Title:The Effect of Different microRNA Backbones on Artificial miRNA Expression and Knockdown Activity Against HIV-1 Replication
Volume: 5
Issue: 2
Author(s): Ahmad Nejati, Masoud Soleimani, Ehsan Arefian, Sayed M. Marashi, Hamideh Tabatabaie, Mohammad Farahmand, Zabihollah Shoja, Mahmood Mahmoodi, Rakhshandeh Nategh and Shohreh Shahmahmoodi
Affiliation:
Keywords:
Artificial miRNA expression, biogenesis, HIV-1, microRNA, RNAi-based gene therapy, terminal loop structure.
Abstract: Background: Artificial microRNAs (miRNAs) are designed to develop
an RNAi-based gene therapy. Recently, it has been suggested that the flanking sequences
and terminal loop structure play a critical role in RNAi biogenesis and target
recognition, but no extensive study regarding the different miRNA backbone for
artificial miRNAs optimization has been conducted.
Objective: We tested three artificial miRNAs with human hsa-miR30a (common
miRNA), hsa-miR150 (T cell specific miRNA), and hsa-miR122 (liver specific
miRNA) backbones in HEK-293T and Jurkat cell lines.
Methods: Artificial miRNA processing and knockdown efficiency were analyzed by
stem-loop RT-PCR, qRT-PCR, luciferase assay and target challenging.
Results: We identified strikingly different RNAi activities among these different artificial miRNAs. Our
results demonstrated that expression and function of art-miR150 was more than art-miR30 and artmiR122
in both HEK-293T and Jurkat cell lines. Since the main difference in these artificial miRNAs
was flanking sequences and terminal loop structure, the change between the expression and function of
artificial miRNAs can be attributed to these structures.
Conclusion: This study showed that expression of cell-specific artificial miRNA in target and nontarget
cells is not different, but variation in flanking sequences and terminal loop can be involved in
expression and function of artificial miRNAs. These results can be important for improving artificial
miRNA design in RNAi-based gene therapy.
