• 1.湖北醫(yī)藥學(xué)院附屬太和醫(yī)院內(nèi)分泌血管外科(湖北十堰 442000);;
  • 2湖北醫(yī)藥學(xué)院基礎(chǔ)醫(yī)學(xué)研究所(湖北十堰 442000);

目的  探討短發(fā)夾RNA(shRNA)靶向沉默DLL4基因?qū)CF-7細(xì)胞凋亡的誘導(dǎo)及對化療藥物多西他賽的增敏作用。
方法  針對DLL4基因的序列設(shè)計具有特異性的shRNA,經(jīng)脂質(zhì)體轉(zhuǎn)染MCF-7細(xì)胞,作為實驗組,空脂質(zhì)體轉(zhuǎn)染的MCF-7細(xì)胞為脂質(zhì)體組,未做任何處理的MCF-7細(xì)胞為對照組。采用免疫細(xì)胞化學(xué)方法檢測3組細(xì)胞DLL4蛋白的表達(dá)情況;采用流式細(xì)胞儀檢測3組細(xì)胞凋亡率及細(xì)胞周期改變;采用噻唑藍(lán)(methyl- thiazoyl-tetrazolium bromide,MTT)法測定3組細(xì)胞的增殖情況及對多西他賽的敏感性。
結(jié)果  實驗組平均光密度值及陽性面積率均低于對照組和脂質(zhì)體組(P<0.01);實驗組細(xì)胞在24h、48h及72h時間點A值均低于對照組和脂質(zhì)體組(P<0.01); 轉(zhuǎn)染后24 h、48 h、72 h及96 h,實驗組細(xì)胞凋亡率高于對照組和脂質(zhì)體組(P<0.05);RNA干擾(RNAi)沉默DLL4基因后,實驗組G2/M期細(xì)胞比例高于對照組和脂質(zhì)體組(P<0.05);實驗組的IC50值較對照組和脂質(zhì)體組低(P<0.05)。
結(jié)論  RNAi技術(shù)抑制DLL4基因的表達(dá)能夠抑制MCF-7細(xì)胞的增殖、誘導(dǎo)細(xì)胞凋亡及增強(qiáng)細(xì)胞對多西他賽作用的敏感性。DLL4可能會成為乳腺癌治療的重要靶點。

引用本文: 王群,余明華,王耕,王明華. RNAi沉默DLL4基因誘導(dǎo)人乳腺癌細(xì)胞凋亡及對多西他賽的增敏作用. 中國普外基礎(chǔ)與臨床雜志, 2013, 20(1): 54-59. doi: 復(fù)制

1. Patel NS, Li JL, Generali D, et al. Up-regulation of delta-like 4 ligand in human tumor vasculature and the role of basal expression in endothelial cell function[J]. Cancer Res, 2005, 65(19):8690-8697.
2. Benedito R, Trindade A, Hirashima M, et al. Loss of notch signalling induced by Dll4 causes arterial calibre reduction by increasing endothelial cell response to angiogenic stimuli[J]. BMC Dev Biol, 2008, 8(16):117.
3. Li JL, Sainson RC, Shi W, et al. Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo[J]. Cancer Res, 2007, 67(23):11244-11253.
4. Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic protein humansurvivin is a direct inhibitor of caspase-3 and -7[J]. Biochemistry,2001, 40(4):1117-1123.
5. 魏愛英, 張慶柱, 張捷. 多西他賽誘導(dǎo)乳腺癌Bcap37細(xì)胞凋亡作用的研究[J]. 中華腫瘤防治雜志, 2006, 13(18):1384-1386.
6. Siekmann AF, Lawson ND. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries[J]. Nature, 2007, 445(7129):781-784.
7. Scehnet JS, Jiang W, Kumar SR, et al. Inhibition of Dll4-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion[J]. Blood, 2007, 109(11):4753-4760.
8. Hellström M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis[J]. Nature, 2007, 445(7129):776-780.
9. Segarra M, Williams CK, Sierra Mde L, et al. Dll4 activation of Notch signaling reduces tumor vascularity and inhibits tumor growth[J]. Blood, 2008, 112(5):1904-1911.
10. Dufraine J, Funahashi Y, Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms[J]. Oncogene, 2008, 27(38):5132-5137.
11. Harrington LS, Sainson RC, Williams CK, et al. Regulation of multiple angiogenic pathways by Dll4 and Notch in humanumbilical vein endothelial cells[J]. Microvasc Res, 2008, 75(2):144-154.
12. Zang S, Ji CH,Qu X, et al. A study on Notch signaling in humanbreast cancer[J]. Neoplasma, 2007, 54(4):304-310.
13. Jubb AM, Soilleux EJ, Turley H, et al. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer[J]. Am J Pathol, 2010, 176(4):2019-2028.
14. 王群, 余明華, 王耕, 等. DLL4和S100A4在不同分子亞型乳腺癌組織中的表達(dá)及意義[J]. 中國普外基礎(chǔ)與臨床雜志, 2012, 19(9):46-50.
15. 王群, 余明華, 王耕, 等. DLL4、COX-2和MMP-2在乳腺癌組織中的表達(dá)及臨床意義[J]. 臨床腫瘤學(xué)雜志, 2012, 17(9):814-817.
16. Indraccolo S, Minuzzo S, Masiero M, et al. Cross-talk between tumor and endothelial cells involving the Notch3-Dll4 interaction marks escape from tumor dormancy[J]. Cancer Res, 2009, 69(4):1314-1323.
17. Hoey T, Yen WC, Axelrod F, et al. DLL4 blockade inhibitstumor growth and reduces tumor-initiating cell frequency[J]. Cell Stem Cell, 2009, 5(2):168-177.
18. Kajiyama H, Shibata K, Terauchi M, et al. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells[J]. Int J Oncol, 2007, 31(2):277-283.
19. Shah AN, Summy JM, Zhang J, et al. Development and characterization of gemcitabine-resistant pancreatic tumor cells[J]. Ann Surg Oncol, 2007, 14(12):3629-3637.
20. Wang Z, Azmi AS, Ahmad A, et al. TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer:involvement of Notch-1 signaling pathway[J]. Cancer Res, 2009, 69(7):2757-2765.
21. Min C, Eddy SF, Sherr DH, et al. NF-kappaB and epithelial to mesenchymal transition of cancer[J]. J Cell Biochem, 2008, 104(7):733-744.
22. Rye PD, Stigbrand T. Interfering with cancer:a brief outline of advances in RNA interference in oncology[J]. Tumour Biol, 2004, 25(5-6):329-336.
23. Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894):244-251.
24. 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.
25. 劉啟志, 王烈. siRNA表達(dá)載體對肝素酶基因的沉默作用[J]. 中國普外基礎(chǔ)與臨床雜志, 2010, 17(2):142-146.
  1. 1. Patel NS, Li JL, Generali D, et al. Up-regulation of delta-like 4 ligand in human tumor vasculature and the role of basal expression in endothelial cell function[J]. Cancer Res, 2005, 65(19):8690-8697.
  2. 2. Benedito R, Trindade A, Hirashima M, et al. Loss of notch signalling induced by Dll4 causes arterial calibre reduction by increasing endothelial cell response to angiogenic stimuli[J]. BMC Dev Biol, 2008, 8(16):117.
  3. 3. Li JL, Sainson RC, Shi W, et al. Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo[J]. Cancer Res, 2007, 67(23):11244-11253.
  4. 4. Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic protein humansurvivin is a direct inhibitor of caspase-3 and -7[J]. Biochemistry,2001, 40(4):1117-1123.
  5. 5. 魏愛英, 張慶柱, 張捷. 多西他賽誘導(dǎo)乳腺癌Bcap37細(xì)胞凋亡作用的研究[J]. 中華腫瘤防治雜志, 2006, 13(18):1384-1386.
  6. 6. Siekmann AF, Lawson ND. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries[J]. Nature, 2007, 445(7129):781-784.
  7. 7. Scehnet JS, Jiang W, Kumar SR, et al. Inhibition of Dll4-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion[J]. Blood, 2007, 109(11):4753-4760.
  8. 8. Hellström M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis[J]. Nature, 2007, 445(7129):776-780.
  9. 9. Segarra M, Williams CK, Sierra Mde L, et al. Dll4 activation of Notch signaling reduces tumor vascularity and inhibits tumor growth[J]. Blood, 2008, 112(5):1904-1911.
  10. 10. Dufraine J, Funahashi Y, Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms[J]. Oncogene, 2008, 27(38):5132-5137.
  11. 11. Harrington LS, Sainson RC, Williams CK, et al. Regulation of multiple angiogenic pathways by Dll4 and Notch in humanumbilical vein endothelial cells[J]. Microvasc Res, 2008, 75(2):144-154.
  12. 12. Zang S, Ji CH,Qu X, et al. A study on Notch signaling in humanbreast cancer[J]. Neoplasma, 2007, 54(4):304-310.
  13. 13. Jubb AM, Soilleux EJ, Turley H, et al. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer[J]. Am J Pathol, 2010, 176(4):2019-2028.
  14. 14. 王群, 余明華, 王耕, 等. DLL4和S100A4在不同分子亞型乳腺癌組織中的表達(dá)及意義[J]. 中國普外基礎(chǔ)與臨床雜志, 2012, 19(9):46-50.
  15. 15. 王群, 余明華, 王耕, 等. DLL4、COX-2和MMP-2在乳腺癌組織中的表達(dá)及臨床意義[J]. 臨床腫瘤學(xué)雜志, 2012, 17(9):814-817.
  16. 16. Indraccolo S, Minuzzo S, Masiero M, et al. Cross-talk between tumor and endothelial cells involving the Notch3-Dll4 interaction marks escape from tumor dormancy[J]. Cancer Res, 2009, 69(4):1314-1323.
  17. 17. Hoey T, Yen WC, Axelrod F, et al. DLL4 blockade inhibitstumor growth and reduces tumor-initiating cell frequency[J]. Cell Stem Cell, 2009, 5(2):168-177.
  18. 18. Kajiyama H, Shibata K, Terauchi M, et al. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells[J]. Int J Oncol, 2007, 31(2):277-283.
  19. 19. Shah AN, Summy JM, Zhang J, et al. Development and characterization of gemcitabine-resistant pancreatic tumor cells[J]. Ann Surg Oncol, 2007, 14(12):3629-3637.
  20. 20. Wang Z, Azmi AS, Ahmad A, et al. TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer:involvement of Notch-1 signaling pathway[J]. Cancer Res, 2009, 69(7):2757-2765.
  21. 21. Min C, Eddy SF, Sherr DH, et al. NF-kappaB and epithelial to mesenchymal transition of cancer[J]. J Cell Biochem, 2008, 104(7):733-744.
  22. 22. Rye PD, Stigbrand T. Interfering with cancer:a brief outline of advances in RNA interference in oncology[J]. Tumour Biol, 2004, 25(5-6):329-336.
  23. 23. Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894):244-251.
  24. 24. 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.
  25. 25. 劉啟志, 王烈. siRNA表達(dá)載體對肝素酶基因的沉默作用[J]. 中國普外基礎(chǔ)與臨床雜志, 2010, 17(2):142-146.