预知子种子提取物对核糖体蛋白抑制HepG2肝癌细胞增殖调控作用研究

Regulation effect of Akebiae fructus seed extract on inhibiting malignant proliferation in HepG2 hepatoma cells

目的预知子是临床常用抗癌中药,具体作用机制仍不明确.本研究观察预知子种子提取物干预HepG2肝癌细胞后,对核糖体蛋白mRNA、蛋白水平及Mdm2-p53信号通路关键蛋白影响,部分明确预知子抑制肿瘤细胞生长的核糖体蛋白相关作用机制.方法 HepG2细胞中分别给予预知子种子提取物对照组0μg/mL、低浓度组375μg/mL和高浓度组750μg/mL,观察对细胞生长活力和周期影响,实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测12个核糖体蛋白及p53 mRNA变化,蛋白质印迹法检测典型核糖体蛋白RPS26、RPSA、Mdm2、p53和细胞周期蛋白变化.结果预知子种子提取物干预后,对照组、低浓度组和高浓度组HepG2细胞活力分别为(100.000±1.509、105.060±1.000和61.130±0.700),差异有统计学意义,F=425.760,P<0.001.PCR法检测结果显示,高浓度组RPSA(F=4959.917,P<0.001)、RPS3A(F=725.900,P<0.001)、RPS8(F=350.515,P<0.001)、RPS13(F=198.316,P<0.001)、RPS16(F=41.693,P<0.001)、RPS29(F=536.835,P<0.001)、RPL6(F=119.226,P<0.001)、RPL10(F=49.339,P<0.001)、RPL15(F=35.603,P<0.001)、RPL17(F=188.221,P<0.001)和RPLP0(F=221.837,P<0.001)等11个核糖体蛋白mRNA表达下调,RPS26(F=330.023,P<0.001)mRNA表达上调.蛋白质印迹法结果显示,RPS26(t=7.365,P=0.002)、RPSA(t=4.654,P=0.010)和Mdm2(t=4.048,P=0.016)表达下调,p53蛋白水平(t=3.185,P=0.033)和mRNA水平(t=16.110,P<0.001)表达上调.干预后HepG2细胞S期比例增多,G0/G1期和G2/M期比例减少,细胞进入S期停滞.细胞周期蛋白CCND2(t=6.069,P=0.004)和CCNB1(t=5.481,P=0.005)大幅度下调,CCNE1小幅度下调(t=2.905,P=0.044).结论预知子可能通过上调或下调核糖体蛋白mRNA或蛋白表达,抑制Mdm2并激活p53,诱导HepG2细胞进入S期停滞,抑制HepG2细胞增殖.

OBJECTIVE Akebiae frutus( Yuzhizi) is a commonly used anti-cancer traditional Chinese medicine, while the specific mechanism was still unclear. This study was to observe the mRNA and protein level of a number of ribosomal proteins and Mdm2-p53 pathway key protein level change in HepG2 hepatoma cells after Chinese medicine Akebiae fructus treatment,for identifying partly mechanism of ribosomal proteins in Akebiae fructus inhibiting hepatoma cell malignant proliferation. METHODS Akebiae fructus seed extract were given to HepG2 cells in control group (0 μg/ml), low concentration group (375 μg/ml) and high concentration group (750 μg/ml),and the effects on cell growth viability and cell cycle were observed. PCR were used to detect the mRNA level change of 12 ribosomal proteins and p53 ,and western blot were used to detect protein level change of typical ribosomal proteins RPS26, RPSA,and Mdm2, p53,and cell cyclin proteins. RESULTS After treatment, the cell viability of control, low concentration and high concentration group were 100. 000±1. 509,105. 060±1. 000 and 61. 130±0. 700,the difference was statistically significant,F= 425. 8,P<0. 001. PCR results showed that mRNA levels in 11 genes including RPSA (F=4 959. 917,P<0. 001),RPS3A (F= 725. 900,P<0. 001),RPS8 (F=350. 515 ,P<0. 001),RPS13 (F=198.316, P<0. 001),RPS16 (F=41. 693, P<0. 001),RPS29 (F=536. 835,P<0. 001),RPL6 (F=119. 226,P<0. 001),RPL10 (F = 49. 339, P<0. 001), RPL15 (F=35. 603, P< 0. 001),RPL17 (F= 188. 221,P<0. 001) and RPLP0 (F=221. 837,P<0. 001) were downregulated,while RPS26 (F= 330. 023,P<0. 001) was upregulated. Western blot results showed that RPS26 (t=7. 365 , P = 0. 002),RPSA (t=4. 654, P = 0. 010),Mdm2 (t= 4. 048,P = 0. 016) proteins levels were downregulated,and protein level (t=3. 185, P = 0. 033) and mRNA level (t= 16. 110,P<0. 001) of p53 was upregulate. After treatment,the proportion of S phase in HepG2 cells increased,and the proportion of G0/G1 phase and G2/M phase decreased,cells were induced into S phase arrest. Cell cyclin protein CCND2 (t= 6. 069,P=0. 004) and CCNB1(t=5. 481,P = 0. 005) were downregulated largely,while CCNE1 (t= 2. 905,P = 0. 044) was downregulated slightly. CONCLUSION Akebiae fructus may inhibit HepG2 cell proliferation by up-regulating or down-regulating ribosomal protein mRNA or protein expression,inhibiting Mdm2 and activating p53,and inducing HepG2 cells to enter S phase arrest.