摘要
背景:
肺癌是全球最常见的恶性肿瘤之一,近年来一些研究发现Livin在肺癌中表达增高,推测其可能参与了肺癌的发生及进展。本课题通过RNA干扰技术抑制Livin基因表达,观察其对肺腺癌SPC-A-1细胞增殖的影响,并在此基础上进一步行体内实验,探讨沉默Livin基因表达对肺腺癌裸鼠移植瘤生长的影响,观察其能否成为肺癌基因治疗的一个有效靶点。
目的:
利用RNA干扰技术沉默凋亡抑制蛋白基因Livin基因在人肺腺癌细胞株SPC-A-1中的表达,探讨Livin表达沉默后对SPC-A-1裸鼠移植瘤生长的影响。
方法:
以慢病毒为载体,将携带针对Livin基因的短发夹RNA(Short hairpin RNA,shRNA)及无关序列片段的shRNA转染SPC-A-1细胞,转染成功后将慢病毒转染Livin shRNA的SPC-A-1细胞(实验组)、慢病毒转染无关序列的SPC-A-1细胞(阴性载体对照组)及未转染任何载体的SPC-A-1细胞(空白对照组)分别接种至裸鼠右腋皮下,构建SPC-A-1裸鼠皮下移植瘤模型,观察各组细胞在裸鼠体内的成瘤时间、成瘤率、测量瘤体体积、瘤体重量,并绘制肿瘤生长曲线及计算抑瘤率;RT-PCR、免疫组织化学法分别检测Livin基因及蛋白表达情况,原位末端转移酶标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测移植瘤组织凋亡情况。
结果:
1、与空白对照、阴性载体对照组相比,实验组裸鼠移植瘤成瘤时间晚,瘤体生长缓慢,体积抑瘤率达(59.5±3.4)%(P<0.05),瘤重也明显减轻,瘤重抑瘤率达(71.1±5.6)% (P<0.01)。
2、实验组LivinαmRNA表达水平为(37.2±1.6)%,LivinβmRNA表达水平为(29.4±1.1)%,均明显低于空白对照组及阴性载体对照组(P值均<0.05)。
3、实验组Livin蛋白表达水平为(15..3±2.8)%,明显低于空白组(51.3±2.1)%和阴性载体对照组(52.5±2.5)%(P<0.05)。
4、实验组瘤组织细胞凋亡明显增多,凋亡率为(35.4±3.2)%,明显高于空白组(5.4±1.3)%和阴性载体对照组(8.6±1.5)%(P<0.05)。
结论:
沉默SPC-A-1细胞中Livin基因表达能有效抑制人肺癌裸鼠移植瘤的生长,并使移植瘤组织细胞凋亡增多,Livin有望成为肺癌治疗的靶点之一。
Background
Lung cancer is one of the most common malignancies worldwide ,Recent studies have suggested that Livin is highly expressed in lung cancer. It may be involved in tumorigenesis and development of lung cancer.In this study,we used RNA interference technology to silence Livin expression,then observed the effects of the proliferation of adenocarcinoma of lung cell SPC-A1, and then we established the tumor-bearing nude mice model of SPC-A-1, explored the possible mechanism in vivo.
Objective:
To explore the effect of proliferation of transplantation tumor induced by SPC-A1 in vivo after silencing Livin gene by RNA interference technology.
Methods:
Livin shRNA was transfected into SPC-A-1 cells by lentivector, then three different nude mice models were established by inoculating different disposal SPC-A1 cells into three nude mice groups subcutaneously,one group were inoculated with blank SPC-A-1 cells(the blank control group), one were inoculated with cells transfected with non-transfected lentivirus vectors ( the negative group), another were inoculated with cells with lentivirus-delivered LivinshRNA(the experimental group). Following on, the formation time,the formation rate, the tumor volume and weight of these tumors were measured in different time points;the curve of tumor growth were described; and the inhibition rate was calculated. Livin gene expression in these tumor tissues were detect by RT-PCR and Inmunohistochemistry assay. Cell apoptosis of tumor tissues was detected by TUNEL assay.
Results:
The study showed a slower tumor growth , a smaller tumor volume and a lighter tumor weight in experimental group than the blank and negative groups (P<0.05). The volume inhibition rate was ( 59.5±3.4 ) %(P<0.05), and the weight inhibition rate was (71.1±5.6)%(P<0.01). LivinаmRNA and LivinβmRNA expression in the experimental group were about (37.2±1.6)%and (29.4±1.1)% respectively by RT-PCR assay, which were significantly lower than two control groups (P<0.05). Livin protein expression level was significantly lower than the blank groupand the negative group too[(15.3±2.8)% versu(s51.3±2.1)% ,(52.5±2.5)% (P<0.05)]. The apoptosis rate by TUNEL assay in the experimental group was significantly higher than these two control groups as well [(35.4±3.2)% versus(5.4±1.3)%and(8.6±1.5)%(P<0.05)].
Conclusions:
The lentivirus-delivered LivinshRNA can inhibite the proliferation of transplantation tumor of lung carcinoma effectively,Livin may be a potent target of gene therapy in lung cancer.
引文
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