敲除C3G对H9C2心肌细胞增殖及凋亡的影响

Effects of C3G Knockout on Proliferation and Apoptosis in H9C2 Cardiomyocytes

该研究构建了NT CRISPR/Cas9(阴性对照)及C3G CRISPR/Cas9质粒,分别将其包装成重组慢病毒,并感染H9C2心肌细胞,以研究敲除C3G(Crk SH3域结合鸟嘌呤核苷酸交换因子)对H9C2心肌细胞增殖和凋亡的影响及其机制。将实验分为NT CRISPR/Cas9组、C3G CRISPR/Cas9组、NT CRISPR/Cas9低氧组和C3G CRISPR/Cas9低氧组。通过RT-PCR检测C3G m RNA的表达;Western blot检测相关蛋白表达;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡。结果显示,C3G CRISPR/Cas9组和C3G CRISPR/Cas9低氧组的C3G m RNA和蛋白无表达;分别与NT CRISPR/Cas9组和NT CRISPR/Cas9低氧组比较,C3G CRISPR/Cas9组和C3G CRISPR/Cas9低氧组的p-ERK1/2和Bcl-2蛋白以及细胞增殖水平均降低(P<0.05),Bax蛋白及细胞凋亡水平均增加(P<0.05);与NT CRISPR/Cas9组相比,NT CRISPR/Cas9低氧组C3G m RNA和蛋白表达均降低(P<0.05),p-ERK1/2和Bcl-2蛋白及细胞增殖水平均降低(P<0.05),Bax蛋白及细胞凋亡水平均增加(P<0.05);与C3G CRISPR/Cas9组相比,C3G CRISPR/Cas9低氧组的p-ERK1/2和Bcl-2蛋白及细胞增殖水平均降低(P<0.05),Bax蛋白及细胞凋亡水平均增加(P<0.05)。以上结果表明,敲除C3G能通过调控p-ERK1/2、Bcl-2及Bax抑制H9C2心肌细胞增殖并促进其凋亡。

The NT CRISPR/Cas9（non-target）, C3 G CRISPR/Cas9 plasmids were constructed and packaged into lentiviruses respectively. H9 C2 cardiomyocytes were infected with above lentiviruses respectively to investigate the effects of C3 G [Crk SH3-domain-binding guanine nucleotide exchange factor] knockout on proliferation and apoptosis in H9 C2 cardiomyocytes and their underlying mechanisms. The experiments were divided into NT CRISPR/Cas9, C3 G CRISPR/Cas9, NT CRISPR/Cas9＋Hypoxia and C3 G CRISPR/Cas9＋Hypoxia groups. C3 G m RNA was detected by RT-PCR. C3 G, p-ERK1/2, Bcl-2 and Bax proteins were tested by Western blot. Cell proliferative rate was examined by CCK-8. Apoptotic rate was determined by flow cytometry. The results showed that the expression of C3 G m RNA and protein were absent in C3 G CRISPR/Cas9 and C3 G CRISPR/Cas9＋Hypoxia groups. Compared with the NT CRISPR/Cas9 and NT CRISPR/Cas9＋Hypoxia groups, the expression of p-ERK1/2 and Bcl-2 proteins and cell proliferative rate were decreased（P〈0.05）, while the expression of Bax protein and the apoptotic rate were increased in the C3 G CRISPR/Cas9 and C3 G CRISPR/Cas9＋Hypoxia groups（P〈0.05）. Compared with the NT CRISPR/Cas9 group, the expression of C3 G m RNA and protein（P〈0.05）, p-ERK1/2 and Bcl-2 proteins and the proliferative rate were decreased in the NT CRISPR/Cas9＋Hypoxia group（P〈0.05）, while the expression of Bax protein and cell apoptotic rate were increased（P〈0.05）. Compared with the C3 G CRISPR/Cas9 group, the expression of p-ERK1/2 and Bcl-2 proteins and the proliferative rate were decreased in the C3 G CRISPR/Cas9＋Hypoxia（P〈0.05）, while the expression of Bax protein and cell apoptotic rate were increased（P〈0.05）. The above results demonstrated that C3 G knockout can inhibit the proliferation and promote the apoptosis of H9 C2 cardiomyocytes through regulation of p-ERK1/2, Bcl-2 and Bax.