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miRNAs as one of the potential therapeutic agents have been recently considered for cancer treatment. AS1411 (aptNCL) is a DNA aptamer specifically binding to nucleolin protein on the cancer cell surface with antiproliferative effect. The aim of the study was to develop a conjugate consisting of aptNCL (as targeted delivery of therapeutic agent) and miRNA let-7d (as a tumor suppressor) using two different linking methods and also to evaluate the potential effect of the conjugates on the proliferation of gastric cancer (MKN-45) cell line compared to negative control cell line of human dermal fibroblast (HDF). Conjugation was performed covalently by SM (PEG)2 as a bifunctional crosslinker (conjugate-1) and noncovalently, using 19bp complementary sticky end sequences (conjugate-2). Nucleolin positive MKN-45 and nucleolin negative HDF cells were cultured and treated with the conjugates. Then, the changes in let-7d expression and cell proliferation were determined using Real-time PCR and MTT methods, respectively. In MKN-45 cells, the conjugates caused significant increase in let7-d uptake compared with HDF cells (P = 0.0001). The conjugate-1, likely due to its higher stability compared with the conjugate-2, led to significantly more increase in intracellular let-7d in MKN-45 cells (30 fold versus 15 fold, respectively, P = 0.0001). The conjugates revealed more potent antiproliferative effect against gastric cancer cells compared with aptNCL alone (P = 0.0001). It was found that the aptNCL-let-7d conjugate efficiently carried let-7d into the cancer cells. Also, it appears that in the setting of aptNCL-let-7d conjugate, let-7d and aptNCL moieties could cooperate and synergistically exhibit the antiproliferative effect on cancer cells.

Aptamer-based Targeted Delivery of miRNA let-7d to Gastric Cancer Cells as a Novel Anti-Tumor Therapeutic Agent