Hao H, Xin T, Nancai Y, Yanxia W, Qian L, Wei M, Yandong Y, Hanju H
Department of Pathogenic Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Int J Gynecol Cancer. 2008 Jan-Feb;18(1):36-42. doi: 10.1111/j.1525-1438.2007.00955.x. Epub 2007 Apr 26.
Proliferating cell nuclear antigen (PCNA) is an important protein for DNA polymerase delta in the nucleus, and shown to have a fundamental role in cellular proliferation. It is overexpressed to support cell growth in cervical carcinoma. To study its role in stress response, we design and use short hairpin RNA (shRNA) to inhibit PCNA expression in HeLa cells and validate its effect on cell proliferation. In this study, three PCNA-shRNA expression vectors are constructed and introduced into HeLa cells, and the cell cycle is analyzed by flow cytometry. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay), and caspase cleavage is studied also. Expression of PCNA is assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it is found that expression of PCNA decreased in shRNA-transfected cells, downregulation of PCNA inhibit cell growth and induce apoptosis in HeLa cells. PCNA downregulation also increase cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in HeLa cells effectively and, therefore, could be used as a new potential anticancer tool for therapy of human cervical carcinoma.
增殖细胞核抗原（PCNA）是细胞核中DNA聚合酶δ的一种重要蛋白质，在细胞增殖中发挥着重要作用。它在宫颈癌中过度表达以支持细胞生长。为了研究其在应激反应中的作用，我们设计并使用短发夹RNA（shRNA）抑制HeLa细胞中PCNA的表达，并验证其对细胞增殖的影响。在本研究中，构建了三种PCNA-shRNA表达载体并导入HeLa细胞，通过流式细胞术分析细胞周期。通过单细胞凝胶电泳试验（彗星试验）检测凋亡细胞，并研究半胱天冬酶的裂解情况。通过实时逆转录-聚合酶链反应和蛋白质免疫印迹分析评估PCNA的表达。在用编码shRNA的质粒进行瞬时转染后，发现shRNA转染的细胞中PCNA表达降低，PCNA的下调抑制HeLa细胞的生长并诱导其凋亡。PCNA的下调还增加了G0-G1期的细胞数量。总之，我们的研究结果表明，shRNA可以有效抑制HeLa细胞中的DNA复制并诱导其凋亡，因此可作为治疗人类宫颈癌的一种新的潜在抗癌工具。
