RNA干扰技术抑制HLX1基因表达对滋养细胞生物学性能的影响

Suppression of homeobox gene HLX1 by siRNA transfection inhibits trophoblast proliferation and invasion

目的:研究小分子干扰RNA(siRNA)技术抑制人类同源异型盒基因HLX1的表达对胎盘滋养细胞株HTR-8/SVneo细胞生物学性能的影响。方法:常规体外培养HTR-8/SVneo细胞,设实验组(转染HLX1基因的特异性siRNA)、阴性对照组(转染阴性对照siRNA)及空白对照组(除不含siRNA片段,余试剂与另两组相同)3组分别进行瞬时转染。用流式细胞术测定siRNA转染效率,应用实时定量PCR技术和蛋白印迹法测定转染后各组HTR-8/SVneo细胞中HLX1 mRNA和蛋白的表达;应用MTT比色实验、Matrigel侵袭实验分别检测转染后各组细胞的增殖能力与侵袭能力的变化。结果:(1)转染24h后测得siRNA转染效率达(86.3±2.6)%。(2)转染48h后HLX1 mRNA的表达水平下调(77.0±1.2)%(P<0.01),转染72h后HLX1蛋白的表达水平下调(82.6±1.2)%(P<0.01),与HLX1 mRNA的表达水平下调相一致。(3)转染HLX1 siRNA 24h后,HTR-8/SVneo细胞的增殖活性已受到抑制,转染72h后细胞增殖抑制最显著,抑制率达到(58.1±4.4)%(P<0.01)。(4)转染HLX1 siRNA后,HTR-8/SVneo细胞侵袭Matrigel基质胶的能力受到显著抑制,其穿膜细胞数为(29±3)个,与空白对照组(穿膜细胞数为[53±8]个)相比,差异有统计学意义(P<0.01)。结论:HLX1基因表达水平的下降可以显著抑制滋养细胞的增殖活性与侵袭能力;HLX1基因表达异常可能通过影响滋养细胞增殖、侵袭等过程参与IFGR、子痫前期等疾病的发生。

Objective：To observe the suppression effects on homeobox gene HLX1 by small interference RNA（siRNA） and investigate the changes of biological performance of HTR-8/SVneo cell line.Method：Transfection of siRNA using DharmaFECTTM1 was conducted to silence homeobox gene HLX1 expression.Transfection efficiency was assessed by flow cytometry（FCM） at 24hours of transfection,after that expression levels of HLX1 mRNA and protein were assessed by Real-time PCR at 48hours of transfection and western blot at 72hours of transfection respectively.Cell proliferation was assayed by methyl thiazolyl tetrazolium（MTT） after 24,48,72hours of siRNA transfection respectively.Transwell chamber assay was performed to determine the effects on cell invasion capacity.Results：（1） Transfection efficiency was up to（86.3±2.6） % at 24hours of Transfection.（2） HLX1 siRNA transfection resulted in a significant（77.0±1.2）% decrease of HLX1 mRNA level and a（82.6±1.2）% decrease expression level of HLX1 protein（both P〈0.01）.（3） HLX1 siRNA resulted in a significant decrease of（58.1±4.4）% in trophoblast proliferation at 72hours of transfection（P〈0.01）.（4） HTR-8/SVneo cell invasion was significantly suppressed with a invasion number of（29±3）,compared with the control group with a invasion number of（53±8）（P〈0.01）.Conclusion：Aberrant HLX1 expression may impair both proliferation and invasion capacities of trophoblasts and is probably involved in the development of placental defects such as idiopathic fetal growth restriction（IFGR）and pre-eclampsia.