摘要目的 探讨微小RNA(microRNA,rniR)-15a/16-1重组慢病毒对人鼻咽癌CNE-2Z细胞生物特性的影响,并探讨其可能机制.方法 重组慢病毒和空载体慢病毒转染CNE-2Z细胞后筛选绿色荧光蛋白阳性细胞,实时定量PCR (RT-qPCR)检测miR-15a、miR-16-1表达水平；将实验分为对照组、转染组、放射线照射组、转染联合放射线照射组,四甲基偶氮唑蓝法分析各组细胞增殖情况；流式细胞术检测各组细胞凋亡情况；克隆形成实验分析对照组和转染组对放射线敏感性的变化；RT-qPCR、Western blot法检测bcl-2 mRNA及其蛋白的表达水平；分光光度法检测Caspase-2、Caspase-3的活化程度.结果 转染组miR-15a、miR-16-1相对表达量((x)±s,下同)分别为524.80±40.79、466.11±40.96,同对照组比较,差异有统计学意义(t=494.611,t=386.840,P=0.000)；转染联合放射线照射组细胞增殖抑制最明显(F =424.255,P=0.000).对照组、转染组、放射线照射组及转染联合放射线照射组的细胞凋亡率分别为(2.2±1.4)％、(9.6±0.8)％、(2.9±1.1)％、(18.6±0.7)％,差异有统计学意义(F=158.519,P=0.000).转染组同等剂量下(2、4、6、8 Gy)存活分数、平均致死剂量(mean lethal dose,Do)、准阈剂量(quasi-threshold dose,Dq)均低于对照组.二者放射增强比(sensitization enhancement ratios,SER) (Do)、SER(Dq)分别为1.473、1.581.转染组、放射线照射组、转染联合放射线照射组bcl-2 mRNA表达相对量组间比较差异无统计学意义(P＞0.05).转染联合放射线照射组bcl-2蛋白表达下降最明显,转染组次之；对照组、放射线照射组、转染组、转染联合放射线照射组的Caspase-2活化程度分别为0.12 ±0.01、0.24±0.04、0.35±0.02、0.44±0.04(F=115.500,P=0.000),Caspase-3的活化程度分别为0.13±0.01、0.27±0.01、0.43±0.02、0.83±0.06(F =439.921,P=0.000).结论 重组慢病毒LV-miR15a/16-1可提高CNE-2Z细胞中miR-15a、miR-16-1的表达水平,抑制CNE-2Z细胞的增殖；促进CNE-2Z细胞的凋亡,增强CNE-2Z细胞的放射线敏感性.其可能通过抑制CNE-2Z细胞中bcl-2蛋白的表达,促进Caspase-2、Caspase-3的活化以实现对鼻咽癌细胞CNE-2Z生物学特性的调控.

abstractsObjective To study the influence of recombinant lentiviral vector encoding miR-15a/16-1 on biological features of human nasopharyngeal carcinoma CNE-2Z cells and underlying mechanisms.Methods GFP-positive CNE-2Z cells transfected with recombinant lentiviral vector were selected.The experiment was divided into control group,transfected group,radiotherapy group,transfected-radiotherapy group.Cell proliferation was analyzed by MTT.Apoptosis was detected by flow cytometry.Radiotherapy sensitivity of the cells in control group and transfected group was evaluated by colony forming experiment.The expressions of miR-15a,miR-16-1 and bcl-2 mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR).The expression of bcl-2 protein was detected by Western blot.The activation of Caspase-2 and Caspase-3 was evaluated by spectrophotometry.Results Relative expression quantities of miR-15a and miR-16-1 in infected group were 524.80 ±40.79 (t =494.611,P =0.000) and 466.11 ±40.96 (t =386.8,P =0.000),rcspcctively.Thc prolifcration of the ccells in transfected-radiotherapy group was the most obvious,followed by the cells in radiotherapy group and transfected group (F =424.3,P =0.000).The apoptosis rates of control group,transfected group,radiotherapy group and transfectedradiotherapy group were (2.2 ± 1.4)％,(9.6 ± 0.8)％,(2.9 ± 1.1)％,and (18.6 ± 0.7)％respectively(F =158.5,P =0.000).Clonogenic assay showed that the values of SF2,Do (1.473) and Dq (1.581) in transfected group were lower than those in control group.The relative expression levels of bcl-2 mRNA in transfected group,radiotherapy group,and transfected-radiotherapy group had no significant difference (P ＞ 0.05).Decrease in the expression of bcl-2 protein in transfeeted-radiotherapy group was most significantly,followed by that in transfected group.The percentages of activated Caspase-2 in control group,radiotherapy group,transfected group and transfected-radiotherapy group were 0.12 ± 0.01,0.24 ±0.04,0.35 ± 0.02,and 0.44 ± 0.04,respectively (F =115.500,P =0.000).The percentages of activatedCaspase-3 in the groups were 0.13 ±0.01,0.27 ± 0.01,0.43 ±0.02,and 0.83 ± 0.06,respectively (F =439.921,P =0.000).Conclusions Recombinant lentiviral vector LV-miR15a/16-1 could improve the expression of miR-15a and miR-16-1 in CNE-2Z cells,inhibit the proliferation of CNE-2Z cells,promote apoptosis and enhance the sensitivity of the cells to radiotherapy probably by inhibiting bcl-2 expression,activating Caspase-2 and Caspase-3.