Gu Cong-Min, Zhu You-Kai, Ma Yi-Hui, Zhang Meng, Liao Bing, Wu Hong-Yang, Lin Han-Liang
Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
Neoplasma. 2006;53(3):206-12.
Survivin is the smallest member of mammalian IAP (inhibitor of apoptosis) family. It is ubiquitous during embryonic development but is not expressed in normal post-natal tissues, except the thymus, colonic epithelial cells and CD34+ hematopoietic stem cells. However, its expression is upregulated during neoplastic transformation in both solid organ and hematological malignancies, including leukemia and lymphoma. In this study, we used RNA interference with short hairpin RNA (shRNA) technique to inhibit survivin expression in a Burkitt's lymphoma cell line Raji and validated its effects on apoptosis and cell proliferation. A survivin-shRNA expression vector were constructed and introduced into Raji cells. Expression of survivin mRNA and protein was assessed by RT-PCR and western blot analysis. Apoptosis index of transfected cells was quantified by flow cytometry and cell proliferation was enumerated by trypan blue exclusion. In Raji cells treated with survivin-shRNA expression vector, survivin mRNA levels were significantly reduced by 67.14% (transient transfection) and 64.28% (stable transfection) respectively, compared with control-shRNA treated group and PBS treated group (p<0.05). The levels of survivin protein were significantly reduced by 62.50% (transient transfection) and 60.93% (stable transfection), compared with the two control groups (p<0.05). Apoptosis index was significantly increased during transient transfection and stable transfection, respectively 31.20+/-2.45% and 29.40+/-1.72% (p<0.05). Survivin-shRNA inhibited the proliferation of Raji cells of stable transfection. In conclusion, the vector-based survivin-shRNA can effectively reduce the expression of survivin gene and induce apoptosis and growth inhibition of transfected Raji cells. We suggest that survivin can be regarded as an ideal target for new anticancer intervention of NHL.
存活素是哺乳动物凋亡抑制蛋白（IAP）家族中最小的成员。它在胚胎发育过程中普遍存在，但在出生后的正常组织中不表达，除了胸腺、结肠上皮细胞和CD34+造血干细胞。然而，在实体器官肿瘤和血液系统恶性肿瘤（包括白血病和淋巴瘤）的肿瘤转化过程中，其表达会上调。在本研究中，我们使用短发夹RNA（shRNA）技术进行RNA干扰，以抑制伯基特淋巴瘤细胞系Raji中的存活素表达，并验证其对细胞凋亡和增殖的影响。构建了一个存活素-shRNA表达载体并将其导入Raji细胞。通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析评估存活素mRNA和蛋白质的表达。通过流式细胞术对转染细胞的凋亡指数进行定量，并通过台盼蓝排斥法计算细胞增殖情况。与对照-shRNA处理组和PBS处理组相比，用存活素-shRNA表达载体处理的Raji细胞中，存活素mRNA水平分别显著降低了67.14%（瞬时转染）和64.28%（稳定转染）（p<0.05）。与两个对照组相比，存活素蛋白水平分别显著降低了62.50%（瞬时转染）和60.93%（稳定转染）（p<0.05）。在瞬时转染和稳定转染过程中，凋亡指数分别显著增加，为31.20±2.45%和29.40±1.72%（p<0.05）。存活素-shRNA抑制了稳定转染的Raji细胞的增殖。总之，基于载体的存活素-shRNA可有效降低存活素基因的表达，并诱导转染的Raji细胞凋亡和生长抑制。我们认为存活素可被视为非霍奇金淋巴瘤新的抗癌干预的理想靶点。
