NEK2对肝癌细胞株HepG2迁移侵袭的影响及其机制的研究

Effects of NEK2 on migration and invasion of HepG2 in hepatoma cells and the related mechanism

目的探讨NEK2基因对人肝癌细胞株Hep G2迁移侵袭能力的影响及其机制。方法通过实时荧光定量PCR及Western blot法检测4种人肝癌细胞系Hep G2、Huh7、SMMC、7402中NEK2的表达水平,并选用正常肝细胞株LO2作为参考。设计si RNA序列转染Hep G2细胞,将Hep G2细胞分为3组:空白对照组(细胞未做任何处理)、阴性对照组(细胞转染无义序列)及干扰组(细胞转染NEK2-si RNA序列)。观察转染前后Hep G2细胞中的NEK2表达水平变化。利用Transwell小室观察转染前后细胞迁移及侵袭能力的改变。应用蛋白印迹技术检测MMP2、MMP9、TIMP-1蛋白的表达情况。结果 5种细胞株中,NEK2在Hep G2细胞中的表达量最高,与LO2细胞比较差异有高度统计学意义(P<0.01)。Hep G2细胞转染NEK2-si RNA后,与空白对照组及阴性对照组比较,干扰组NEK2的表达水平显著降低,差异均有统计学意义(P<0.05或P<0.01)。Transwell小室检测细胞迁移,空白对照、阴性对照的穿膜细胞数分别为(415.00±6.36)、(411.75±9.90),与干扰组比较(99.50±7.07),差异均有高度统计学意义(P<0.01)。Transwell小室检测细胞侵袭,空白对照组、阴性对照组的穿膜细胞数分别为(220.50±6.40)、(219.75±3.54),与干扰组(38.50±3.54)比较,差异均有高度统计学意义(P<0.01);转染NEK2-si RNA 48 h后,Hep G2细胞中TIMP-1蛋白表达较空白对照组、阴性对照组明显上调,而MMP2及MMP9蛋白表达明显下调,差异均有统计学意义(P<0.05)。结论 NEK2可能通过TIMP-1、MMP2及MMP9调控人肝癌Hep G2细胞的侵袭转移。

Objective To identify the effects of NEK2 on migration and invasion and the related mechanism in human liver cancer cells of HepG2. Methods Real-time PCR and Western blot were used to detect the NEK2 expression in HepG2, Huh7, SMMC, 7402 cell lines and L02 as the control. To design siRNA sequence to transfect into the HepG2 cells. The experiment was divided into three groups： blank control （cells with no disposes）; negative control （cells were transfeeted with meaningless sequence） and interference group （cell were transfected with NEK2-siRNA sequence）. The expression level of NEK2 was observed before and after transfection. The transwell chambers was employed to detect a- bility of migration and invasion of HepG2 cells. Western blot approach was used to detect the expression levels of MMP2, MMP9 and TIMP-1. Results In the five kinds of cell lines, the expression of NEK2 was the highest in HepG2 cells, compared with the LO2 cell the difference was significant （P 〈 0.01）. After transfection of HepG2 cells with NEK2-siRNA, the expression level of NEK2 was attenuated, compared with blank control group and negative control group, the differences were statistically significant （P 〈 0.05 or P 〈 0.01）. Transwell chamber was used to detect cell migration capacity, the transmembrane cell numbers in the blank control group, negative control group were （415.00~ 6.36）, （411.75＋9.90）, compared with the NEK2-siRNA group （99.50±7.07）, the differences were statistically significant （P 〈 0.01）. Transwell chamber was used to detect cell invasion capacity, the transmembrane cell numbers in the blank control group, negative control group were （220.50±6.40）, （219.75 ±3.54）, compared with the NEK2-siRNA group （38.50±3.54）, the differences were statistically significant （P 〈 0.01）. The protein expression of TIMP-1 in the NEK2- siRNA group was increased significantly, and the protein expression of MMP2 and MMP9 was decreased, compared with that in the blank control group and negative control group, the differences were significant （P 〈 0.05）. Conclusion NEK2 may regulate invasion and migration of HepG2 cells via TIMP-1, MMP2 and MMP9.