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目的探讨膜-细胞骨架联接蛋白Ezrin对骨肉瘤细胞株UMR106成瘤及转移能力的影响。方法为获得较好的干扰效果,针对大鼠Villin2基因(Ezrin编码基因)设计2个发夹式RNA(shRNA)。合成后克隆入载体pGenSil-1,扩增并中量提取质粒,应用脂质体LipofectamineTM 2000转染进UMR106细胞。采用逆转录聚合酶链反应(RT-PCR)和West-ern印迹检测Ezrin的表达。选取效果最好的shRNA转染UMR106细胞并行G418筛选,传代扩增。扩增后细胞原位接种到SD大鼠胫骨结节,并设立正常细胞组和阴性对照组,观察记录肿瘤形成和大鼠存活时间,计数肺表面转移灶数目,镜检定位增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)在原发灶和肺转移灶内的表达。结果①shRNA1干扰作用最明显,下调达90%。②移植时稳定转染细胞数约50%。③实验组与对照组均100%成瘤,成瘤时间正常细胞组为(10.25±1.07)d,阴性对照组为(10.08±0.88)d,实验组为(10.30±1.08)d,三者间没有明显差别(P>0.05);实验组存活时间(42.30±17.25)d明显长于正常细胞组(20.60±11.70)d(P<0.01)和阴性对照组(22.40±7.42)d(P<0.01);实验组肺转移灶数目明显低于正常细胞组(P<0.01)和阴性对照组(P<0.01),分别为(39.40±51.86)个、(124.60±39.78)个和(117.60±36.48)个;阴性对照组中原发灶和肺转移灶内均有EGFP表达,而实验组肺转移灶内未见EGFP表达。结论Ezrin蛋白是骨肉瘤肺转移中的一个关键因子,干扰Ezrin蛋白表达能抑制其肺转移的发生,可以作为预防肺转移的靶点。
Objective To investigate the effect of membrane-cytoskeleton connexin Ezrin on the tumorigenesis and metastasis of osteosarcoma cell line UMR106. Methods To obtain better interference effects, two hairpin RNAs (shRNAs) were designed for the rat Villin2 gene (Ezrin encoding gene). After synthesis, the plasmid pGenSil-1 was cloned into the vector pGenSil-1. The plasmid was amplified and medium-sized. The liposome LipofectamineTM 2000 was used to transfect the plasmid into UMR106 cells. Ezrin expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and West-ern blotting. Select the best shRNA transfected UMR106 cells parallel G418 screening, passage amplification. After inoculation, the cells were inoculated into the tibial tubercle of SD rats, and normal cell group and negative control group were set up. Tumor formation and survival time were recorded. The number of lung metastases, the number of enhanced green fluorescent protein (enhanced green fluorescence protein, EGFP) in primary and lung metastases. Results ① The most obvious interference effect of shRNA1, down 90%. ② transplanted cells stably transfected about 50%. ③ The experimental group and the control group all formed 100% tumor, the time of tumorigenesis was (10.25 ± 1.07) d, the negative control group was (10.08 ± 0.88) d, the experimental group was (10.30 ± 1.08) d, (P> 0.05). The survival time of the experimental group (42.30 ± 17.25) d was significantly longer than that of the normal control group (20.60 ± 11.70) d and (22.40 ± 7.42) d ; The number of lung metastases in the experimental group was significantly lower than that in the normal group (P <0.01) and the negative control group (P <0.01), and were (39.40 ± 51.86), (124.60 ± 39.78) and (117.60 ± 36.48) ; Negative control group, both primary and lung metastases have EGFP expression, but no experimental group of lung metastases EGFP expression. Conclusion Ezrin protein is a key factor in lung metastasis of osteosarcoma. Interfere with Ezrin protein expression can inhibit the occurrence of lung metastasis, which can be used as a target to prevent lung metastasis.