原花青素提高人舌癌细胞系放射敏感性及机制研究

Effect and mechanism of grape seed proanthocyanidin on improving radiosensitivity of tongue cancer cells

目的探讨原花青素(grapeseedproanthocyanidins,GSP)提高人舌癌细胞系放射敏感性及机制。方法2019年3月至2020年2月,将对数期生长的人舌癌Tca8113细胞分为对照组(TcaCON),Tca8113细胞加10 mg/LGSP组(TcaGSP-10 mg/L)、Tca8113细胞加20 mg/LGSP组(TcaGSP-20 mg/L)、Tca8113细胞照射组(TcaIR)、Tca8113细胞照射加10 mg/LGSP组(TcaIR+GSP-10 mg/L)、Tca8113细胞照射加20 mg/LGSP组(TcaIR+GSP-20 mg/L)。MTT法检测加入不同浓度GSP对舌癌细胞Tca8113增殖的影响;流式细胞术检测6 MVX线直线加速器照射后(6 Gy)GSP对Tca8113细胞凋亡的影响;实时荧光定量逆转录聚合酶链反应(qRT-PCR)实验检测在加入不同浓度GSP作用下对照射后Tca8113细胞微小RNA(miR)-124表达的影响;蛋白质印迹法(Westernblotting)检测加入不同浓度GSP作用下对照射后Tca8113细胞B细胞淋巴瘤-2(Bcl-2)和胱天蛋白酶-9(caspase-9)蛋白水平的影响。结果MTT法结果显示,TcaIR组、TcaIR+GSP-10 mg/L组及TcaIR+GSP-20 mg/L组Tca8113细胞存活率分别为(63.21±2.97)%、(41.19±4.33)%及(19.61±2.51)%,GSP加强了照射后对人舌癌Tca8113细胞的抑制,且随药物浓度的增大,抑制效果增强(F=126.30,P<0.001);流式细胞术结果显示,TcaIR组、TcaIR+GSP-10 mg/L组及TcaIR+GSP-20 mg/L组Tca8113细胞凋亡率分别为(7.62±0.27)%、(9.61±0.42)%及(17.50±0.37)%,GSP显著提高了照射后Tca8113细胞的凋亡率(F=55.77,P=0.001);qRT-PCR结果显示,TcaIR组、TcaIR+GSP-10 mg/L组及TcaIR+GSP-20 mg/L组Tca8113细胞miR-124的表达水平分别为(1.43±0.29)、(2.64±0.45)及(3.81±0.44),照射给药组(加10 mg/LGSP组、加20 mg/LGSP组)Tca8113细胞miR-124的表达有明显上调(F=26.54,P=0.001);蛋白质印迹法结果显示,与单纯照射组[Bcl-2:(0.77±0.25),caspase-9:(1.97±0.33)]相比,照射给药组[10 mg/LGSPBcl-2及caspase-9分别为(0.43±0.23)、(2.58±0.43),20 mg/LGSPBcl-2及caspase-9分别为(0.31±0.17)、(2.94±0.52)],Bcl-2表达明显下调(F=7.10,P=0.006),而caspase-9的表达明显上调(F=7.67,P=0.005)。结论GSP可能通过上调miR-124的表达和增强细胞凋亡提高舌癌Tca8113细胞的放射敏感性。

Objective To investigate the effect and mechanism of proanthocyanidin on improving radiosensitivity of tongue cancer cells.Methods From March 2019 to February 2020,human tongue cancer Tca8113 cells growing in logarithmic phase were assigned into control group(Tca CON),Tca8113 cells plus 10 mg/L GSP group(Tca GSP-10 mg/L),Tca8113 cells plus 20 mg/L GSP group(Tca GSP-20 mg/L),Tca8113 cell irradiation group(Tca IR),Tca8113 cell irradiation plus 10 mg/L GSP group(Tca IR+GSP-10 mg/L),and Tca8113 cell irradiation plus 20 mg/L GSP group(Tca IR+GSP-20 mg/L).MTT experiments were performed to detect the effects of GSP with different concentrations on the proliferation of Tca8113cells;flow cytometry was performed to detect the effects of GSP after 6 MV X-ray linear accelerator irradiation(6 Gy)on the apoptosis of Tca8113 cells;real-time fluorescence quantitative reverse transcrip⁃tion polymerase chain reaction(qRT-PCR)experiments were performed to detect the effects of GSP with different concentrations on the expression of miR-124 in Tca8113 cells after irradiation;the Western blotting test was performed to detect the effects of GSP with dif⁃ferent concentrations on the levels of Bcl-2 and caspase-9 protein in Tca8113 cells after irradiation.Results MTT results showed that the survival rates of Tca8113 cells in Tca IR group,Tca IR+GSP-10 mg/L group and Tca IR+GSP-20 mg/L group were(63.21±2.97)%,(41.19±4.33)%and(19.61%±2.51)%.GSP enhanced the inhibition of human tongue cancer Tca8113 cells after irradiation,and the in⁃hibitory effect was enhanced with the increase in drug concentration(F=126.30,P<0.001).The results of flow cytometry showed that the apoptosis rates of Tca8113 cells in Tca IR group,Tca IR+GSP-10 mg/L group and Tca IR+GSP-20 mg/L group were(7.62±0.27)%,(9.61±0.42)%and(17.50±0.37)%,respectively.GSP significantly increased the apoptosis rate of Tca8113 cells after irradiation(F=55.77,P=0.001).qRT-PCR results showed that the expression levels of cellular miR-124 in Tca8113 cells in Tca IR group,Tca IR+GSP-10 mg/L group and Tca IR+GSP-20 mg/L group were(1.43±0.29),(2.64±0.45)and(3.81±0.44),respectively.The expression lev⁃els of miR-124 in Tca8113 cells in irradiation plus drug administration group(plus 10 mg/L GSP group and 20 mg/L GSP group)were significantly up-regulated(F=26.54,P=0.001).Western blotting results showed that compared with the irradiation group[Bcl-2:(0.77±0.25),caspase-9:(1.97±0.33)],the irradiation plus drug administration group[plus 10 mg/L GSP group:Bcl-2 and caspase-9 were(0.43±0.23),(2.58±0.43),respectively;plus 20 mg/L GSP group:Bcl-2 and caspase-9 were(0.31±0.17),(2.94±0.52),respectively]had a significantly down-regulated expression of Bcl-2(F=7.10,P=0.006)and a significantly up-regulated expression of caspase-9(F=7.67,P=0.005).Conclusion The radiosensitivity of tongue cancer Tca8113 cells was improved with GSP by up-regulating the expression of miR-124 and enhancing apoptosis.