RNA干扰技术是通过小的双链RNA高效、特异的阻断体内特定基因的表达,促使mRNA降解,诱使细胞表现出特定基因缺失表达的技术,是目前沉默某些致病基因功能的有效手段。本实验通过构建针对Survivin基因的siRNA真核表达载体转入人喉癌HEP2细胞中。结果显示:阴性对照序列表达载体转染HEP2细胞后对Survivin表达无影响;而pRNATSurvivin转染HEP2细胞后显著抑制了Survivin的mRNA及蛋白表达(P<0.01),其中 pRNATSurvivin组中 Survivin mRNA表达水平比空白对照组最多下降了75.43%,而蛋白表达量比空白对照组最多下降了79.27%,提示本研究构建的siRNA真核表达载体的确能够在细胞内持续地表达siRNA,高效、特异的抑制Survivin基因的表达。本实验通过MTT法检测转染前后化疗药物抑制细胞生长的情况表明:稳定转染Survivin siRNA 真核表达载体后5FU、优福定对细胞生长抑制作用增加。其中在转染Survivin siRNA和5FU联合作用下细胞生长抑制率最高达到(67.71±4.58)%,转染Survivin siRNA和优福定联合作用下细胞生长抑制率最高达到(74.54±4.43)%,相比未转染Survivin siRNA的空白对照组和阴性对照组有明显提高(P<0.01)。以上研究结果显示:用RNA干扰技术抑制Survivin表达造成肿瘤细胞对化疗药物的耐受性降低。
过表达Survivin可能并非激活肿瘤细胞化疗药物耐受性和抗凋亡能力所必需,但抑制Survivin表达可作为有效的调控信号,提高肿瘤细胞化疗敏感性, 导致肿瘤细胞凋亡。本研究通过RNA干扰技术的应用为基于调控细胞凋亡的生物治疗与化疗联合治疗喉癌提供了新的实验依据。
【参考文献】
[1]Reed JC.Bcl2:prevention of apoptosis as a mechanism of drug resistance[J].Hematol Oncol Clin North Am,1995,9(2):451-473.
[2]Li F.Survivin study:what is the next wave?[J].J Cell Physiol,2003,197(1):8-29.
[3]Nishimura G,Tsukuda M,Mikami Y,et al.Efficacy of concurrent chemoradiotherapy for T1 and T2 laryngeal squamous cell carcinoma regarding organ preservation[J].Anticancer Res,2009,29(2):661-666.
[4]Rengan R,Pfister DG,Lee NY,et al.Longterm neck control rates after complete response to chemoradiation in patients with advanced head and neck cancer[J].Am J Clin Oncol,2008,31(5):465-469.
[5]Schmitt CA.Senescence, apoptosis and therapycutting the lifelines of cancer[J].Nat Rev Cancer,2003,3(4):286-295.
[6]Chen N,Chen CC,Lau LF.Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin alpha 6 beta 1 and cell surface heparan sulfate proteoglycans[J].J Biol Chem,2000,275(32):24 953-24 961.
[7]Spaulding B,Pan D,Ghadersohi A,et al.Characterization of the 12C4 survivin monoclonal antibody and insight into the expression of survivin in human adult tissues[J].Histopathology,2006,49(6):622-633.
[8]Marioni G,D′Alessandro E,Bertolin A,et al.Survivin multifaceted activity in head and neck carcinoma:current evidence and future therapeutic challenges[J].Acta Otolaryngol,2009,25:1-6.