Ago HITS-CLIP expands microRNA-mRNA interactions in nucleus and cytoplasm of gastric cancer cells

Xinyi Zhang,Yalei Cui,Bo Shen

doi:10.1186/s12885-018-5246-0

Xinyi Zhang, Yalei Cui + Show 1 more

Open Access

https://doi.org/10.1186/s12885-018-5246-0

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Abstract

BackgroundIntensive investigations have identified a collection of microRNAs (miRNAs) and their functional machineries in cytoplasm. However, a comprehensive view of miRNAs and mRNAs in cytoplasm and nucleus has not been explored. This study aims to reveal the mechanisms of miRNA-RNA interactions in nucleus and cytoplasm.MethodsIn this study, the miRNAs and their target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were characterized using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP). Then, the selected miRNAs were verified by Northern blot. The target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were analyzed through Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis.ResultsThe results revealed that there were 243 miRNAs and 265 miRNAs in the Ago2 complexes of nucleus and cytoplasm, respectively. The majority of mature miRNAs existed in cytoplasm. The analysis of miRNA targetome from the Ago2 complexes indicated that a lot of mRNAs with high expression level existed in nucleus. The target genes of miRNAs in the Ago2 complexes of nucleus and cytoplasm played important roles in cell proliferation, cell differentiation, innate immune response and tumorigenesis.ConclusionsmicroRNA-mRNA interactions occur in nucleus and cytoplasm of gastric cancer cells.Therefore, our study demonstrated that miRNA-mRNA interactions not only took place in cytoplasm but also in nucleus.

Highlights

Intensive investigations have identified a collection of microRNAs and their functional machineries in cytoplasm
In order to explore the miRNA-mRNA interaction in nucleus and cytoplasm, the miRNAs and mRNAs in the Ago complexes of nucleus and cytoplasm of gastric cancer cells (MGC-803) were characterized using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in this study
Acquirement of Ago2 complexes from cytoplasm and nucleus of gastric cancer cells To identify miRNAs and mRNAs targeted by miRNAs in Ago2 complex of cytoplasm and nucleus, coimmunoprecipitation (Co-IP) assays with Ago2-specific antibody were performed using gastric cancer cells (MGC-803)

Summary

Introduction

Intensive investigations have identified a collection of microRNAs (miRNAs) and their functional machineries in cytoplasm. This study aims to reveal the mechanisms of miRNA-RNA interactions in nucleus and cytoplasm. MicroRNA (miRNA) as one kind of non-coding small RNA (~ 22 nucleotide) plays an important role in the regulation of gene expression [1, 2]. After the primary miRNA (primiRNA) is transcribed by RNA Pol II, it needs a series of processing to form the mature miRNA [1, 2]. The Drosha protein (~ 160 kDa) cleaves pri-miRNAs to initiate miRNA processing and maturation, and forms ~ 65 nt (nucleotide) precursor miRNAs with hairpin structures (pre-miRNAs) [3]. The Dicer protein can act on the pre-miRNA and cleave the end loop of the pre-miRNA to generate a double-stranded RNA with a length of approximately 22 base pairs (bp) [1, 2]. The miRNAs in nucleus have not been intensively explored

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