利用Crispr/Cas9构建POMP基因组原位荧光报告系统及其应用

Construction of m Cherry reporter system of POMP by Crispr/Cas9 and its application

目的利用Crispr/Cas9系统建立蛋白酶体成熟蛋白(proteasome maturation protein,POMP)基因组原位荧光报告系统。方法构建POMP-2A-mCherry-T载体并转染HEK293(human embryonic kdiney 293 cell)细胞;流式细胞仪分选获取m Cherry阴阳性细胞;实时荧光定量PCR(real-time fluorescent quantitative PCR)检测mCherry阴阳性细胞中POMP基因转录水平表达差异;Western blot检测mCherry阴阳性细胞中POMP蛋白表达差异以及Huh7细胞中JNK1/JNK2磷酸化水平;蛋白酶体活性试剂盒检测mCherry阴阳性细胞中蛋白酶体活性差异。结果 mCherry阳性细胞POMP蛋白表达水平比mCherry阴性细胞高1.3倍(P<0.01),但其基因转录水平差异并不显著(P>0.05);同时mCherry阴性细胞中泛素化水平比mCherry阳性细胞高1.2倍(P<0.01);另外mCherry阳性细胞中蛋白酶体活性也显著高于阴性细胞(P<0.01);低表达POMP的Huh7细胞中,JNK1和JNK2的磷酸化水平分别升高1.5倍和1.4倍(P<0.01)。结论成功构建POMP荧光报告系统;将该系统用于Huh7细胞,发现POMP可改变JNK1、JNK2磷酸化水平,其可能通过JNK信号通路来影响细胞的增殖以及凋亡。

Objective To construct m Cherry reporter system of proteasome maturation protein（ POMP） by Crispr/Cas9 and study the impact of POMP on cancer cells. Methods POMP-2A-m Cherry-T vector was constructed and transferred into human embryonic kidney 293 cells（ HEK293 cells）. The m Cherrypositive cells and m Cherry-negative cells were sorted by flow cytometry. Real-time quantitative PCR（ RTq PCR） was used to detect the POMP gene transcription level in the m Cherry-positive cells and m Cherrynegative cells. Western blotting was adopted to detect the POMP protein expression and JNK1 /2phosphorylation in Huh7 cells. Proteasome activity of m Cherry-positive cells and m Cherry-negative cells was detected with proteasome activity detection kit. Results The protein expression of POMP in the m Cherrypositive cells was increased by 1. 3 times（ P〈0. 01） compared with the m Cherry-negative cells,but the POMP gene transcription level had no significant difference between the 2 kinds of cells（ P〈0. 05）. The level of ubiquitin in the m Cherry-negative cells was obviously elevated by 1. 2 times than that of m Cherrypositive cells（ P〈0. 01）. The proteasome activity was relatively high in the m Cherry-positive cells（ P〈0. 01）. The JNK1 and of JNK2 phosphorylation levels were increased by 1. 5 times and 1. 4 times,respectively,in the Huh7 cells with low expression of POMP（ P〈0. 01）. Conclusion The POMP luciferase reporter system is successfully constructed. After the system is applied to Huh7 cells,POMP can change JNK1 /2 phosphorylation levels,so it may affect the cell proliferation and apoptosis through JNK signaling pathway.