吉非替尼对敲低ANXA7的HepG2细胞增殖和凋亡的影响

Influence of Gefitinib on Proliferation and Apoptosis of HepG2 Cells with ANXA7 Knockdown

该文目的为研究吉非替尼对敲低膜联蛋白A7(annexin A7,ANXA7)的人肝癌Hep G2细胞增殖及凋亡的影响,为肝癌的治疗提供参考依据。应用MTT法检测0.1、0.5、1.0、5.0、10.0、50.0μmol/L等浓度的吉非替尼作用72 h后Hep G2细胞的增殖情况,计算IC50为5μmol/L;将Hep G2细胞分为敲低加药组、单加药组、敲低组、阴性对照组和空白对照组,采用RNA干扰技术将靶向ANXA7的si RNA和阴性对照si RNA通过脂质体转染法分别转染入Hep G2细胞。转染72 h后采用Western blot法对ANXA7的抑制效果进行鉴定;用MTT法和流式细胞术检测Hep G2细胞增殖和凋亡情况。结果显示,吉非替尼浓度越大,对Hep G2细胞增殖的抑制作用越明显;靶向ANXA7的si RNA能显著抑制ANXA7的表达;敲低加药组、单加药组、敲低组、阴性对照组和空白对照组的生长抑制率分别为73.88%、48.67%、54.33%、1.02%和0.02%,早期凋亡率分别为8.48%、3.06%、2.93%、1.72%和1.64%。与阴性对照组和空白对照组相比,敲低加药组、单加药组和敲低组细胞的增殖抑制率和凋亡率均明显增加(P<0.05),且敲低加药组较敲低组对抑制细胞增殖、促进细胞凋亡的效果更为显著。由此说明,敲低ANXA7后联合药物吉非替尼能抑制Hep G2细胞的增殖,促进其凋亡,可能是一种临床治疗的方法。

The paper aimed at investigating the influences of Gefitinib on the proliferation and apoptosis of HepG2 cells with annexin A7 （ANXA7） knockdown in order to developing a potential therapy for hepatocellar carcinoma. The human hepatoblastoma cell line HepG2 cells were incubated for three days with Gefitinib at differ- ent concentrations ranging from 0.1 to 50, and then studied by MTT to measure 50% inhibitory concentration （ICs0） of Gefitinib, the IC50 was 5 μmol/L. The cells were divided into A7si combine Gefitinib group, Gefitinib group, A7si group, negative control group and blank control group. RNA interference technology was used to transfect the siRNA targeted ANXA7, scrambled siRNA into HepG2 cells with lipofectine 2000. After 72 hours, the suppression of ANXA7 on protein level was assured by Western blot. The inhibitory rate was tested using MTT assay and theapoptosis ratio was detected by flow cytometry. The results showed that HepG2 cells proliferation was significantly suppressed with Gefitinib concentration increasing in a does-dependent manner. The suppression of ANXA7 on protein level was assured by Western blot. It also showed by MTT assay and Flow cytometry that the inhibition rate and apoptotic rate of the HepG2 cells in the A7si combine Gefitinib group, Gefitinib group and A7si group, were much higher than that in the negative control group and the blank control group （P〈0.05）. When compared A7si combine Gefitinib group with Gefitinib group and A7si group respectively, the A7si combine Gefitinib group had the greatest effect of proliferation inhibition and apoptosis promotion. Taken together, our results suggested that Gefitinib and ANXA7 knockdown can synergistically inhibit HepG2 cell proliferation, and promote cell apoptosis which might be a potential method for clinic treatment.