长链非编码RNA HOX转录反义RNA通过调节微小RNA-148a-3p/转录因子DP-2轴影响结直肠癌细胞增殖

摘要

目的 探讨HOX转录反义RNA (HOTAIR)调节结直肠癌(CRC)细胞增殖的机制.方法 实时定量反转录聚合酶链反应(RT-qPCR)检测人CRC细胞系及人正常结肠黏膜上皮细胞FHC中HOTAIR表达.用HOTAIR小干扰RNA(siRNA)降低CRC细胞中HOTAIR水平,细胞计数试剂盒(CCK-8)实验检测沉默HOTAIR对CRC细胞增殖的影响.流式细胞术分析沉默HOTAIR对细胞凋亡及周期的影响.Western blot检测凋亡相关蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3/Caspase-7的表达.荧光素酶报告系统检测HOTAIR的作用靶点.结果 HOTAIR及转录因子DP-2(TFDP2)在CRC细胞中高表达(P＜0.05),而微小RNA(miRNA,miR) 148a-3p在CRC细胞中低表达(p＜0.05).荧光素酶报告结果显示,miR-148a-3p是HOTAIR的直接靶点,TFDP2是miR-148a-3p的直接作用靶点.HOTAIR下调后细胞吸光度值在培养第2天时(HCT-116:0.425±0.002,SW480:0.437±0.007)即明显低于对照组(HCT-116:0.468±0.002,SW480:0.518±0.007) (P ＜0.05),细胞凋亡比例增加(HOTAIR下调比对照:HCT-116:16.417±1.407比6.002±0.301;SW480:16.493±0.653比6.240±0.346)以及发生G0/G1期阻滞(HOTAIR下调比对照:HCT-116:72.267 ±0.834比66.050±0.687;SW480:71.857±1.174比65.833±1.103) (P＜0.05).下调miR-148a-3p可以逆转沉默HOTAIR对CRC细胞增殖、凋亡及周期阻滞的影响.结论 HOTAIR通过调节miR-148 a-3 p/TFDP2轴影响CRC细胞增殖.%Objective To investigate the mechanism of HOX antisense intergenic RNA (HOTAIR) in regulating cell proliferation in colorectal cancer (CRC).Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect HOTAIR expression in human CRC cell lines and human normal colonic mucosal epithelial cells FHC.HOTAIR small interfering RNA (siRNA) was used to reduce HOTAIR levels in CRC cells,and cell counting kit-8 (CCK-8)assay was used to detect the effect of HOTAIR on CRC cell proliferation.Flow cytometry was used to analyze the effect of HOTAIR on apoptosis and cycle of CRC cells.Western blotting was used to detect the expression of apoptosis-related protein Caspase-3/Caspase-7.The luciferase reporter system detects direct targets of HOTAIR.Results HOTAIR and transcription factor DP-2 (TFDP2) were highly expressed in CRC cells (P ＜ 0.05),while microRNA (miRNA,miR) 148a-3p was down-regulated in CRC cells (P ＜0.05).Luciferase report showed that miR-148a-3p is a direct target of HOTAIR,and TFDP2 is a direct target of miR-148a-3p.The cell absorbance value of HOTAIR was significantly lower than that of the control group (HCT-116:0.425 ±0.002,SW480:0.518 ±0.007) (P＜0.05).Increased proportion of apoptosis (HOTAIR down-regulated vs.control:HCT-116:16.417 ± 1.407 vs.6.002 ±0.301;SW480:16.493 ±0.653 vs.6.240 ±0.346) and Go/G1 arrest (HOTAIR down-regulation vs.control:HCT-116:72.267 ±0.834 vs.66.050 ±0.687;SW480:71.857 ± 1.174 vs.65.833 ± 1.103) (P ＜0.05).Down-regulation of miR-148a-3p reversed the effect of silent HOTAIR on CRC cell proliferation,apoptosis and cell cycle arrest.Conclusion HOTAIR affects CRC cell proliferation by regulating miR-148a-3p/TFDP2 axis.