RNA干扰Livin基因对甲状腺乳头状癌的增殖及侵袭抑制作用及其分子机制

Inhibition on Proliferation and Invasion of Human Thyroid Papillary Cancer Cells by RNA Interference Livin Gene Expression and Its Possible Molecular Mechanism

目的:探讨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sh RNA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-sh RNA后细胞Livin m RNA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-sh RNA载体成功抑制TPC-1细胞中Livin m RNA和蛋白的表达;转染Livin-sh RNA后TPC-1细胞的增殖能力受到明显抑制(P<0.01),转染Livin-sh RNA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。

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 respeetively.MTT, FCM, transwell and Western blot were utilized to analyze the result and specific molecular mechanism of Livin on TPC-1 cancer cells by transfeeted. 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.