抗肿瘤肽Mt5对人肝癌细胞增殖和凋亡的影响及机制

Effect of antitumor peptide Mt5 on proliferation and apoptosis of human hepatoma cells and its mechanism

目的探讨抗肿瘤肽Mt5通过磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT)信号通路对人肝癌细胞增殖与凋亡的影响。方法采用pymol构建Mt5的三维可视化结构示意图,Prot Param分析Mt5的正电荷和疏水性,NPS分析Mt5的α-螺旋所占比例;运用实时无标记细胞分析(RTCA)技术检测不同浓度Mt5(50 mg/L、100mg/L、200 mg/L、400 mg/L)作用后的HepG2细胞活力;通过实时荧光定量PCR法(qRT-PCR)法检测不同浓度Mt5(25 mg/L、50 mg/L、100 mg/L)作用后HepG2细胞中PI3K、AKT信使RNA(mRNA)表达;以Western blot法检测不同浓度Mt5(25 mg/L、50 mg/L、100 mg/L)作用后凋亡相关蛋白B细胞淋巴瘤-2蛋白(Bcl-2)、Bcl-2相关X蛋白(bax)和PI3K、AKT蛋白表达。结果生物学信息结果表明,Mt5的正电荷为+8,疏水性为0.588,α-螺旋度为84.62%;RTCA结果显示Mt5不同浓度处理组作用后,HepG2细胞指数随着Mt5浓度的增大和时间的延长逐渐降低至0以下;qPCR结果显示,Mt5组HepG2细胞中PI3K和AKT mRNA较阴性对照组表达降低(P<0.05),bax蛋白表达较阴性对照组升高(P<0.05),且呈浓度依赖性;Western blot结果显示,Mt5组HepG2细胞中Bcl-2、PI3K及AKT蛋白表达较阴性对照组降低(P<0.05),且呈浓度依赖性。结论Mt5可抑制人肝癌细胞增殖及诱导细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。

Objective To investigate the effect of antitumor peptide Mt5 on the proliferation and apoptosis of human hepatoma cells via the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(AKT)signaling pathway.Method The 3D visual structure diagram of Mt5 was generated using pymol,and the positive charge and hydrophobicity of Mt5 were analyzed through Prot Param.Additionally,the proportion of α-helix in Mt5 was determined using NPS.The viability of HepG2 cells was assessed using RTCA following exposure to varying concentrations of Mt5(50 mg/L,100 mg/L,200 mg/L,and 400 mg/L).The mRNA levels of PI3K and AKT in HepG2 cells were assessed using qRT-PCR following treatment with different concentrations of Mt5(25 mg/L,50 mg/L,and 100 mg/L).Western blot was utilized to analyze the expression levels of apoptosis-related proteins B-cell lymphoma-2 protein(Bcl-2),Bcl2-associated X protein(bax),PI3K,and AKT following exposure to various concentrations of Mt5(25 mg/L,50 mg/L,and 100 mg/L).Results The biological information indicated that the Mt5 protein had a positive charge of+8,a hydrophobicity value of 0.588,and an α-helicity of 84.62%.The RTCA results demonstrated a gradual decrease in HepG2 cell index to below 0 with the increase of Mt5 concentration and time extension of treatment.The qPCR results revealed that the mRNA expressions of PI3K and AKT in HepG2 cells in the Mt5 group were significantly lower than those in the negative control group(P<0.05),and this decrease in mRNA expression was found to be concentration-dependent.Western blot indicated a concentration-dependent decrease in the protein expressions of Bcl-2,PI3K,and AKT in HepG2 cells treated with Mt5 compared with those in the negative control group(P<0.05).Conclusion The Mt5 is able to suppress the proliferation and induce apoptosis of human hepatoma cells,which may be related to the inhibition of PI3K/AKT signaling pathway.