MiR-300对骨肉瘤细胞增殖与凋亡作用研究

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48 h,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=o.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24 h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;48 h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.

MiR-300 regulating the proliferation and apoptosis of osteosarcoma cells

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 cancers.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 transfected to the osteosarcoma cell Saos-2 to promote the amount of miR-300.miRZip lent-shMiR-300 was transfected to Saos-2 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±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 transfected Saos-2 had 39.42％ 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) . Compared to the control,miR-300 group of Saos-2 had the protein level of Bax,Bak,and cleavaged caspase-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 0=3.218,P=0.002).In miRZip lentshMiR-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=0.024;t=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 control;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 caspase-3 were 6.25±4.23(t=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 ostcosarcoma cells to a certain extent.This research offers a new direction to study the pathomechanism of osteosarcoma.