Hsa-miR-874-3p Reduces Endogenous Expression of RGS4-1 Isoform In Vitro

Abstract

Background: The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3'untranslated regions (3'UTRs) are known to regulate the levels of their mRNA splice variants.

Methods: We constructed recombinant pmir-GLO vectors with a truncated 3' regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs.

Results: The dual-luciferase reporter assay found that regions +401-+789, +789-+1152, and +1562-+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789-+1152 region in the 3'UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853-+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells.

Conclusions: Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853-+859.

Keywords: 3′UTR; RGS4 gene; RGS4-1 isoform; hsa-miR-874-3p; schizophrenia.

Figure 1

The truncated 3′ regulatory region of the RGS4 gene recombined into pmir-GLO vectors.

Figure 2

The predicted miR-874-3p binding site in the RGS4 mRNA 3′-UTR and the deletion sequences in m3R1. The red bases are the deleted ones in m3R1.

Figure 3

The relative fluorescence intensities of recombined pmir-GLO vectors (3R1–3R6) in HEK-293, U87, and SK-N-SH cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

The relative fluorescence intensities of 3R1 and m3R1 co-transfected with hsa-miR-874-3p mimic or NC. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

Western blot assay measuring the endogenous protein levels of the RGS4-1 inform after transfection of NC (1, 2, 3), the hsa-miR-874-3p mimic (4, 5, 6), an NC inhibitor (7, 8, 9), and an hsa-miR-874-3p inhibitor (10, 11, 12), in HEK-293 (A,D), SK (B,E), and U87 (C,F). ** p ≤ 0.01; *** p ≤ 0.001, (A–C) each panel summarizes data from two separate western blots.