Zhang Yuhong, Wu Jun, Wang Zihang, Xia Daozi, Li Guangsen, You Yue, Huang Dongmei, Bo Huaying, Hu Bin, Tang Jianwu
Department of Dignostic Ultrasound, Second Affiliated Hospital of Dalian Medical University, 116023 Dalian, PR China.
Department of Dignostic Ultrasound, Second Affiliated Hospital of Dalian Medical University, 116023 Dalian, PR China.
Biomed Pharmacother. 2016 Mar;78:1-7. doi: 10.1016/j.biopha.2015.12.026. Epub 2016 Jan 8.
The inhibitory effects on expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion were mediated by ultrasound-targeted microbubble destruction (UTMD).
The best shRNA vector was built and screened. The hepatocellular carcinoma cell lines were cultured in vitro and divided into five groups: the group of normal Hca-F cells, the group of shRNA plasmid (already selected from the above procedure), the group of Lipofectamine, the group of UTMD (ultrasound microbubbles combined with ultrasound exposure) and the group of Lipofectamine and UTMD. The transfection rate was observed by inverted fluorescence microscope. The expression levels of JNK1 mRNA and protein were evaluated by fluorescence quantitative PCR and Western Blot respectively. The cell proliferation was detected by CCK-8. The ability of migration and invasion in vitro was detected by transwell assay.
The best shRNA vector was established. The comparison of the transfection rate: The group of Lipofectamine and UTMD was larger than that of the groups of shRNA plasmid, Lipofectamine lipofection and UTMD (all P<0.05). There was no significant difference between the group of Lipofectamine and the group of UTMD (P>0.05). The comparison of the expression levels of JNK1 mRNA and protein: Both of the mRNA and protein expression levels were lowest in the group of Lipofectamine and UTMD (all P<0.05). CCK-8 showed that cell viability decreased most in the group of Lipofectamine and UTMD (all P<0.05); Transwell assay showed that the abilities of migration and invasion decreased most in the group of Lipofectamine and UTMD (all P<0.05).
The transfection rate of JNK1 shRNA can be improved through the combination of lipofection and UTMD in mouse hepatocellular carcinoma cell lines, therefore enhancing the inhibitory effects of gene expression. The inhibitory effects of cell proliferation, migration and invasion can also be enhanced.
探讨超声靶向微泡破坏（UTMD）介导对小鼠肝癌细胞系中JNK1表达及细胞迁移和侵袭的抑制作用。
构建并筛选最佳shRNA载体。体外培养肝癌细胞系，分为五组：正常Hca - F细胞组、shRNA质粒组（已从上述过程中筛选）、脂质体转染组、UTMD组（超声微泡联合超声照射）、脂质体转染与UTMD联合组。通过倒置荧光显微镜观察转染率。分别采用荧光定量PCR和蛋白质免疫印迹法评估JNK1 mRNA和蛋白的表达水平。采用CCK - 8检测细胞增殖情况。采用Transwell实验检测体外迁移和侵袭能力。
建立了最佳shRNA载体。转染率比较：脂质体转染与UTMD联合组大于shRNA质粒组、脂质体转染组和UTMD组（均P<0.05）。脂质体转染组与UTMD组之间差异无统计学意义（P>0.05）。JNK1 mRNA和蛋白表达水平比较：脂质体转染与UTMD联合组的mRNA和蛋白表达水平均最低（均P<0.05）。CCK - 8结果显示，脂质体转染与UTMD联合组细胞活力下降最为明显（均P<0.05）；Transwell实验显示，脂质体转染与UTMD联合组迁移和侵袭能力下降最为明显（均P<0.05）。
在小鼠肝癌细胞系中，脂质体转染与UTMD联合可提高JNK1 shRNA的转染率，从而增强基因表达的抑制作用。同时也可增强对细胞增殖、迁移和侵袭的抑制作用。
