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目的骨肉瘤具有局部高度侵袭能力以及快速转移潜能,因而致死率较高。研究指出,miR-300的表达异常与多种肿瘤的发病相关。miR-300对骨肉瘤细胞是否存在调控作用却属未知。本研究探讨miR-300对骨肉瘤细胞Saos-2增殖与凋亡调控的作用。方法将miR-300转染进骨肉瘤细胞Saos-2中,实现miR-300在Saos-2细胞内的高表达。通过荧光定量PCR测定miR-300在细胞内的表达水平;分别采用MTT活细胞测试,细胞周期实验和克隆形成实验检测骨髓瘤细胞的增殖情况;通过蛋白质印迹法测定Bcl-2、Bax和Bak,裂解型Caspase-3蛋白水平来鉴定细胞凋亡。结果转染miR-300的Saos-2细胞,其miR-300表达量为对照组的(10.23±1.04)倍,t=3.613 8,P=0.000 6。MTT实验结果显示,在转染后24和48h,miR-300组的Saos-2相对活细胞百分比相对对照组分别为[(174.35±28.46)%,t=2.219,P=0.03]和[(225.73±24.62)%,t=2.738,P=0.008]。miR-300组Saos-2细胞处在G1期的百分比为39.42%,低于对照组的47.58%,t=2.366,P=0.021;而处在G2期的细胞百分比为19.12%,显著高于对照组的10.55%,t=2.987,P=0.004。miR-300组Saos-2细胞克隆形成率为(52.53±30.21)%,显著高于对照组的(24.67±2.05)%,t=2.711,P=0.008 6。miR-300组Saos-2细胞中Bax、Bak和裂解型Caspase-3表达水平分别为对照组的(0.25±0.02)倍(t=2.785,P=0.007)、(0.31±0.03)倍(t=3.223,P=0.002)和(0.36±0.03)倍(t=3.006,P=0.003 8),差异有统计学意义。Bcl-2蛋白水平为对照组的(6.32±0.57)倍,t=3.218,P=0.002。转染miRZip lent-shMiR-300的Saos-2细胞,其miR-300数量为对照组的(0.28±0.03)倍,t=3.531,P=0.000 78。转染24h后,miR-300组Saos-2相对活细胞数为对照组的(64.46±5.32)%,t=2.324,P=0.024;48h后为对照组的(48.14±4.38)%,t=3.009,P=0.004。miR-300组Saos-2细胞处在G1期的百分比为55.71%,高于对照组的46.78%,t=2.108,P=0.039;处在G2期的细胞百分比为2.81%,低于对照组的11.46%,t=3.224,P=0.002。miR-300组的Saos-2细胞的克隆形成率为(17.63±3.89)%,显著低于对照组的(26.84±2.94)%,t=2.989,P=0.004。miR-300组Saos-2细胞中Bax表达水平为对照组的(6.25±4.23)倍,t=3.138,P=0.002 6;Bak表达水平为对照组的(6.03±4.27)倍,t=3.395,P=0.001 2;裂解型Capase-3表达水平为对照组的(5.35±0.37)倍,t=3.017,P=0.003 7;而Bcl-2蛋白水平为对照组的(0.36±0.02)倍,t=2.769,P=0.007 4。结论 miR-300在骨肉瘤细胞的增殖与凋亡调控中具有重要作用,抑制miR-300表达可以一定程度上减少骨髓瘤细胞的增殖,促进其凋亡,为骨肉瘤的病理机制研究提供了新研究方向。
The purpose of osteosarcoma with a high degree of local invasion and rapid metastatic potential, resulting in higher mortality. The study pointed out that the expression of miR-300 abnormalities associated with the incidence of a variety of tumors. The role of miR-300 in the regulation of osteosarcoma cells is unknown. This study was aimed to investigate the role of miR-300 in the regulation of proliferation and apoptosis of osteosarcoma cell line Saos-2. Methods miR-300 was transfected into osteosarcoma cell line Saos-2 to express miR-300 in Saos-2 cells. The expression level of miR-300 in cells was determined by real-time PCR. The proliferation of myeloma cells was detected by MTT assay, cell cycle assay and clonogenic assay respectively. The expressions of Bcl-2, Bax and Bak were detected by Western blotting. Cleavage of Caspase-3 protein levels to identify apoptosis. Results Saos-2 cells transfected with miR-300 showed an expression level of miR-300 of (10.23 ± 1.04) times that of the control group, t = 3.613 8, P = 0.0006. The results of MTT showed that the percentage of viable cells of Saos-2 in miR-300 group was [(174.35 ± 28.46)%, t = 2.219, P = 0.03] and [(225.73 ± 24.62%, t = 2.738, P = 0.008]. The percentage of Saos-2 cells in G1 phase in miR-300 group was 39.42%, which was lower than 47.58% in control group, t = 2.366, P = 0.021. The percentage of cells in G2 phase was 19.12% Group 10.55%, t = 2.987, P = 0.004. The clonogenic rate of Saos-2 cells in miR-300 group was (52.53 ± 30.21)%, which was significantly higher than that in control group (24.67 ± 2.05)%, t = 2.711, P = 0.008 6. The expression levels of Bax, Bak and lytic type Caspase-3 in Saos-2 cells of miR-300 group were (0.25 ± 0.02) times (t = 2.785, P = 0.007) and (0.31 ± 0.03) 3.223, P = 0.002) and (0.36 ± 0.03) times (t = 3.006, P = 0.003 8), the difference was statistically significant. Bcl-2 protein level (6.32 ± 0.57) times the control group, t = 3.218, P = 0.002. Saos-2 cells transfected with miRZip lent-shMiR-300 had a miR-300 count of (0.28 ± 0.03) times that of the control group, t = 3.531, P = 0.00078. The number of viable cells of Saos-2 in miR-300 group was (64.46 ± 5.32)% of control group, t = 2.324, P = 0.024 after transfection for 24 h and 48.14 ± 4.38% 3.009, P = 0.004. The percentage of Saos-2 cells in G1 phase in miR-300 group was 55.71%, higher than 46.78% in control group, t = 2.108, P = 0.039; the percentage of cells in G2 phase was 2.81% 11.46%, t = 3.224, P = 0.002. The clonogenic rate of Saos-2 cells in miR-300 group was (17.63 ± 3.89)%, which was significantly lower than that in control group (26.84 ± 2.94)%, t = 2.989, P = 0.004. The expression level of Bax in Saos-2 cells of miR-300 group was (6.25 ± 4.23) times of the control group, t = 3.138, P = 0.002 6; Bak protein expression level was (6.03 ± 4.27) P = 0.001 2; The expression level of cleaved Capase-3 was (5.35 ± 0.37) times that of the control group, t = 3.017, P = 0.003 7, while the level of Bcl-2 protein was (0.36 ± 0.02) = 2.769, P = 0.007 4. Conclusion miR-300 plays an important role in the regulation of proliferation and apoptosis of osteosarcoma cells. Inhibition of miR-300 expression can reduce the proliferation and promote the apoptosis of myeloma cells to a certain extent, providing a new mechanism for the study of the pathological mechanism of osteosarcoma research direction.