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

Jingda LI; Chengjie YU; Renjun Wang; Changzhen FU; Zhilong Xiu; Qingping Liu

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

Author: Jingda LI ¹ ; Chengjie YU ² ; Renjun WANG ² ; Changzhen FU ² ; Zhilong XIU ¹ ; Qingping LIU ²
Author Information

1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China.
2. Key Laboratory of Glucolipid Metabolism in Liaoning Province & College of Life Science and Technology, Dalian University, Dalian 116600, Liaoning, China.
Publication Type:Journal Article
Keywords: CD36 receptor; caveolin-1; macrophage; oxidized low density lipoprotein; shRNA gene silence
From: Chinese Journal of Biotechnology 2018;34(1):110-121
CountryChina
Language:Chinese
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.