. Int J Biochem Cell Biol, 2008； 40(3): 530542.
2. Reiland J, Sanderson RD, Waguespack M, et al. Heparanase degrades syndecan1 and perlecan heparan sulfate functional implications for tumor cell invasion [J]. J Biol Chem, 2004； 279(9): 80478055.
3. Xu X, Ding J, Rao G, et al. Estradiol induces heparanase1 expression and heparan sulphate proteoglycan degradation in human endometrium [J]. Hum Reprod, 2007； 22(4): 927937.
4. Sanderson RD, Yang Y, Suva LJ, et al. Heparan sulfate proteoglycans and heparanase-partners in osteolytic tumer growth and metastasis [J]. Matrix Biol, 2004； 23(6): 341352.
5. Cohen I, Pappo O, Elkin M, et al. Heparanase promotes growth, angiogenesis and survival of primary breast tumors [J]. Int J Cancer, 2005； 118(7): 16091617.
6. Ilan N, Elkin M, Vlodavsky I. Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis [J]. Int J Biochem Cell Biol, 2006； 38(12): 20182039.
7. Doviner V, Maly B, Kaplan V, et al. Spatial and temporal heparanase expression in colon mucosa throughout the adenomacarcinoma sequence [J]. Mod Pathol, 2006； 19(6): 878888.
8. 肖毅，冯云路，钱家鸣，等. 乙酰肝素酶在结直肠癌中的表达及意义 [J]. 中国普外基础与临床杂志， 2007； 14（4）： 442447.
9. Shinyo Y, Kodama J, Hongo A, et al. Heparanase expression is an independent prognostic factor in patients with invasive cervical cancer [J]. Ann Oncol, 2003； 14(10): 15051510.
10. Hannon GJ. RNA interference [J]. Nature, 2002； 418(6894): 244251.
11. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002; 296(5567): 550553.
12. Zhang ZH, Chen Y, Zhao HJ, et al. Silencing of heparanase by siRNA inhibits tumor metastasis and angiogenesis of human breast cancer in vitro and in vivo [J]. Cancer Biol Ther, 2007； 6(4): 587595.
13. Sommerfeldt N, Beckhove P, Ge Y, et al. Heparanase: a new metastasisassociated antigen recognized in breast cancer patients by spontaneously induced memory T lymphocytes [J]. Cancer Res, 2006； 66(15): 77167723.
14. Sambrook J， Russell DW著. 黄培堂译. 分子克隆实验指南 [M]. 第4版. 北京：科学出版社， 2005： 2628.
15. 王守立，姚辉华，董亮，等. 脂质体介导TGF1 shRNA转染的方法 [J]. 苏州大学学报（医学版）， 2008； 28（2）： 222225.
16. 任亮，朱宝芹，张轶博，等. 利用软件Primer Premier 5.0 进行PCR引物设计的研究 [J]. 锦州医学院学报， 2004； 25（6）： 4345.
17. 翟中会，陈希南，王娟. 利用Primer Premier 5.0进行引物设计 [J]. 西北医学教育， 2008； 16（4）： 695698.
18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2－△△CT method [J]. Methods, 2001； 25(4): 402408.
19. 唐永凯，贾永义. 荧光定量PCR数据处理方法的探讨 [J]. 生物技术， 2008； 18（3）： 8991.
20. Kelly T, Miao HQ, Yang Y, et al. High heparanase activity in multiple myeloma is associated with elevated microvessel density [J]. Cancer Res, 2003； 63(24): 87498756.
21. Vlodavsky I, Ilan N, Nadir Y, et al. Heparanase, heparin and the coagulation system in cancer progression [J]. Thromb Res, 2007； 120(Suppl 2): S112S120.
22. Maxhimer JB, Quiros RM, Stewart R, et al. Heparanase1 expression is associated with the metastatic potential of breast cancer [J]. Surgery, 2002； 132(2): 326333.
23. Gtte M， Yip GW. Heparanase, hyaluronan, and CD44 in cancers: a breast carcinoma perspective [J]. Cancer Res, 2006； 66(21): 1023310237.
24. 童强，舒晓刚，卢晓明，等. STAT3基因短发夹RNA表达质粒的构建及其对胃癌细胞生长和侵袭的抑制作用 [J]. 中国普外基础与临床杂志， 2009； 16（1）： 611.
25. 刘利平，陈孝平，杨盛力，等. 沉默缺氧诱导因子1 对肝癌细胞化疗敏感性影响及相关机理的研究 [J]. 中国普外基础与临床杂志， 2008； 15（4）： 234239.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Silencing of Heparanase Gene-Targeted by Small Interfering RNA

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