Novel lncRNA UGGT1-AS1 Regulates UGGT1 Expression in Breast Cancer Cell Line

Abstract

Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides long that do not encode proteins. Although many lncRNAs remain uncharacterized, they are known to play diverse regulatory roles in gene expression. A group of lncRNAs called natural antisense transcripts can form double-stranded structures with their sense partners due to sequence complementarity. These duplexes can become substrates for A-to-I RNA editing, an epitranscriptomic modification mediated by ADAR enzymes. RNA editing is known to influence transcript splicing, affect the resulting gene expression product or alter RNA stability, all of which can impact cancer cell biology. Here, we show a novel natural antisense transcript, UGGT1-AS1, that we have identified and characterized in terms of its cellular localization and sense partner interactions. Furthermore, we demonstrate that UGGT1-AS1 affects cell proliferation and regulates the stability of the UGGT1 sense transcript. Finally, using publicly available RNA sequencing data, we identify A-to-I RNA editing events in the protein-coding gene UGGT1 and further confirm them by RT-PCR and Sanger sequencing in MCF7 cell lines. We hypothesize that UGGT1-AS1 may act as a triggering factor for the A-to-I RNA editing process in its sense partner. Our findings highlight the regulatory role of UGGT1-AS1 and suggest its involvement in RNA editing and cancer biology.

Keywords: A-to-I RNA editing; breast cancer; lncRNA; natural antisense transcript.

Figure 1

Comparison of the number of edited sites across different conditions. The graph shows the numbers of edited RNA sites detected in two breast cancer subtypes, ER+ breast cancer and triple-negative breast cancer (TNBC), comparing the tumor tissues to their adjacent non-cancerous tissues, normalized for sequencing depth, which indicates that observed differences were not artifacts from sequencing depth, ensuring comparability between samples. The graph represents statistically significant differences in the number of edited sites between the cancerous and non-cancerous samples.

Figure 2

The natural antisense transcript of the UGGT1 gene. (a) Genomic context of the sense (UGGT1) and antisense (UGGT1-AS1) transcripts. Dark blue: exons; light blue: untranslated regions; thin black lines: introns (diagonal lines denote gaps in the intronic sequence, used to simplify the graph. (b) Results of RT-PCR on the MCF7 cells material, showing that UGGT1 and UGGT1-AS1 are expressed. (c) Results of UGGT1 Sanger sequencing, visualized in Chromas viewer. DNA and RNA were isolated from the same MCF7 cells. RNA was reverse transcribed to cDNA and both acids were used in the PCRs. Two loci predicted to be edited in breast cancer were checked, and the predictions of A-to-I RNA editing events were confirmed in vitro.

Figure 3

Subcellular localization of UGGT1-AS1 and its protein interactome. (a) RT-PCR results showing UGGT1-AS1 expression in different cell fractions. MALAT1 and ACTB were used as markers for the chromatin and cytoplasmic fractions, respectively. GAPDH was used as a reference gene in reverse transcription. (b) RT-qPCR results showing the relative expression of the UGGT1-AS1 gene with two control genes, ACTB and MALAT1. The expression of the genes was normalized to the total cell fraction and to the GAPDH reference gene expression (**** p < 0.0001, ns not significant).

Figure 4

UGGT1-AS1 regulates MCF7 cell proliferation and its sense partner expression. (a) Relative expression of the UGGT-AS1 gene after transfection with GapmeRs. The transfection was performed using a lipofectamine reagent, which alone also served as a negative control. The second negative control was carried out with custom negative control GapmeRs (NEG control). GapmeR UGGT1-AS1—cells transfected with gapmeR against UGGT1-AS1. (b) Relative expression of MALAT1 gene after GapmeR treatment. The knock-down of MALAT1 was used as a positive control for GapmeRs. (c) Impact of UGGT1-AS1 silencing on the expression levels of the UGGT1 gene. The silencing of the UGGT1-AS1 gene with GapmeRs was performed at two different concentrations (30 nM and 50 nM). The experiments were conducted with three biological and three technical replicates. (d) MCF7 cell proliferation after UGGT1-AS1 GapmeR treatment. Absorbance was measured at OD = 570 nm in the MCF7 cells after the UGGT-AS1 and negative control (NEG) GapmeR treatments. An increase in cell proliferation was observed at 72 h post-treatment in the UGGT1-AS1 knock-down group (** p < 0.01; **** p < 0.0001).

Figure 5

UGGT1 and UGGT1-AS1 form a duplex that increases sense gene stability. (a) Electrophoresis gel of the UGGT1, UGGT1-AS1 and MALAT1 genes after the RAP-RNA experiment. RNA probes (biotinylated antisense oligos) were designed to hybridize with UGGT1-AS1. The presence of a sense transcript in the last stage of the experiment (final RNA) confirms RNA:RNA interactions between the sense and antisense transcripts. (b) Relative expression of the ACTB control gene after actinomycin D treatment, demonstrating no effect of UGGT1-AS1 silencing on ACTB stability. (c) Relative expression of the UGGT1-AS1 gene after actinomycin D treatment. The vertical line shows the half-lives of the UGGT1-AS1 transcripts. (d) Relative expression of the UGGT1 gene after actinomycin D treatment, indicating a significant decreased stability of the UGGT1 transcripts after UGGT1-AS1 knock-down. The vertical lines represent the half-lives of the UGGT1 transcripts in two conditions (ns not significant; * p < 0.05).