IGF-1R基因沉默对L02细胞过氧化损伤时Caspase12表达的影响

Effects of IGF-1R gene silencing on the expression of Caspase12 in L02 cells with peroxidation damage

目的:成功构建稳定沉默表达IGF-1R基因质粒,并转染至L02细胞(人正常肝细胞),观察IGF-1R基因沉默对L02细胞过氧化损伤时经内质网途径特异性凋亡蛋白Caspase12表达的影响。方法:构建针对L02细胞IGF-1R基因的短发夹状RNA(shRNA),将4种不同序列的Sh-H-IGF1R-1108、Sh-H-IGF1R-2797、Sh-H-IGF1R-3215、Sh-H-IGF1R-3787质粒(设为实验组)以及含与IGF-1R无关序列的Sh-H-IGF1R-V1质粒(设为阳性对照组)和空白质粒Sh-H-IGF1R-V2(设为阴性对照组)分别转染L02细胞,24 h后用RTPCR方法检测IGF-1R表达水平,筛选出稳定沉默IGF-1R基因的质粒。随后进行下一步实验分组:空白对照组(A组)、过氧化损伤组(B组)、阴性质粒转染组(C组)、目的基因转染组(D组)。12 h后收集细胞,采样待测,用Western Blot法检测各组Caspase12表达。结果:各实验组与Sh-H-IGF1R-V1组及Sh-H-IGF1R-V2组比较扩增倍数均降低(P<0.05),其中Sh-H-IGF1R-3215组(0.02±0.01)与Sh-H-IGF1R-3787(0.05±0.05)沉默效果最稳定,但后组重复性更好。B组(2.41±0.03)、C组(2.45±0.02)、D组(3.60±0.06)Caspase12表达均较A组(0.34±0.02)升高(P<0.01),C组较B组略升高(P>0.05),D组较B、C组显著升高(P<0.01)。结论:4种针对IGF-1R基因沉默质粒均构建成功,并成功转染至人L02细胞(人正常肝细胞),沉默效果最理想的质粒为Sh-H-IGF1R-3787。IGF-1R靶向shRNA质粒可通过沉默L02细胞IGF-1R表达加重细胞凋亡,间接验证了IGF-1/IGF-1R信号通路对L02细胞过氧化损伤时经内质网途经的细胞凋亡程序活化有一定的调控作用。

Objective To observe the effects of silencing IGF-1 receptor on the expression of Caspase12, a endoplasmie reticulum specificity apoptosis protein of the peroxidation damage in L02 ceUs. Methods Four the recombined plasmids expressing short hairpin RNA （shRNA） specific for sileincing IGF-1R gene were constructed and these shRNAs were potentially targeted on different mRNA sequences of the IGF-1 receptor gene （Sb- H-IG1R- 1108, Sh-H-IGF1R-2797, Sh-H-IGF1R-3215 and Sh-H-IGF1R-3787）. These constructs were classed as the experimental group. Another two constructs containing an irrespective sequence and no sequence were defined as a positive control group a negative control group, respectively. All these constructs were transfeeted into the L02 ceils by Lipefectamine 2000. The interference effect was detected by RT-PCR after 24 h and the plasmid stably silencing the expression of IGF-1 receptor was selected for further analysis which was divided into four groups： the blank control group（Group A）, the peroxidation damage group（Group B）, the negative plasmid transfeetion group （Group C） and the positive plasmid transfeetion group （Group D）. The expression of Caspasel2 was detected by Westen Blot after 12 h. Results The amplification folds of experimental groups were statistically significantly lower than those of the Sh-H-IGF1R-V1 group and Sh-H-IGF1R-V2 group （P 〈 0.05）. The silence effect of the Sh-H- IGF1R-3215 group （0.02 ± 0.01） and the Sh-H-IGF1R-3787 group （0.05 ±0.05） were most stable and the repeatability of the later was better. The expressions of Caspasel2 in Group B （2.41 ± 0.03）, Group C （2.45 ± 0.02） and Group D （3.60 ±0.06） were higher than Group A （0.34 ± 0.02） （P 〈 0.01）. The Group Cexpression was higher than that of the Group B （P 〉 0.05）. The Caspase12 expression in Group D was higher than that in Group B and C （P 〈 0.01）. Conclusion （1） Of all the four constructs in this study, Sh-H-IGF1R-3787 is the most suitable for silencing IGF-1 receptor. （2） Silencing IGF 1R expression using shRNA plasmids can aggravate the L02 cells apoptosis, suggesting that the IGF-1/IGF 1R signal pathway might be involved in regulating the activation of apoptosis in L02 cells with peroxidation damag via endoplasmic retieulum stress.