慢病毒介导CD36基因沉默细胞株的构建及其对caveolin-1蛋白表达的影响

Construction of CD36 gene silencing cell lines by lentivirus-mediated RNA interference and the effect on protein expression of caveolin-1

摘要:

CD36作为重要的清道夫受体密切参与了巨噬细胞对氧化低密度脂蛋白的摄取作用，为了进一步研究CD36的功能，本文利用慢病毒介导的shRNA干扰技术，构建了CD36基因沉默巨噬细胞 (J774A.1) 株，并以此为模型分析了CD36在caveolin-1蛋白表达过程中的作用。首先，针对CD36基因序列设计合成5条shRNA片段，并构建得到pLKO.1-CD36-shRNA慢病毒干扰载体，测序鉴定后与psiCHECK-Ⅱ-CD36载体共转染入293T细胞中，筛选出有效的CD36-shRNA。将慢病毒干扰载体与病毒包装质粒共转染入293T细胞，包装得到慢病毒颗粒，之后感染J774A.1细胞，经嘌呤霉素筛选后得到CD36基因沉默稳转细胞株。Western blotting及激光共聚焦检测结果表明CD36基因沉默效率达90%，并且伴随着CD36的基因沉默，与之结合的DiI-oxLDL也随之大幅降低，证明构建成功具有良好生物学活性的CD36基因沉默细胞株。最后，抑制剂处理及oxLDL给药刺激实验结果表明，CD36的基因沉默能够显著降低JNK及ERK的磷酸化水平，进而抑制了caveolin-1的蛋白表达，表明CD36能够经由JNK及EKR信号传导调节caveolin-1的蛋白表达。

Abstract:

CD36, the major scavenger receptor, is intimately involved in the uptake of oxLDL in macrophages. To further study the function of CD36 in macrophages, we constructed CD36 gene silence cell lines (J774A.1) by lentivirus-mediated RNA interference technique, and analyzed the effect of CD36 in caveolin-1 protein expression. At first, 5 shRNA fragments were designed and synthesized according to the coding sequence (CDS) region of CD36 gene. Next, the CD36-shRNA was inserted into lentiviral vector to yield pLKO.1-CD36-shRNA plasmid. After DNA sequencing, the pLKO.1-CD36-shRNA plasmid and psiCHECK-II-CD36 were co-transfected into the 293T cells to screen the efficient CD36-shRNA. The efficient CD36-shRNA plasmid and the helper plasmid were co-transfected into the 293T cells to package the lentivirus, and then infected the J774A.1 cells. After screening by puromycin, CD36 gene silence cell lines (J774A.1) was established. Western blotting and confocal fluorescence microscopy results showed that the CD36 silencing efficiency in the gene silence cell line was 90%. Accompanied by a decrease in CD36 protein on cell surface, oxLDL binding to CD36 was significantly inhibited, indicating that the CD36 gene silence cell line is successfully established. Finally, the oxLDL stimulation and inhibitor experiments results showed that the CD36 knockdown significantly suppresses the phosphorylation of JNK and ERK, thereby inhibiting the oxLDL-induced caveolin-1 protein expression, demonstrating that CD36 modulates the caveolin-1 protein expression through the JNK/ERK-mediated signaling transduction.