长链非编码RNA核仁小RNA宿主基因8通过靶向微小RNA-224-3p调控肾癌细胞增殖、侵袭迁移以及顺铂敏感性

Long non-coding RNA small nucleolar RNA host gene 8 regulates renal cancer cell proliferation,invasion and migration,and cisplatin sensitivity by targeting microRNA-224-3p

目的探讨长链非编码RNA核仁小RNA宿主基因8(lncRNA SNHG8)对肾癌细胞增殖、侵袭、迁移和顺铂敏感性的影响及其分子机制。方法实时荧光定量聚合酶链反应(RT-qPCR)检测SNHG8和微小RNA(miR)-224-3p表达水平;蛋白质印迹(Western blot)法检测基质金属蛋白酶-2(MMP-2)、细胞增殖核抗原Ki-67(Ki-67)、裂解半胱氨酸天冬氨酸蛋白酶-3(cleaved Caspase-3)和多药耐药基因1(MDR1)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞存活率和顺铂半数抑制浓度(IC50);Transwell检测细胞迁移和侵袭;流式细胞术检测细胞凋亡;荧光素酶报告实验检测SNHG8和miR-224-3p的靶向关系。两组比较采用t检验。结果肾癌细胞系ACHN、786-O和A498中SNHG8高表达(1.05±0.09比4.58±0.47、6.19±0.52、5.37±0.48,t=22.130、29.219、26.538,P<0.05),miR-224-3p低表达(1.02±0.08比0.53±0.07、0.41±0.06、0.47±0.08,t=13.829、18.300、14.584,P<0.05)。与si-con组相比,si-SNHG8组,肾癌细胞786-O中MMP-2、Ki-67、MDR1表达降低,cleaved Caspase-3表达升高,细胞存活率降低[(69.86±6.37)%比(96.42±9.26)%,t=7.089,P<0.05],迁移和侵袭数[(175.64±12.20)个比(292.70±20.45)个、(92.81±8.38)个比(147.24±15.93)个,t=14.748、9.072,P<0.05]降低,凋亡率升高[(19.48±2.28)%比(4.28±0.64)%,t=19.256,P<0.05],顺铂IC50值降低(1.47±0.14比7.81±0.22,t=19.454,P<0.05)。过表达miR-224-3p后,肾癌细胞786-O中MMP-2、Ki-67、MDR1表达降低,cleaved Caspase-3表达升高,细胞存活率降低[(67.36±8.65)%比(98.24±9.53)%,t=7.198,P<0.05],迁移和侵袭数[(155.81±16.67)个比(274.73±28.61)个、(72.96±8.35)个比(156.61±14.28)个,t=10.774、15.170,P<0.05]降低,凋亡率升高[(18.49±2.28)%比(4.67±0.63)%,t=17.527,P<0.05],顺铂IC50值降低(1.79±0.15比6.72±0.26,t=49.273,P<0.05)。结论沉默SNHG8可抑制肾癌细胞786-O增殖、迁移、侵袭,促进细胞凋亡,增强其对顺铂的敏感性;其机制可能与miR-224-3p有关。

Objective To investigate the effects and mechanism of long non-coding RNA small nucleolar RNA host gene 8(lncRNA SNHG8)on the proliferation,invasion,migration and cisplatin sensitivity of renal cell carcinoma.Methods Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)to detect the expression levels of SNHG8 and microRNA(miR)-224-3p;Western blotting was used to detect matrix metalloproteinase-2(MMP-2),nuclear associated antigen Ki-67(Ki-67),and cleaved cysteine-containing aspartate-specific proteases-3(cleaved Caspase-3)and multidrug resistance gene 1(MDR1)protein expression;tetramethylazozolium salt colorimetric assay(MTT)for cell viability and the half-inhibitory concentration of cisplatin on cells(IC50);Transwell for cell migration and invasion;flow cytometry to detect apoptosis;luciferase reporter assay to detect the targeting relationship between SNHG8 and miR-224-3p.Statistical analysis was performed with SPSS 20.0 software,and the two groups were compared by t test.Results SNHG8 was highly expressed in renal cancer cell lines ACHN,786-O and A498(1.05±0.09 vs.4.58±0.47,6.19±0.52,5.37±0.48,t=22.130,29.219,26.538,P<0.05),miR-224-3p waslow expression(1.02±0.08 vs.0.53±0.07,0.41±0.06,0.47±0.08,t=13.829,18.300,14.584,P<0.05).Compared with the si-con group,in the si-SNHG8 group,the expressions of MMP-2,Ki-67 and MDR1 in renal cancer cell 786-O were decreased,the expression of cleaved Caspase-3 wasincreased,and the cell survival rate was decreased[(69.86±6.37)%vs.(96.42±9.26)%,t=7.089,P<0.05],the number of migration and invasion[(175.64±12.20)vs.(292.70±20.45),(92.81±8.38)vs.(147.24±15.93),t=14.748,9.072,P<0.05]were decreased,apoptosis rate was increased[(19.48±2.28)%vs.(4.28±0.64)%,t=19.256,P<0.05],cisplatin IC50 value was decreased(1.47±0.14 vs.7.81±0.22,t=19.454,P<0.05).After overexpression of miR-224-3p,the expressions of MMP-2,Ki-67,MDR1 in renal cancer cell 786-O were decreased,the expression of cleaved Caspase-3 wasincreased,and the cell survival rate was decreased[(67.36±8.65)%vs.(98.24)±9.53)%,t=7.198,P<0.05],migration and invasion numbers[(155.81±16.67)vs.(274.73±28.61),(72.96±8.35)vs.(156.61±14.28),t=10.774,15.170,P<0.05]weredecreased,apoptosis rate was increased[(18.49±2.28)%vs.(4.67±0.63)%,t=17.527,P<0.05],cisplatin IC50 value was decreased(1.79±0.15 vs.6.72±0.26,t=49.273,P<0.05).SNHG8 targets miR-224-3p,and interfering with miR-224-3p partially reverses the effect of silencing SNHG8 on proliferation,migration,invasion inhibition and apoptosis promotion of 786-O cells.Conclusion Silencing of SNHG8 can inhibit the proliferation,migration and invasion of renal cell carcinoma 786-O,promote apoptosis and enhance its sensitivity to cisplatin.The mechanism may be related to miR-224-3p.