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目的研究表明,Kv1.5钾离子通道参与了多种肿瘤的生物学行为,但其在骨肉瘤中的表达和作用尚未明确。本研究拟检测Kv1.5钾离子通道在骨肉瘤中的表达,并探讨小干扰RNA(small interfering,siRNA)沉默Kv1.5表达后对骨肉瘤细胞增殖、周期和凋亡的影响及可能的调控机制。方法实验分组,Kv1.5-siRNA转染细胞为实验组,controlsiRNA转染细胞为对照组,未处理细胞为空白组。采用siRNA抑制Kv1.5的表达,并分别采用CCK-8法、克隆形成率、流式细胞仪和Tunel法检测MG-63细胞增殖、生长、周期和凋亡的变化。采用qRT-PCR和蛋白质印迹法检测骨肉瘤细胞中周期和凋亡相关基因的表达水平。结果 Kv1.5在骨肉瘤细胞和组织中均异常高表达。CCK-8法检测结果示,Kv1.5-siRNA组细胞增殖水平为(53.87±5.91)%,与control-siRNA组的(99.14±7.87)%相比差异有统计学意义,Z=-3.49,P<0.001;与空白组(100.00±8.37)%相比差异有统计学意义,Z=-3.57,P<0.001。克隆形成实验结果示,Kv1.5-siRNA组克隆形成数为164.00±7.66,与control-siRNA组(223.20±11.41)相比,Z=-2.61,P=0.008;与空白组(228.20±12.62)相比,Z=-2.40,P=0.016。流式细胞周期结果示,Kv1.5-siRNA组G0/G1期所占比例为(68.81±0.55)%,与control-siRNA组(41.22±0.61)%相比,Z=-2.15,P=0.031;与空白组(40.79±0.52)%相比,Z=-2.31,P=0.029。流式细胞凋亡结果示,Kv1.5-siRNA组(30.23±1.74)%与control-siRNA组(14.10±1.27)%相比,Z=-2.04,P=0.039;与空白组(12.85±1.02)%相比,Z=-2.09,P=0.035。Tunel法结果示,Kv1.5-siRNA组凋亡指数为(35.20±3.14)%,与control-siRNA组(8.03±1.50)%相比,Z=-2.41,P=0.015;与空白组(8.22±1.32)%相比,Z=-2.25,P=0.019。沉默Kv1.5表达可明显下调骨肉瘤中Cyclin A、Cyclin D1、Cyclin E、Bcl-2和Bik基因的表达,同时上调p21、p27、Bax、Bcl-XL和Caspase-3基因的表达,与对照组相比,P值均<0.05。结论 Kv1.5钾离子通道参与了骨肉瘤细胞增殖、周期和凋亡的过程,其作用机制可能与调控周期依赖激酶和Bcl-2家族的相关因子有关。
The purpose of the study shows that Kv1.5 potassium channel involved in a variety of tumor biological behavior, but its expression and role in osteosarcoma has not yet been identified. This study was designed to investigate the expression of Kv1.5 potassium channel in osteosarcoma and to explore the effect of small interfering (siRNA) silencing Kv1.5 expression on the proliferation, cycle and apoptosis of osteosarcoma cells and its possible regulation mechanism. Methods The experimental group, Kv1.5-siRNA transfected cells for the experimental group, controlsiRNA transfected cells for the control group, untreated cells as a blank group. The siRNA was used to inhibit the expression of Kv1.5. The proliferation, growth, cycle and apoptosis of MG-63 cells were detected by CCK-8 method, clone formation rate, flow cytometry and Tunel method respectively. QRT-PCR and Western blotting were used to detect the expression of cycle-related and apoptosis-related genes in osteosarcoma cells. Results Kv1.5 was highly expressed in osteosarcoma cells and tissues. The results of CCK-8 showed that the proliferation of Kv1.5-siRNA group was (53.87 ± 5.91)%, which was significantly different from that of control-siRNA group (99.14 ± 7.87)%, Z = -3.49, P <0.001; compared with the blank group (100.00 ± 8.37)%, the difference was statistically significant, Z = -3.57, P <0.001. The result of clone formation showed that the number of clone formation in Kv1.5-siRNA group was 164.00 ± 7.66, Z = -2.61, P = 0.008 compared with control-siRNA group (223.20 ± 11.41) In contrast, Z = -2.40, P = 0.016. The results of flow cytometry showed that the ratio of G0 / G1 phase was (68.81 ± 0.55)% in Kv1.5-siRNA group and was Z = -2.15 and P = 0.031 compared with 41.22 ± 0.61% in control-siRNA group ; Compared with the blank group (40.79 ± 0.52)%, Z = -2.31, P = 0.029. The results of flow cytometry showed that Z = -2.04 and P = 0.039 in Kv1.5-siRNA group (30.23 ± 1.74)% and control-siRNA group (14.10 ± 1.27) )%, Z = -2.09, P = 0.035. Tunel method showed that the apoptotic index in Kv1.5-siRNA group was (35.20 ± 3.14)%, which was significantly lower than that in control group (Z = -2.41, P = 0.015) ± 1.32)%, Z = -2.25, P = 0.019. Silencing Kv1.5 expression significantly reduced the expression of Cyclin A, Cyclin D1, Cyclin E, Bcl-2 and Bik genes in osteosarcoma and up-regulated the expressions of p21, p27, Bax, Bcl-XL and Caspase- P values were all <0.05. Conclusions The Kv1.5 potassium channel is involved in the proliferation, cycle and apoptosis of osteosarcoma cells. The mechanism may be related to the regulation of cyclin dependent kinase and Bcl-2 family members.