Montaño-Samaniego Mariela, Sánchez-Cedillo Jorge, Lucas-González Amellalli, Bravo-Estupiñan Diana M, Alarcón-Hernández Ernesto, Rivera-Gutiérrez Sandra, Balderas-López José Abraham, Ibáñez-Hernández Miguel
Laboratorio de Terapia Génica, Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11340, Mexico.
Laboratorio de Técnicas Fototérmicas, Departamento de Ciencias Básicas, Unidad Politécnica Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, Mexico City 07340, Mexico.
Curr Issues Mol Biol. 2023 Aug 24;45(9):7043-7057. doi: 10.3390/cimb45090445.
Atherosclerosis is the leading cause of cardiovascular diseases in Mexico and worldwide. The membrane transporters ABCA1 and ABCG1 are involved in the reverse transport of cholesterol and stimulate the HDL synthesis in hepatocytes, therefore the deficiency of these transporters promotes the acceleration of atherosclerosis. MicroRNA-33 (miR-33) plays an important role in lipid metabolism and exerts a negative regulation on the transporters ABCA1 and ABCG1. It is known that by inhibiting the function of miR-33 with antisense RNA, HDL levels increase and atherogenic risk decreases. Therefore, in this work, a genetic construct, pPEPCK-antimiR-33-IRES2-EGFP, containing a specific antimiR-33 sponge with two binding sites for miR-33 governed under the PEPCK promoter was designed, constructed, and characterized, the identity of which was confirmed by enzymatic restriction, PCR, and sequencing. Hep G2 and Hek 293 FT cell lines, as well as a mouse hepatocyte primary cell culture were transfected with this plasmid construction showing expression specificity of the PEPCK promoter in hepatic cells. An analysis of the relative expression of miR-33 target messengers showed that the antimiR-33 sponge indirectly induces the expression of its target messengers (ABCA1 and ABCG1). This strategy could open new specific therapeutic options for hypercholesterolemia and atherosclerosis, by blocking the miR-33 specifically in hepatocytes.
动脉粥样硬化是墨西哥乃至全球心血管疾病的主要病因。膜转运蛋白ABCA1和ABCG1参与胆固醇的逆向转运，并刺激肝细胞中高密度脂蛋白（HDL）的合成，因此这些转运蛋白的缺乏会促进动脉粥样硬化的加速发展。微小RNA-33（miR-33）在脂质代谢中起重要作用，并对转运蛋白ABCA1和ABCG1发挥负调控作用。已知通过用反义RNA抑制miR-33的功能，HDL水平会升高，动脉粥样硬化风险会降低。因此，在本研究中，设计、构建并鉴定了一种基因构建体pPEPCK-antimiR-33-IRES2-EGFP，其包含一个特定的抗miR-33海绵体，带有两个受磷酸烯醇丙酮酸羧激酶（PEPCK）启动子调控的miR-33结合位点，通过酶切、聚合酶链反应（PCR）和测序确认了其身份。用该质粒构建体转染Hep G2和Hek 293 FT细胞系以及小鼠肝细胞原代培养物，结果显示PEPCK启动子在肝细胞中具有表达特异性。对miR-33靶信使RNA的相对表达分析表明，抗miR-33海绵体间接诱导其靶信使RNA（ABCA1和ABCG1）的表达。通过在肝细胞中特异性阻断miR-33，该策略可为高胆固醇血症和动脉粥样硬化开辟新的特异性治疗选择。
