miR-155对乙型肝炎病毒复制及PTEN表达的影响

The effect of miR-155 on HBV replication and PTEN expression in vivo

目的构建mmu-miR-155真核过表达载体pmR-155，探讨其在乙型肝炎病毒（HBV）转基因小鼠中对HBV复制及PTEN基因表达的调控作用。方法PCR扩增mmu-miR-155的前体基因片段（pre-mmu-miR-155），双酶切后连接到pmR-mCherry载体上，通过菌落PCR、双酶切和测序验证重组载体的准确性。将重组载体采用高压尾静脉法注入HBV转基因小鼠体内作为实验组，同时设空质粒组（注入pmR-mCherry空质粒）、空白组（注入等体积磷酸盐缓冲液），7d后qPCR检测各组细胞miR-155的表达情况，14d采用酶联免疫吸附法检测干扰素（IFN）Y表达量；1个月后qPCR检测肝内细胞因子信号抑制物（SOCS）1、PTEN基因mRNA表达量及Westernblot检测肝内socs1、PTEN、HBX蛋白表达水平。两组数据比较采用独立样本t检验；三组数据比较采用方差分析，进一步两两比较采用LSD法。结果经菌落PCR、双酶切和测序证实，pre-mmu-miR-155基因片段成功插入pmR-mCherry载体中。注入质粒7d后，实验组表达miR-155的水平明显升高（t=8．90，P〈0．01）；注入质粒14d后，实验组IFNy表达量较空白组升高（F=26．58，P〈0．01）；注入质粒30d后，实验组肝内SOCSl、PTENmRNA（FSOCSI mRNA=19．72，P〈0．01；FpTEN。RNA=7．38，P〈0．05）及蛋白表达水平（Fsocs1=50．30，P〈0．01；FPTEN=129．00，P〈0．01）均下降，同时HBX表达水平下降（FHBX=77．97，P〈0．01）。结论成功构建mmu-miR-155真核过表达载体pmR-155，该重组载体可稳定表达mmu-miR-155；miR-155可以通过下调SOCSl表达，进而促进IFNy的表达，增强HBV转基因小鼠的抗HBV能力。与此同时，miR-155可下调PTEN的表达，有潜在的促进肝癌发生的可能。

Objective To construct the mmu-miR-155 eukaryotic overexpression vector pmR- 155 and to investigate its effect on HBV replication and expression of PTEN in vivo. Methods The mmu- mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR- mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR, double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice（Experimental Group）with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid, PBS were respectively transfected into the mice as Empty plasmid Group, Blank Group. The concentration of IFN-y in the serum was detected by ELISA. The expression of SOCS 1, PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1, PTEN, HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student＇s t-test, or one-way analysis of variance and the least significant difference test. Results the colony PCR, double enzyme digestion and sequencing verified that the gene was inserted into the pmR- mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased（t - 8.90, P 〈 0.01）; the concentration of IFN-7 in the Experimental Group was significantly increased（F = 26.58, P 〈 0.01）; the mRNA（Fsocs1 mRNA = 19.72, P 〈 0.01; FPTEN mRNA = 7.38, P 〈 0.05） and protein（Fsocs1= 50.30, P 〈 0.01; FPVEN -129.00, P 〈 0.01） expression of COCS1, PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly（FHBx = 77.97, P 〈 0.01）. Conclusion The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs 1, PTEN gene expression and up-regulate the expression of IFN-y, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.