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

Document Type: Research article

Authors

1 Medical Biotechnology Research Center, School of paramedicine, Guilan University of Medical Sciences, Rasht, Iran.

2 Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

3 Department of Medical Microbiology, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran.

Abstract

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 consisted 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), respectively. 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 significantly 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.

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