【摘要】 目的观察miR-20b在人骨肉瘤细胞系中的表达及对Saos-2细胞增殖、侵袭能力的影响,探讨其机制。方法采用qRT-PCR法检测人骨肉瘤细胞系Saos-2、U20S、143B及人正常成骨细胞系hFOB1.19中miR-20b。将Saos-2细胞分成miR-20b沉默组和阴性对照组,利用Lipofectamine^TM 2000分别转染miR-20b干扰序列miR-20b inhibitor和阴性对照序列scramble,采用qRT-PCR法检测转染后两组细胞中miR-20b,采用MTT法检测转染后两组细胞培养0、24、48、72、96、120 h时的光密度值（OD值,以OD值表示细胞增殖能力）,采用Transwell实验观察转染后两组细胞穿膜细胞数（以穿膜细胞数表示细胞侵袭能力）,采用Western blotting法检测转染后两组细胞PTEN蛋白。结果人骨肉瘤细胞系Saos-2、U20S、143B及人正常成骨细胞系hFOB1.19中miR-20b的相对表达量分别为3.31±0.26、2.57±0.23、2.03±0.21和1.00±0.02,人骨肉瘤细胞系Saos-2、U20S、143B中miR-20b的相对表达量与正常成骨细胞系hFOB1.19相比,P均<0.01。miR-20b沉默组Saos-2细胞中miR-20b相对表达量为0.12±0.01,阴性对照组为1.00±0.03,两组相比,P<0.01。miR-20b沉默组培养0、24、48、72、96、120 h时的OD值分别为0.29±0.02、0.39±0.03、0.62±0.06、0.89±0.07、1.34±0.11、2.02±0.17,阴性对照组培养0、24、48、72、96、120 h时的OD值分别为0.28±0.02、0.49±0.05、1.05±0.08、1.63±0.12、2.57±0.19、3.69±0.25,两组细胞培养48、72、96、120 h时的OD值相比,P均<0.05。miR-20b沉默组穿膜细胞数为（32.5±3.7）个,阴性对照组穿膜细胞数为（73.6±6.8）个,两组相比,P<0.01。miR-20b沉默组细胞PTEN蛋白相对表达量为2.37±0.15,阴性对照组细胞PTEN蛋白相对表达量为1.00±0.02,两组相比,P<0.01。结论 miR-20b在人骨肉瘤细胞系中高表达;沉默miR-20b可抑制人骨肉瘤细胞系Saos-2的增殖和侵袭能力,其机制可能与上调PTEN蛋白表达有关。更多还原

【Abstract】 Objective To explore the expression of miR-20 b in human osteosarcoma cells and the effect of miR-20 b on the cell proliferation and invasion, as well as its molecular mechanism in Saos-2 cell line. Methods Fluorescent quantitative PCR(qPCR) was used to assess the expression levels of miR-20 b in the osteosarcoma cells lines(Saos-2, U2 OS, and 143 B) and human normal osteoblast cell line hFOB1.19. The Saos-2 cell line was divided into two groups: the miR-20 b inhibitor group and NC group, which were transfected with miR-20 b inhibitor and miR-20 b scramble by LipofectamineTM 2000. The miR-20 b in two groups after transfection was detected by qRT-PCR. MTT was used to detect the optical density value(OD value, with OD value indicating cell proliferation ability) at 0, 24, 48, 72, 96 and 120 h after transfection. Transwell assay was used to observe the number of cells penetrating the membrane(with the number of cells penetrating the membrane indicating cell invasive ability) after transfection. Western blotting was used to detect phosphatase and tensin homolog deleted on chromosome ten(PTEN) expression of the two groups after transfection. Results The relative expression of miR-20 b in human osteosarcoma cell lines Saos-2, U2 OS, 143 B and human normal osteoblast line hFOB1.19 was 3.31±0.26, 2.57±0.23, 2.03±0.21 and 1.00±0.02, respectively. The relative expression of miR-20 b in human osteosarcoma cell lines Saos-2, U2 OS and 143 B was lower than that in the normal osteoblast line hFOB1.19(P<0.01). The relative expression of miR-20 b was 0.12±0.01 in Saos-2 cells of miR-20 b silence group and 1.00±0.03 in negative control group, P<0.01. The OD values at 0, 24, 48, 72, 96, 120 h were 0.29±0.02, 0.39±0.03, 0.62±0.06, 0.89±0.07, 1.34±0.11, 0.02±0.17, respectively, in the miR-20 b silence group, versus 0.28±0.02, 0.49±0.05, 1.05±0.08, 1.63±0.12, 2.57±0.19,and 3.69±0.25, respectively, in the negative control group, with statistically significant difference(all P<0.05). The number of transmembrane cells in the miR-20 b silence group was 32.5±3.7, and that in negative control group was 73.6±6.8, with statistically significant difference(P<0.01). The relative expression of PTEN protein was 2.37±0.15 in miR-20 b silence group and 1.00±0.02 in negative control group, with statistically significant difference(P<0.01). Conclusion MiR-20 b is highly expressed in the osteosarcoma cell line, silencing the expression of miR-20 b can inhibit the proliferation and invasion of osteosarcoma cells, possibly by up-regulating the PTEN protein expression.更多还原