肝癌干细胞外泌体miR-196a增强肝癌细胞对多柔比星的耐药性

Exosomal miR-196a derived from liver cancer stem cell enhances liver cancer cells resistance to doxorubicin

目的筛选肝癌干细胞来源外泌体中差异表达的miRNA并分析其对肝癌细胞恶性生物学特征的影响。方法采用miRNA表达谱芯片分析肝癌干细胞来源外泌体中差异表达的miRNA并鉴定miRNA对肝癌干细胞恶性表型的作用。采用不同浓度(0、150、300μmol/L)多柔比星处理细胞,采用实时定量PCR(qRT-PCR)检测miR-196a表达水平的变化;分别采用肝癌干细胞来源外泌体(Exo-NC组)、转染miR-196a抑制剂处理后的外泌体(Exo-Inhibitor组)培养肝癌细胞,检测肝癌细胞的凋亡情况以及caspase3/7的活性。按照随机数字表法将裸鼠分为Do-PBS组、Do-Exo-Inhibitor组、Do-Exo-NC组,每组各5只,采用裸鼠移植瘤实验分析miR-196a对肝癌细胞裸鼠移植瘤模型的作用。结果本研究分离纯化了CD133^+Huh7干细胞培养上清中的外泌体,发现miR-7162-3p、miR-1910-5p、miR-3613-3p、miR-196a、miR-155-5p表达上调,miR-1246和miR-3613-5p表达下调。外泌体中miR-7162-3p、miR-196a和miR-155-5p对肝癌干细胞的自我更新能力具有重要影响;miR-1910-5p、miR-196a和miR-155-5p对肝癌干细胞的侵袭能力具有重要影响,其中miR-196a的抑制效果最显著。在反应24 h时,多柔比星浓度在0、150、300μmol/L时miR-196a的表达量分别为0.96±0.05、1.23±0.05和2.33±0.03,差异具有统计学意义(F=996.90,P<0.001);48 h时,多柔比星浓度在0、150、300μmol/L时miR-196a的表达量分别为1.02±0.07、2.35±0.05和2.89±0.55,差异具有统计学意义(F=303.00,P<0.001)。Exo-NC组肝癌细胞在多柔比星浓度为0和300μmol/L时的细胞凋亡率分别为9.37%±0.19%和11.64%±0.27%,Exo-Inhibitor组分别为18.80%±1.91%和22.79%±1.57%,差异均具有统计学意义(t=4.41,P=0.048;t=4.96,P=0.038)。未使用多柔比星处理时,Exo-NC组在24 h和48 h的caspase3/7比值分别为0.94±0.08和0.97±0.09,Exo-Inhibitor组分别为1.56±0.01和1.58±0.01,差异均具有统计学意义(t=11.41,P=0.008;t=6.07,P=0.026)。使用300μmol/L多柔比星处理细胞后,Exo-NC组在24 h和48 h的caspase3/7比值分别为0.95±0.07、1.36±0.08,Exo-Inhibitor组分别为2.84±0.08、3.20±0.14,差异均具有统计学意义(t=24.20,P=0.002;t=15.78,P=0.004)。Do-PBS组、Do-Exo-Inhibitor组和Do-Exo-NC组3组裸鼠的移植瘤体积依次增大,分别为(1051.86±89.90)mm^3、(1310.91±86.66)mm^3和(2185.14±352.34)mm^3,差异具有统计学意义(F=30.28,P<0.001);3组移植瘤重量依次增加,分别为(0.36±0.10)g、(0.39±0.12)g和(0.76±0.16)g,差异具有统计学意义(F=11.81,P=0.002);转染miR-196a抑制剂后裸鼠移植瘤肿瘤内miR-196a的表达显著降低,3组表达量分别为1.05±0.16、0.38±0.08和2.17±0.26,差异具有统计学意义(F=48.93,P<0.001)。结论肝癌干细胞分泌的外泌体可通过其中的miR-196a增强肝癌细胞对多柔比星的耐药性。

Objective To screen the differentially expressed exosomal miRNAs derived from liver cancer stem cells(LCSCs)and its effect on the malignant biological characteristics of liver cancer cells.Methods miRNA expression profile chip was used to analyze the differentially expressed exosomal miRNA derived from LCSCs.The effects of miRNA on malignant phenotypes of LCSCs were identified.The cells were further treated with doxorubicin at different concentrations(0,150,300μmol/L),and the expression level of miR-196a was detected by quantitative real-time PCR(qRT-PCR).The apoptosis of liver cancer cells cultured by exosomes derived from LCSCs(Exo-NC group)and exosomes derived from miR-196a inhibited LCSCs(Exo-Inhibitor group)and the activity of caspase3/7 under the action of exosomes from LCSCs were detected.Nude mice were randomly divided into Do-PBS group,Do-Exo-Inhibitor group and Do-Exo-NC group using random number table method,with 5 mice in each group,and the effect of miR-196a on nude mice xenograft tumor model with liver cancer cells was analyzed.Results In this study,exosomes were isolated and purified from CD133^+Huh7 stem cell culture supernatant.miR-7162-3p,miR-1910-5,miR-3613-3p,miR-196a and miR-155-5p were up-regulated,while miR-1246 and miR-3613-5p were down-regulated.miR-7162-3p,miR-196a and miR-155-5p in exosomes had important effects on the self-renewal ability of LCSCs.miR-1910-5p,miR-196a and miR-155-5p had important effects on the invasion ability of liver cancer stem cells,among which miR-196a had the most significant inhibitory effect.Treatment for 24 h,the miR-196a expression level of the 0,150 and 300μmol/L doxorubicin was 0.96±0.05,1.23±0.05 and 2.33±0.03 respectively,with a statistically significant difference(F=996.90,P<0.001).Treatment for 48 h,the miR-196a expression level of the 0,150 and 300μmol/L doxorubicin were 1.02±0.07,2.35±0.05 and 2.89±0.55 respectively,with a statistically significant difference(F=303.00,P<0.001).When the concentration of doxorubicin was 0 and 300μmol/L,the apoptosis rates of the Exo-NC group were 9.37%±0.19%and 11.64%±0.27%,and those of the Exo-Inhibitor group were were 18.80%±1.91%and 22.79%±1.57%,with statistically significant differences(t=4.41,P=0.048;t=4.96,P=0.038).When doxorubicin was not used,the ratios of caspase3/7 in the Exo-NC group at 24 h and 48 h were 0.94±0.08 and 0.97±0.09,and those in the Exo-Inhibitor group were 1.56±0.01 and 1.58±0.01,with statistically significant differences(t=11.41,P=0.008;t=6.07,P=0.026).Under 300μmol/L doxorubicin,the ratios of caspase3/7 in the Exo-NC group at 24 h and 48 h were 0.95±0.07 and 1.36±0.08,and those in the Exo-Inhibitor group were 2.84±0.08 and 3.20±0.14,with statistically significant differences(t=24.20,P=0.002;t=15.78,P=0.004).The results of xenograft tumor in nude mice showed that the tumor volumes of Do-PBS,Do-Exo-Inhibitor and Do-Exo-NC groups increased successively,which were(1051.86±89.90)mm^3,(1310.91±86.66)mm^3 and(2185.14±352.34)mm^3 respectively,with a statistically significant difference(F=30.28,P<0.001).The weights of the transplanted tumors in the 3 groups increased successively,which were(0.36±0.10)g,(0.39±0.12)g and(0.76±0.16)g respectively,with a statistically significant difference(F=11.81,P=0.002).The expression of miR-196a in tumors was significantly decreased after miR-196a inhibitor transfection.The expression levels of the 3 groups were 1.05±0.16,0.38±0.08 and 2.17±0.26,with a statistically significant difference(F=48.93,P<0.001).Conclusion The exosomal secreted by LCSCs can enhance the resistance of liver cancer cells to doxorubicin by miR-196a.