Increase in RNASEL Gene Expression by MiR-29-3p Inhibitors in HEK293T Cells

Background: RNase L is known as a terminal component of antiviral and Interferon (IFN) pathways in mammalian cells. On the other hand, the human miR-29 family of microRNAs (miRs) has three mature members, miR-29a, miR-29b, and miR-29c. miR-29 is encoded by two gene clusters and the family members have multifunctional roles in various biological processes.

Objectives: To determine the potential role of miR-29 in the regulation of RNASEL gene expression by designing inhibitors against its targeting miRNA, miR-29a-3p and evaluate the RNase L expression.

Material And Methods: After selecting miR-29a-3p as a main regulating miRNA for RNASEL in silico, two inhibitors were designed against it and synthesized. Synthesized strands were made double-stranded DNA oligos, treated with T4 polynucleotide kinase (PNK), cloned into the pCDH-CMV-MCS-EF1-cGFP-T2A-Puro vector and transformed into DH5α. Colony PCR and sequencing was done for affirmation. Then the miR-29a-3p inhibitors were transfected into HEK-293T cell line and RNASEL gene expression was analyzed.

Results: The miR-29a-3p inhibitors decreased miR-29a-3p expression in vitro. In addition, miR-29a-3p expression reduction resulted in an increase of RNASEL gene expression.

Conclusions: miR-29a-3p inhibitors could increase in RNASEL gene expression which potentially affects the antiviral/IFN pathway. The inhibitors could be considered as drug candidates in different diseases especially viral infections.