MiR-300对骨肉瘤细胞增殖与凋亡作用研究

MiR-300 regulating the proliferation and apoptosis of osteosarcoma cells

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。

OBJECTIVE Osteosarcoma cells have the highly local invasion ability and rapid transfer potential. Therefore it is a malignancy with high mortality. The study indicated that the abnormal expression of miR-300 is related to the incidence of canc- ers. But we don＇t know whether miR-300 has the regulation of osteosarcoma cells. This study aims to investigate the role of miR -- 300 in the regulation of the proliferation and apoptosis of osteosarcoma cell sao-2. METHODS The miR-300 mimics was trans- fected to the osteosarcoma cell Saos-2 to promote the amount of miR-300, miRZip lent-shMiR-300 was transfected to Sao2 to silence miR-300. Real-time PCR was used to determine the amount of miR-300 in Saos-2 cells. MTT method, cell cycle analysis and colon-forming efficiency assay were employed to detect the cell proliferation. To determine the cell apoptosis, western blot was used to detect the amount of apoptosis associated protein Bcl-2, Bax, Bak, and cleaved caspase-3. RESULTS In the miR-300 transfected Saos-2 cell, gene expression of miR-300 was 10.23___1.04,which was higher than that of control （t=3. 613 8,P=0. 000 6）. After 24 or 48 h of transfection, the viable cell percentage in miR-300 overexpression group were （174. 35_--4-28.46）% （t= 2. 219,P= 0.03） and （225.73± 24.62）% （t= 2. 738, P=0. 008） compared to that of the control. The miR-300 transfect- ed Saos-2 had 39. 420% of the total cells in G1 phase,lower than the 47.58% （t=2. 366, P〈0. 021） in the control group 19.12% of the cell was in G2 phase,higher than the 10.55%（t=2. 987, P=0. 004） in the control, miR-300 group of Saos-2 had the colon formation rate （52.53!30.21）% ,higher than the （24.67±2.05）% in control （t=2. 711,P=0. 008 6）% Com- pared to the control, miR-300 group of Saos-2 had the protein level of Bax, Bak,and cleavaged easpase-3 （0. 25±0.02） fold （t= 2. 785, P= 0. 007）, （0. 31±0.03） fold （t= 3. 223, P= 0. 002） and （0. 36±0. 03） fold （t= 3. 006, P= 0. 003 8） as compared to the control. However, protein level of Bcl-2 was （6.32!0.57） folds as the control （t=3. 218, P=0. 002）. In miRZip lent- shMiR-300 transfected Saos-2 cell, the miR-300 level was （0. 28±0.03） folds as control （t=3. 531 ,P=0. 000 78）. After 24 and 48 h of transfection,in miR-300 transfected Saos-2, viable cell percentage were （64.46±5.32） % and （48. 14±4.38） % （t= 2. 324,P=0. 024t=3. 009,P=0. 004）. Totally 55.71% of the cells were in G1 phase,higher than the 46.78% （t=2. 108,P= 0. 039） in controll 2.81% of the cell was in G2 phase,lower than the 11.46% in control （t=3. 224,P=0. 002）. In miR-300 group, the colon formation rate was （ 17.63 ± 3.89） %, lower than the （26.84 ± 2.94） % （t = 2. 989, P = 0. 004） in control group. In miR-300 group,protein level of Bax, Bak,and cleavage easpase-3 were 6.25±4. 23±3. 138, P=0.0 026） ,6.03± 4. 27 fold （t=3. 395, P=0.0 012） and 5. 35±0.37 fold （t=3. 017, P=0.003 7）. however, protein level of Bcl-2 was 0. 36± 0. 02 folds as the control （t=2. 769, P=0.0 074）. CONCLUSIONS miR-300 plays an important role in the regulation of the proliferation and apotosis of osteosarcoma cells. Inhibition of miR-300 expression can reduce the proliferation and apoptosis of os teosarcoma cells to a certain extent. This research offers a new direction to study the pathomechanism of osteosareoma.