摘要
目的构建筛选最优Caprin⁃1敲除质粒并探讨超声微泡介导其转染对人肝癌HepG2细胞中cyclinD1、cyclinD2的表达及细胞增殖、周期的影响。方法构建Caprin⁃1敲除质粒KO⁃1、KO⁃2,测序鉴定重组质粒,将目的质粒转染入人肝癌HepG2细胞,超声转染条件利用前期研究成果,免疫印迹法(WB)筛选最优质粒,实时荧光定量PCR(qPCR)检测抑制Caprin⁃1表达后cyclinD1及cyclinD2表达情况,细胞增殖实验(CCK⁃8法)及流式细胞术(FCM)检测抑制Caprin⁃1表达对细胞增殖与周期的影响。结果设计Caprin⁃1敲除质粒Caprin⁃1⁃KO⁃1、Caprin⁃1⁃KO⁃2引物序列,提取质粒经过电泳及测序,鉴定质粒构建成功。转染目的质粒后,KO⁃2组Caprin⁃1表达显著降低,与阴性对照组(NC)、KO⁃1组比较,差异有统计学意义(P<0.05);且抑制Caprin⁃1表达明显抑制HepG2细胞的增殖,阻滞了细胞周期,cyclinD1,cyclinD2表达均明显下调,差异均有统计学意义(P<0.05)。结论超声微泡介导Caprin⁃1⁃KO转染人肝癌HepG2细胞,Caprin⁃1⁃KO对人肝癌HepG2细胞增殖有抑制作用并使细胞周期发生阻滞,cyclinD1及cyclinD2表达下降,为进一步研究Caprin⁃1在肝癌发病机理中的作用提供思路。
Objective To construct and screen the optimal Caprin⁃1 knockout plasmid and to explore the effect of cell proliferation,cell cycle andthe expression of cyclinD1 and cyclinD2 in hepatocellular carcinoma HepG2 cellsafterCaprin⁃1⁃KO transfectionmediated byultrasound⁃microbubbles.Method First of all,Caprin⁃1 knockout plasmids KO⁃1 and KO⁃2 were constructed and the recombinant plasmids were identified by sequencing.Secondly,after transfecting the target plasmid into HepG2 cells through ultrasound transfection conditions which explored in the previous research,the best quality granules were screened by WB.Finally,qPCR was selected to detect the expression of cyclinD1 and cyclinD2.CCK8 and FCM were used to detect the effect of inhibiting Caprin⁃1 expression on cell proliferation and cycle.Results In this study,the primers of Caprin⁃1 knockout plasmid KO⁃1 and KO⁃2 were designed.The extracted plasmids were constructed successfully after electrophoresis and sequenc⁃ing.The results showed that the expression of Caprin⁃1 in NC group and KO⁃1 group were higher compared to the KO⁃2 group(P<0.05).After inhibiting the expression of Caprin⁃1,the proliferation of HepG2 cells was significantly inhibited,and the cell cycle was blocked;the expression of cyclinD1 and cyclinD2 was significantly down⁃regulat⁃ed(P<0.05).Conclusion Caprin⁃1⁃KO were transfected into HepG2 cells by UTMD.The expression of Caprin⁃1⁃KO can inhibit the cell proliferation、block the cycle and decrease the expression of cyclinD1 and cyclinD2.This study is expected to provide ideas for further research on the role of Caprin⁃1 in the pathogenesis of liver cancer.
作者
陈佳琳
林翠燕
赖美燕
林健如
张慧
过新民
CHEN Jialin;LIN Cuiyan;LAI Meiyan;LIN Jianru;ZHANG Hui;GUO Xinmin(Department of Ultrasound Medicine,Guangzhou Red Cross Hospital,Ji′nan University,Guangzhou 510220,China)
出处
《实用医学杂志》
CAS
北大核心
2021年第10期1250-1256,共7页
The Journal of Practical Medicine
基金
广东省科技计划项目(编号:2016A020215015)。
