miR-122 targeting with LNA/2'-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates

Abstract

MicroRNAs are small noncoding RNAs that regulate many cellular processes in a post-transcriptional mode. MicroRNA knockdown by antisense oligonucleotides is a useful strategy to explore microRNA functionality and as potential therapeutics. MicroRNA-122 (miR-122) is a liver-specific microRNA, the main function of which has been linked with lipid metabolism and liver homeostasis. Here, we show that lipofection of an antisense oligonucleotide based on a Locked Nucleic Acids (LNA)/2'-O-methyl mixmer or electroporation of a Peptide Nucleic Acid (PNA) oligomer is effective at blocking miR-122 activity in human and rat liver cells. These oligonucleotide analogs, evaluated for the first time in microRNA inhibition, are more effective than standard 2'-O-methyl oligonucleotides in binding and inhibiting microRNA action. We also show that microRNA inhibition can be achieved without the need for transfection or electroporation of the human or rat cell lines, by conjugation of an antisense PNA to the cell-penetrating peptide R6-Penetratin, or merely by linkage to just four Lys residues, highlighting the potential of PNA for future therapeutic applications as well as for studying microRNA function.

FIGURE 1.

Transfection of a synthetic double-stranded miR-122 mimic leads to mRNA destabilization in both Huh7 (A) and primary rat hepatocytes (B). ([–] Control) Nontransfected cells.

FIGURE 2.

Northern blot of total RNA from Huh7 cells (A), or primary rat hepatocytes (B), transfected by lipofection with miR-122 inhibitors and controls. U6 RNA was used as loading control. ([–] Control) Nontransfected cells, (*) RNA ladder band corresponding to 100 nucleotides.

FIGURE 3.

miR-122 inhibition by antisense oligonucleotides leads to intracellular accumulation of negatively regulated mRNAs. (A) GYS1 mRNA levels in Huh7 cells. (B) GTF2b mRNA levels in primary rat hepatocytes. (C) GYS1 mRNA levels in Huh7 cells transfected by lipofection with increasing amounts of OMe ON. ([–] Control) Nontransfected cells.

FIGURE 4.

MicroRNA inhibitors based on peptide nucleic acids (PNAs). (A) Northern blots from total RNA for samples electroporated with K-PNA-K3 and K-SCRPNA-K3 in either Huh7 cells (left) or primary rat hepatocytes (right). (B) Aldolase A mRNA levels in primary rat hepatocytes electroporated with 2 μg of the indicated ONs. ([–] Control) Nontransfected cells.

FIGURE 5.

Cell delivery of K-PNA-K3 and R₆Pen-K-PNAK3 oligonucleotides into Huh7 cells in the absence of transfecting agents. (A) Northern blot of total RNA from cells incubated with 1 μM ONs. (B) Northern blot of total RNA from cells incubated with 1 μM electroneutral K-PNA-E, Antagomir-122, and controls. (C) mRNA levels of miR-122-regulated genes in Huh7 cells and primary rat hepatocytes incubated with the R₆-Penetratin conjugate. (D) Aldolase A mRNA levels in primary rat hepatocytes incubated with K-PNA-K3 in the absence of transfecting agent, at 1 μM. ([–] Control) Nontransfected cells.

FIGURE 6.

Northern blots of wild-type miR-122 and of its equimolar mixes with all ON inhibitors tested. (A) Fifty picomoles of the indicated ONs were mixed, diluted in loading buffer containing 20% formamide/8 M urea, and incubated 5 min at 95°C before loading gels. (B) ONs were mixed as described above and extracted with 150 μL of Trizol prior to PAGE and Northern blot analysis. (C) Inhibitor amounts corresponding to 50 nM (or 1 μM for K-PNA-K3) transfections were spiked into a Huh7 cell lysate (one lysate equals one well on a six-well plate) before Trizol extraction (1 mL). Total RNA was subjected to PAGE and Northern blot. ([–] Control) Total RNA not spiked with ONs.