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目的:以乙酰肝素酶(HPA)基因为靶点,构建短发夹RNA(sh RNA)慢病毒表达载体,研究慢病毒携带的sh RNA对乳腺癌HPA基因表达的抑制作用。方法:以乳腺癌HPA基因为靶基因,根据RNA干扰(RNAi)序列设计原则,通过构建3对sh RNA重组慢病毒的表达载体,转染乳腺癌MDA-MB-231细胞,在体外采用蛋白质印迹(Western blot)法检测HPA基因蛋白表达水平;在体内构建乳腺癌模型,通过免疫组化检测HPA基因蛋白的表达。结果:在体外实验中,HPAsh RNA-1组和HPA-sh RNA-2组均有效抑制了人乳腺癌MDA-MB-231细胞HPA的表达。在体内实验中,HPA-sh RNA-2组HPA基因蛋白的表达明显低于阴性对照组及空白对照组,HPAsh RNA-2组阳性率与空白对照组比较,差异有统计学意义(x2=12.504,P<0.05)。表明在体内实验中HPA-sh RNA-2组可以下调HPA基因蛋白表达。结论:以HPA为靶点构建sh RNA重组慢病毒载体,体内外实验均能下调乳腺癌HPA基因的表达,可为乳腺癌靶向治疗提供新的靶点。
Abstract：Objective: To construct short hairpin RNA(sh RNA) lentiviral expression vector by choosing HPA genes as the targeting point so as to investigate the inhibitive effect of lentiviral vector carryed sh RNA for the expression level of heparanase(HPA) genes of breast cancer. Methods: The HPA genes were chosen as target gene, 3 pairs expression vectors of recombinant lentivirus carried sh RNA were constructed according to the sequence designed principle of interfering RNA(RNAi) were transfected in MDA-MB-231 cell of breast cancer. Western Blot was used to detect the expression level of HPA gene in vitro, and the breast cancer model was constructed for vivo verification, and then the expression level of HPA gene was detected by using immunohistochemistry. Results: In vitro experiment, both of the HPA-sh RNA-1 group and HPA-sh RNA-2 group effectively inhibited the expression of HPA gene in MDA-MB-231 cells of breast cancer. In vivo experiment, the expression of HPA gene of HPA-sh RNA-2 group was obviously lower than that of negative group and blank group. The difference of positive rate between HPA-sh RNA-2 group and blank group was statistically significant(x2=12.504, P<0.05). And these results revealed that the expression of HPA gene in HPA-sh RNA-2 group could be down-regulated in vivo experiment. Conclusion: The HPA is chosen as the targeting point to construct recombinant lentiviral vector carried sh RNA, and the results of vitro and vivo experiment reveals that the expression of HPA gene can be down-regulated in the two groups. This result verifies that the new method can provide new targeting point for targeted therapy of breast cancer.
[1]Barash U,Cohen-Kapaln V,Dowek I,et al.Proteoglycans in health and disease:New concepts for heparanase function in tumor progression and metastasis[J].FEBS J,2010,277(19):3890-3903.
[2]Varchalama E,Rodolakis A,Strati A,et al.Quantitative analysis of heparanase gene expression in normal cervical,cervical intraepithelial neoplastic,and cervical carcinma tissues[J].Int JGynecol Cancer,2009,19(9):1614-1619.
[3]Inamine M,Nagal Y,Hirakawa M,et al.Heparanase expression in endometrial cancer:analysis of immunohistochemistry[J].J Obstet Gynaecol,2008,28(6):634-637.
[4]Roy M,Marchetti D.Cell Surface Heparan Sulfate Released by Heparanase Promotes Melanoma Cell Migration and Angiogenesis[J].J Cell Biochem,2009,106(2):200-209.
[5]Theodoro TR,De Matos LL,Sant Anna AV,et al.Heparanase expression in circulating lymphocytes of breast cancer patients depends on the presence of the primary tumor and/or systemic metastasis[J].Neoplasia,2007,9(6):504-510.
[6]Purushothaman A,Babitz SK,Sanderson RD.Heparanase enhances the insulin receptor signaling pathway to activate extracellular signalregulated kinase in multiple myeloma[J].J Bilo Chem,2012,287(49):41288-41296.
[7]Park MS,Dong SM,Kim BR,et al.Thioridazine inhibits angiogenesis and tumor growth by targeting the VEGFR-2/PI3K/mT OR pathway in ovarian cancer xenografts[J].Oncotarget,2014,5(13):4929-4934.
[8]Chen Z,Xu S,Xu W,et al.Expression of cluster of differentiation 34 and vascular endothelial growth factor in breast cancer,and their prognostic significance[J].Oncol Lett,2015,10(2):723-729.
[9]Lv B,Zhang B,Hu XY,et al.Heparanase regulates in vitro VEGF-C expression and its clinical significance to pancreatic ductal cell adenocarcinoma[J].Oncol Lett,2016,11(2):1327-1334.
[10]Hammond E,Khurana A,Shridhr V,et al.The Role of Heparanase and Sulfatases in the Modification of Heparan Sulfate Proteoglycans within the Tumor Microenvironment and Opportunities for Novel Cancer Therapeutics[J].Front Oncol,2014,4:195-210.
[11]Benedetto MD,Toullec A,H Buteaulozano,et al.MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics[J].Br J Cancer,2015,113(5):773-785.
[12]Liu L,Tong Q,Liu S,et al.ZEB1 Upregulates VEGFExpression and Stimulates Angiogenesis in Breast Cancer[J].PloS One,2016,11(2):e0148774.
[13]Zachary I.VEGF signalling:integration and multitasking in endothelial cell biology[J].Biochem Soc Trans,2003,31(6):1171-1177.
[14]Purushothaman A,Uyama T,Kobayashi F,et al.Heparanase-enhanced shedding of syndecan-1 by myeloma cells promotes endothlial invasion and angiogenesis[J].Blood,2010,115(12):2449-2457.
[15]Qiang B,Lim SY,Lekas M,et al.Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia[J].Can J Cardiol,2014,30(11):1444-1451.
[16]van Wijk XM,van Kuppevelt TH.Heparan sulfate in angiogenesis:a target for therapy[J].Angiogene sis,2014,17(3):443-462.
[17]Gingisvelitski S,Zetser A,Flugelamn MY,et al.Heparanase induces endothelial cell migration via protein kinase B/Akt activation[J].J Biol Chem,2004,279(24):23536-23541.
[18]Zetser A,Bashenko Y,Edovitsky E,et al.Heparanase induces vascular endothelial growth factor expression:correlation with p38 phosphorylation levels and Src activation[J].Cancer Res,2006,66(3):1455-1463.
[19]Purushothaman A,Chen L,Yang Y,et al.Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma[J].J Biol Chem,2008,283(47):32628-32636.
[20]Purushothaman A,Uyama T,Kobayashi F,et al.Heparanase-enhanced shedding of syndecan-1 by myeloma cells promotes endothelial invasion and angiogenesis[J].Blood,2010,115(12):2449-2457.
[21]Waisberg J,Theodoro TR,Matos LL,et al.Immunohistochemical expression of heparanase isoforms and syndecan-1 proteins in colorectal adenomas[J].Eur J Histochem,2016,60(1):2590.
[22]Ho JY,Hsu RJ,Wu CH,et al.Reduced miR-550a-3p leads to breast cancer initiation,growth,and metastasis by increasing levels of ERK1 and 2[J].Oncotarget,2016,7(33):53853-53868.
[23]Becker MA,Ibrahim YH,Oh AS,et al.Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer[J].PLo S One,2016,11(3):e0150564.
[24]Shaw LM.The insulin receptor substrate(IRS)proteins:At the intersection of metabolism and cancer[J].Cell Cycle,2011,10(11):1750-1756.
[25]Choi SK,Kim HS,Jin T,et al.Overexpression of the miR-141/200c cluster promotes the migratory and invasive ability of triple-negative breast cancer cells through the activation of the FAKand PI3K/AKT signaling pathways by secreting VEGF-A[J].BMC Cancer,2016,16(1):570-584.
[26]Gao SL,Wang SL,Liu HY,et al.mi R-200a inhibits tumor proliferation by targeting AP-2γin neuroblastoma cells[J].Asian Pac J Cancer Prev,2014,15(11):4671-4676.
[27]Zhao X,Guan JL.Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis[J].Adv Drug Deliv Rev,2011,63(8):610-615.
[28]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.
[29]Fuxl L,Ilan N,Sanderson ED,et al.Heparanase:busy at the cell surface[J].Trends Biochem Sci,2009,34(10):511-519.
[30]Masoud N,Karimi A,Allahbakhshian Farsani M.Production,purification and titration of a lentivirus-based vector for gene delivery purposes[J].Cytotechnology,2014,66(6):1031-1038.
[31]Vannucci L,Lai M,Chiuppesi F,et al.Viral vectors:a look back and ahead on gene transfer technology[J].New Microbiol,2013,36(1):1-22.
[32]Ozcan G,Ozpolat B,Coleman RL,et al.Preclinical and clinical development of sh-RNA-based therapeutics[J].Adv Drug Deliv Rev,2015,87:108-119.
[33]Khatri N,Rathi M,Baradia D,et al.In vivo delivery aspects of miR NA,shR NA and shR NA[J].Crit Rev Ther Drug Carrier Syst,2012,29(6):487-527.
基本信息:
中图分类号:R737.9
引用信息:
[1]陈国利,郑志方,张学军,等.慢病毒携带shRNA对乳腺癌HPA基因表达的抑制作用[J].中国医学装备,2017,14(06):150-155.
基金信息:
承德市科学技术研究与发展计划项目(20151029)“探讨以HPA为靶点治疗乳腺癌可行性”
2017-06-16
2017-06-16
2017-06-16