薛栋　靳继海　夏修良　王建强　王雯　成丕光　李新军　巩本刚

【摘要】 目的：探討Livin对甲状腺乳头状癌TPC-1细胞增殖、凋亡及侵袭的影响以及可能的调控分子机制。方法：构建靶向Livin基因的慢病毒shRNA载体，转染甲状腺乳头状癌细胞株TPC-1，实验分为干扰组、阴性对照组和空白组。应用RT-PCR和Western印迹检测转染Livin-shRNA后细胞Livin mRNA和蛋白表达水平的变化。应用MTT、流式细胞仪、Western印迹与Transwell侵袭实验检测Livin对甲状腺乳头状癌细胞增殖、凋亡及侵袭的影响。并接种于裸鼠模型中，进一步确定Livin在体内对甲状腺乳头状癌细胞的影响。结果：慢病毒Livin-shRNA载体成功抑制TPC-1细胞中Livin mRNA和蛋白的表达；转染Livin-shRNA后TPC-1细胞的增殖能力受到明显抑制（P<0.01），转染Livin-shRNA组、阴性对照组和空白组的凋亡率分别为（15.72±0.21）%，（4.54±0.13）%和（4.87±0.22）%，三组比较差异均有统计学意义（P<0.01）。干扰组穿膜细胞数明显少于阴性对照组和空白组（P<0.05）。与空白组和阴性对照组相比，干扰组细胞AKT、p-AKT及PDK1蛋白表达量均下调（P<0.05），而PTEN蛋白明显上调（P<0.05）。甲状腺乳头状癌裸鼠模型结果表明干扰组肿瘤的生长速度、肿瘤质量[（1.11±0.08）g]明显小于空白组[（1.90±0.15）g]和阴性对照组[（2.10±0.12）g]。结论：沉默Livin基因能抑制甲状腺乳头状癌细胞的增殖、侵袭及体内生长，促使甲状腺乳头状癌细胞凋亡的发生，可能是通过抑制PI3K-AKT信号转导通路来发挥作用。

【关键词】 甲状腺肿瘤； Livin； RNA干扰； 侵袭； 迁移； 动物模型

Inhibition on Proliferation and Invasion of Human Thyroid Papillary Cancer Cells by RNA Interference Livin Gene Expression and Its Possible Molecular Mechanism/XUE Dong，JIN Ji-hai，XIA Xiu-liang，et al.//Medical Innovation of China，2017，14（14）：004-008

【Abstract】 Objective：To investigate the effect of Livin on the proliferation，apoptosis and invasion of human thyroid papillary cancer cell line TPC-1，and its possible molecular mechanism.Method：Livin was silenced by using RNA interference.In this experiment，three groups were designed：the experimental group，the negative control group and the blank control group.The expression of Livin at mRNA and protein levels were detected by reverse transcription PCR and Western blot respectively.MTT，FCM，transwell and Western blot were utilized to analyze the result and specific molecular mechanism of Livin on TPC-1 cancer cells by transfected.The expressions of AKT，p-AKT，PDK1 and PTEN in TPC-1 cells were detected after transfection.Nude mice models inoculated TPC-1 cells were used to further clarify the effect of Livin on thyroid papillary cancer cells in vivo.Result：The stable Livin silencing cells line was successfully established.The expression of Livin at mRNA and protein levels was reduced significantly（P<0.05），leading to the inhibition of cell proliferation in the experimental group compared with the negative control group and the blank control group（P<0.05）.The apoptosis index of tumor cells in the above three groups were （15.72±0.21）%，（4.54±0.13）% and （4.87±0.22）%，respectively.Compared to the negative control group and the blank control group，the experimental group was significantly increased（P<0.01）.The transwell chamber assay showed that transmembrane cells of the experimental group were less than the negative control group and the blank control group after incubation 48 hours.Meanwhile，western blot analysis revealed that cells with stably knock-down of Livin showed decreased expression levels of AKT，p-AKT and PDK1 compared to the negative control group and the blank control group，but PTEN was highly expressed（P<0.05）.Nude mice models indicated that the growth and weight （1.11±0.08）g of the experimental group were significantly lower than （1.90±0.15）g of the blank control group and （2.10±0.12）g of the negative control group（P<0.05）.Conclusion：Scilencing the expression of Livin can inhibit the proliferation of thyroid papillary cancer cell and growth in vivo，and then accelerate the apoptosis of TPC-1 cancer cells by cutting down the activity of PI3K-AKT signal pathway.

【Key words】 Thyroid neoplasm； Livin； RNA interference； Invasion； Migration； Animal model

First-authors address：The Peoples Hospital of Binzhou，Binzhou 256610，China

doi：10.3969/j.issn.1674-4985.2017.14.002

甲状腺乳头状癌的发病率正逐年上升。Livin与肿瘤的发生、恶性侵袭和转移具有密切相关性[1-2]。笔者前期研究结果已发现Livin在甲状腺乳头状癌组织中呈高表达状态，并且Livin与肿瘤的临床分期、淋巴结转移都明显有关[3]。磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/AKT）信号转导通路在肿瘤细胞的侵袭转移过程之中发挥着重要的作用，3-磷酸肌醇依赖性蛋白激酶1（PDK1）及第10号染色体缺失的磷酸酶及张力蛋白同源的基因（PTEN）是该信号通路的重要基因[4]。新近的研究结果表明，Livin還能够明显促进肺癌及口腔鳞癌细胞的增殖、局部侵袭和转移[5-6]。

本研究通过构建慢病毒载体介导的shRNA沉默甲状腺乳头状癌细胞中Livin基因，观察Livin对甲状腺乳头状癌TPC-1细胞的生物学行为的影响，构建裸鼠成瘤模型，分析Livin促进甲状腺乳头状癌细胞生长及侵袭转移的作用机理。……