Position-specific oxidation of miR-1 encodes cardiac hypertrophy.

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes1. One such modification, 8-oxoguanine2 (o8G), is abundant in RNA3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy4. We find that position-specific o8G modifications are generated in seed regions (positions 2-8) of selective miRNAs, and function to regulate other mRNAs through o8G•A base pairing. o8G is induced predominantly at position 7 of miR-1 (7o8G-miR-1) by treatment with an adrenergic agonist. Introducing 7o8G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o8G-miR-1 function in affected phenotypes; the specific inhibition of 7o8G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o8G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression.

Widespread 8-oxoguanine modifications of miRNA seeds differentially regulate redox-dependent cancer development.

Oxidative stress contributes to tumourigenesis by altering gene expression. One accompanying modification, 8-oxoguanine (o8G) can change RNA-RNA interactions via o8G•A base pairing, but its regulatory roles remain elusive. Here, on the basis of o8G-induced guanine-to-thymine (o8G > T) variations featured in sequencing, we discovered widespread position-specific o8Gs in tumour microRNAs, preferentially oxidized towards 5' end seed regions (positions 2-8) with clustered sequence patterns and clinically associated with patients in lower-grade gliomas and liver hepatocellular carcinoma. We validated that o8G at position 4 of miR-124 (4o8G-miR-124) and 4o8G-let-7 suppress lower-grade gliomas, whereas 3o8G-miR-122 and 4o8G-let-7 promote malignancy of liver hepatocellular carcinoma by redirecting the target transcriptome to oncogenic regulatory pathways. Stepwise oxidation from tumour-promoting 3o8G-miR-122 to tumour-suppressing 2,3o8G-miR-122 occurs and its specific modulation in mouse liver effectively attenuates diethylnitrosamine-induced hepatocarcinogenesis. These findings provide resources and insights into epitranscriptional o8G regulation of microRNA functions, reprogrammed by redox changes, implicating its control for cancer treatment.

AGO-accessible anticancer siRNAs designed with synergistic miRNA-like activity.

Small interfering RNAs (siRNAs) therapeutically induce RNA interference (RNAi) of disease-causing genes, but they also silence hundreds of seed-matched off-targets as behaving similar to microRNAs (miRNAs). miRNAs control the pathophysiology of tumors, wherein their accessible binding sites can be sequenced by Argonaute crosslinking immunoprecipitation (AGO CLIP). Herein, based on AGO CLIP, we develop potent anticancer siRNAs utilizing miRNA-like activity (mi/siRNAs). The mi/siRNAs contain seed sequences (positions 2-7) of tumor-suppressive miRNAs while maintaining perfect sequence complementarity to the AGO-accessible tumor target sites. Initially, host miRNA interactions with human papillomavirus 18 (HPV18) were identified in cervical cancer by AGO CLIP, revealing tumor-suppressive activity of miR-1/206 and miR-218. Based on the AGO-miRNA binding sites, mi/siRNAs were designed to target E6 and E7 (E6/E7) transcript with seed sequences of miR-1/206 (206/E7) and miR-218 (218/E7). Synergistic anticancer activity of 206/E7 and 218/E7 was functionally validated and confirmed via RNA sequencing and in vivo xenograft models (206/E7). Other mi/siRNA sequences were additionally designed for cervical, ovarian, and breast cancer, and available as an online tool (http://ago.korea.ac.kr/misiRNA); some of the mi/siRNAs were validated for their augmented anticancer activity (206/EphA2 and 206/Her2). mi/siRNAs could coordinate miRNA-like activity with robust siRNA function, demonstrating the potential of AGO CLIP analysis for RNAi therapeutics.