Construction of shRNA Eukaryotic Expression Vector Targeting Human Papillomavirus Type 16 E6/E7 Gene and Its Effects on Proliferation and Migration of SiHa Cells
-
摘要: 目的 构建针对人乳头瘤病毒16型E6/E7基因的shRNA真核表达载体，获得稳定表达干扰质粒的人宫颈癌SiHa细胞系并探讨其对SiHa细胞增殖及迁移能力的影响。方法 合成3条特异性干扰HPV16 E6/E7基因的shRNA片段并定向插入psilencer 2.1-U6 hygro载体，构建重组质粒psilencer 2.1-U6 hygro-shE6/E7并转染入人宫颈癌SiHa细胞， Real- time PCR检测转染后细胞E6/E7 mRNA的表达，选择沉默效应最好的重组质粒并用潮霉素B稳筛，获得稳定表达重组质粒的SiHa细胞，并用real time-PCR和Western blot方法进行鉴定；分别运用CCK-8细胞增殖实验和细胞划痕愈合实验检测细胞的增殖及迁移能力。结果 测序证实针对HPV16 E6/E7的shRNA真核表达载体psilencer 2.1-U6 hygro-shE6/E7 构建正确；转染psilencer 2.1-U6 hygro-shE6/E7的SiHa细胞HPV16 E6/E7表达明显受抑，同时其增殖及迁移能力也明显受抑制。结论 psilencer 2.1-U6 hygro-shE6/E7真核表达载体的成功构建并获得稳定沉默表达HPV16 E6/E7的人宫颈癌SiHa细胞系，证实沉默表达HPV16 E6/E7的SiHa细胞增殖及迁移能力会明显受到抑制，这为进一步研究HPV 16 E6/E7基因在宫颈癌发生发展过程中的功能奠定了实验基础。
-
关键词:
- 人乳头瘤病毒16型E6/E7 /
- 载体构建 /
- 细胞增殖 /
- 细胞迁移 /
- 宫颈癌
Abstract: Objective To construct the short hairpin RNA(shRNA) eukaryotic expressing vector targeting human papillomavirus type 16 E6/E7 to establish stable transfected SiHa cell line and to observe its effects on cell proliferation and migration. Methods Three pairs of shRNA fragments which could specifically inhibit HPV16 E6/E7 genes were synthesized and cloned into the shRNA vector psilencer2.1-U6 hygro. The recombinant vectors were transfected into SiHa cells by Lipo2000 and E6/E7 mRNA expression was validated by real-time PCR. The stable psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line was selected with hygromycin B and validated by real-time PCR and Western blot.The CCK-8 cell proliferation assay and cell scratch wound healing assay were performed to examine cell proliferation and migration. Results Sequencing suggested that shRNA eukaryotic expression vector targeting HPV16 E6/E7 gene, psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line, was established successfully with a signifi cantly inhibition of HPV16 E6/E7 expression, cell proliferation and migration. Conclusion Inactivation of HPV16 E6/E7 in cervical cancer cells could reduce cell proliferation and migration. -
-
[1] Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation[J]. Nat Rev Cancer,2010,10(8):550-60. [2] Muñoz N, Bosch FX, de Sanjosé S,et a1.Epidemioologic classification of human papillomavirus types associated with cervical cancer [J]. N Engl J Med,2003,348(6):518-27. [3] McCloskey R,Menges C,Friedman A,et al.Human papillomavirus type 16 E6/E7 upregulation of nucleophosmin is important for proliferation and inhibition of differentiation. [J]. J Virol,2010,84(10):5131-9. [4] Hoenil Jo, Jae Weon Kim. Implications of HPV infection in uterine cervical cancer[J]. Cancer Therapy,2005,3:419-34. [5] McLaughlin-Drubin ME,Münger K.Oncogenic activities of human papillomaviruses[J]. Virus Res,2009,143(2):195-208. [6] Corusi? A, Skrgati? L, Mahovli? V,et al.Cervical cancer as a public health issue--what next? [J]. Coll Antropol, 2010, 34(1):301-7. [7] Woodman CB,Collins SI,Young LS.The natural history of cervical HPV infection: unresolved issues[J]. Nat Rev Cancer,2007,7(1):11-22. [8] Berghella V,Figueroa D,Szychowski JM,et al.17-alphahydroxyprogesterone caproate for the prevention of preterm birth in women with prior preterm birth and a short cervical length[J]. Am J Obstet Gynecol,2010,202(4):351.e1-6. [9] Massimi P, Shai A, Lambert P,et al.HPV E6 degradation of p53 and PDZ containing substrates in an E6AP null background[J]. Oncogene,2008,27(12):1800-4. [10] American College of Obstetricians and Gynecologists. ACOG Committee Opinion. Use of progesterone to reduce preterm birth[J]. Obstet Gynecol,2003,102(5 Pt 1):1115-6. [11] Kisseljov F, Sakharova O, Kondratjeva T. Cellular and molecular biological aspects of cervical intraepithelial neoplasia[J]. Int Rev Cell Mol Biol,2008,271:35-95. [12] Feng SY,Zhang YN,Liu JG.Risk factors and prognosis of nodepositive cervical carcinoma[J].Ai Zheng,2005,24(10):1261-6. [冯 淑瑜,张彦娜,刘建刚.宫颈癌淋巴结转移的高危因素及预后分 析[J] 癌症,2005,24(10):1261-6.] [13] Hellner K,Mar J,Fang F,et al.HPV16 E7 oncogene expression in normal human epithelial cells causes molecular changes indicative of an epithelial to mesenchymal transition[J].Virology,2009,391 (1):57-63.
计量
- 文章访问数: 1681
- HTML全文浏览量: 466
- PDF下载量: 717
下载:
