MiR-300对骨肉瘤细胞增殖与凋亡作用研究

发布时间：2018-04-15 23:35

  本文选题：MiR- + 骨肉瘤　； 参考：《中华肿瘤防治杂志》2017年16期

【摘要】：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。
[Abstract]:Objective Osteosarcoma has high local invasion and rapid metastasis potential, so the fatality rate is high.It is suggested that the abnormal expression of miR-300 is related to the pathogenesis of many kinds of tumors. Whether miR-300 can regulate osteosarcoma cells is unknown.To investigate the effect of miR-300 on proliferation and apoptosis of osteosarcoma cell line Saos-2.Methods miR-300 was transfected into Saos-2 of osteosarcoma cells, and the high expression of miR-300 in Saos-2 cells was realized.The expression of miR-300 was measured by fluorescence quantitative PCR, and the proliferation of myeloma cells was detected by MTT living cell test, cell cycle assay and clone formation assay.Apoptotic cells were identified by Western blotting assay of Bcl-2P Bax and Bak-Bax and cleavage Caspase-3 protein levels.Results the expression of miR-300 in Saos-2 cells transfected with miR-300 was 10.23 卤1.04 times as high as that in the control group. The results of the experiment showed that the expression of miR-300 was 3.6138Pu 0.000 6.MTT.鐨，

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