慢病毒介导星形胶质细胞活化基因-1沉默对葡萄膜黑色素瘤生物学行为的影响

Effects of lentivirus-mediated knockdown of astrocyte elevated gene-1 on the biological behavior of uveal melanoma Mao Ying,Bai Haixia,Chang Ying,Li Bin

目的探讨星形胶质细胞活化基因-1（AEG-1）对葡萄膜黑色素瘤（UM）生物学行为的影响。方法培养不同侵袭转移潜能的MUM-2B和MUM-2C细胞系，根据AEG-1基因序列设计出有效RNA干扰（RNAi）靶点序列及RNAi阴性对照scramble序列进行慢病毒载体的制备和病毒包装。在2株细胞系中，经AEG-1-RNAi慢病毒感染的细胞作为慢病毒感染组，空白慢病毒载体感染的细胞作为阴性对照组，未处理的细胞作为正常对照组。采用Real-time PCR、Western blot法分别检测各组细胞中AEG-1转录水平和蛋白表达水平；采用MTT法、Annexin V-APC法、细胞侵袭试验和细胞Transwell试验检测慢病毒介导AEG-1沉默对人UM细胞系生物学行为的影响。结果成功制备RNAi慢病毒载体而进行病毒包装，然后分别转染对数生长期的MUM-2B、MUM-2C细胞系，2株细胞系中的正常对照组、阴性对照组和慢病毒感染组间细胞中AEG-1 mRNA相对表达量总体比较差异均有统计学意义（F=130.02，P<0.01；F=144.17，P<0.01）。在MUM-2B细胞系中和MUM-2C细胞系中，染组较阴性对照组AEG-1 mRNA相对表达量分别下降约77.1%和79.8%，差异均有统计学意义（均P<0.01）；正常对照组与阴性对照组细胞中AEG-1 mRNA相对表达量比较，差异均无统计学意义（均P>0.05）。Western blot法检测结果显示，AEG-1蛋白表达水平在2株细胞系中的各组比较，差异均有统计学意义（F=146.17，P<0.01；F=156.79，P<0.01），其中慢病毒感染组较阴性对照组蛋白相对表达均显示下调，差异均有统计学意义（均P<0.01）。MTT法检测发现，2株细胞系实验第2～5天，慢病毒感染组细胞增生倍数较阴性对照组明显下降，差异均有统计学意义（t=5.78、30.68、23.99、29.40，均P<0.01；t=7.88、7.09、5.56、6.60，均P<0.01）；Annexin V-APC法检测发现，2株细胞系中慢病毒感染组凋亡率明显高于阴性对照组，差异均有统计学意义（t=54.34，P＜0.01；t=11.68，P＜0.01）；细胞侵袭试验发现，在2株细胞系中慢病毒感染组侵袭倍数明显低于阴性对照组，差异均有统计学意义（t=16.04，P＜0.01；t=13.98，P＜0.01）；细胞Transwell试验发现，在2株细胞系中慢病毒感染组转移倍数均明显低于阴性对照组，差异均有统计学意义（t=12.04，P＜0.01；t=22.43，P＜0.01）。结论慢病毒介导AEG-1沉默后，通过抑制UM细胞AEG-1的转录水平和蛋白表达水平，从而改变UM细胞的生物学行为，一定程度上减缓细胞增生，促进细胞凋亡并降低细胞侵袭和转移的能力。

Objective To investigate the effects of lentivirus-mediated knockdown of astrocyte elevated gene-1(AEG-1)on the biological behavior of uveal melanoma(UM).Methods MUM-2B and MUM-2C cell lines with different invasiveness and metastasis potential were cultured.Based on AEG-1 sequence that designed the effective(RNA interference)RNAi target sequence and the RNAi negative control scramble sequence,and then performed the preparation of the lentiviral vector and viral packaging.In these two kinds of cell lines,the cells infected by AEG-1-RNAi lentivirus were used as lentiviral infection group,the cells of the RNAi negative control of the blank lentivirus infected cells were used as negative control group,and the cells without any processing were used as normal control group.Real-time PCR and Western blot were used to detect the change of AEG-1 transcription and protein expression levels in these two kinds of cell lines'groups,as well as by MTT method,Annexin V-APC method,cell invasion assay,and Transwell cell migration assay that to investigate the effect of lentivirus transfection induced knockdown of AEG-1 on the biological behavior of human UM cell lines.Results The successful preparation of RNAi lentivirus vector and its viral package were transfected to MUM-2B,MUM-2C cell lines of logarithmic growth phase,respectively.The relative expressions of AEG-1 mRNA in normal control groups,negative control groups and lentiviral infection groups were statistically significant(F=130.02,P<0.01;F=144.17,P<0.01).The relative expression of AEG-1 mRNA in lentiviral infection groups were lower than that in the negative control groups,and the AEG-1 gene knockout rates were 77.1%and 79.8%,respectively,the differences were statistically significant(both at P<0.01).There were no significant differences in the relative expression of AEG-1 mRNA between normal control groups and negative control groups(all at P>0.05).Western blot showed that no significant changes were found in the AEG-1 proteins expression levels between normal control groups and negative control groups in the two kinds of cell lines(F=146.17,P<0.01;F=156.79,P<0.01).The expression levels of AEG-1 protein in the lentiviral infection groups were significantly lower than that in negative control groups(all at P<0.01).The proliferation of both cells lines in lentiviral infection groups were significantly lower than that in negative control groups from the second day to the fifth day,the differences were statistically significant(t=5.78,30.68,23.99,29.40,all at P<0.01;t=7.88,7.09,5.56,6.60,all at P<0.01).In both cell lines,the apoptosis rate of lentiviral infection groups were significantly higher than those in the negative control groups,the differences were statistically significant(t=54.34,P<0.01;t=11.68,P<0.01).In both cell lines,the multiple of invasions in lentiviral infection groups were significantly lower than those in negative control groups,and the differences were statistically significant(t=16.04,P<0.01;t=13.98,P<0.01);In both cell lines,the multiple of transfer in lentiviral infection groups were significantly lower than those in negative control groups,and the differences were statistically significant(t=12.04,P<0.01;t=22.43,P<0.01).Conclusions Lentivirus transfection inducing knockdown of AEG-1 inhibits the transcription and protein expression level of AEG-1 in the melanoma cells,which changes the biological behaviors of UM,like slowing down cell proliferation,promoting apoptosis,and reducing the abilities of cells'invasion and metastasis.