processes
Article
ADAR Mediated RNA Editing Modulates MicroRNA
Targeting in Human Breast Cancer
Justin T. Roberts 1 , Dillon G. Patterson 1 , Valeria M. King 1 , Shivam V. Amin 1 ,
Caroline J. Polska 1 , Dominika Houserova 2 , Aline Crucello 1 , Emmaline C. Barnhill 1 ,
Molly M. Miller 1 , Timothy D. Sherman 1 and Glen M. Borchert 1,2, *
1
Department of Biology, University of South Alabama, Mobile, AL 36688-0002, USA;
justin.roberts@ucdenver.edu (J.T.R.); dillon.patterson@emory.edu (D.G.P.);
vmk902@jagmail.southalabama.edu (V.M.K.); sva1002@jagmail.southalabama.edu (S.V.A.);
carolinepolska@gmail.com (C.J.P.); alinecrucello@gmail.com (A.C.);
ecb1621@jagmail.southalabama.edu (E.C.B.); mmm314@jagmail.southalabama.edu (M.M.M.);
tsherman@southalabama.edu (T.D.S.)
2
Department of Pharmacology, USA College of Medicine, Mobile, AL 36688-0002, USA;
dh1001@jagmail.southalabama.edu
* Correspondence: borchert@southalabama.edu; Tel.: +1-251-460-7310
Received: 6 April 2018; Accepted: 21 April 2018; Published: 25 April 2018
Abstract: RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly
being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their
processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target
mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased
in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous
reports have now identified an array of miRNAs that directly contribute to various malignancies
although the majority of their targets remain largely undefined. Here we propose that modulating
the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated
participant in pathology. In order to more accurately characterize the relationship between these
two regulatory processes, this study examined RNA editing events within mRNA sequences of
two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits
could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell
lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were
located in 3 ′ untranslated regions (UTRs). When these locations were screened against the list of
currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant
alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be
misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to
directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these
two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and
physiological responses, we hypothesized that the differential RNA editing of DFFA in these two
cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western
blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but
not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7,
but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be
an underappreciated function of ADAR and may help further elucidate the role of RNA editing in
tumor pathogenicity.
Keywords: ADAR; breast; cancer; inosine; microRNA; microRNA targeting; RNA editing
Processes 2018, 6, 42; doi:10.3390/pr6050042
www.mdpi.com/journal/processes
130
Article
ADAR Mediated RNA Editing Modulates MicroRNA
Targeting in Human Breast Cancer
Justin T. Roberts 1 , Dillon G. Patterson 1 , Valeria M. King 1 , Shivam V. Amin 1 ,
Caroline J. Polska 1 , Dominika Houserova 2 , Aline Crucello 1 , Emmaline C. Barnhill 1 ,
Molly M. Miller 1 , Timothy D. Sherman 1 and Glen M. Borchert 1,2, *
1
Department of Biology, University of South Alabama, Mobile, AL 36688-0002, USA;
justin.roberts@ucdenver.edu (J.T.R.); dillon.patterson@emory.edu (D.G.P.);
vmk902@jagmail.southalabama.edu (V.M.K.); sva1002@jagmail.southalabama.edu (S.V.A.);
carolinepolska@gmail.com (C.J.P.); alinecrucello@gmail.com (A.C.);
ecb1621@jagmail.southalabama.edu (E.C.B.); mmm314@jagmail.southalabama.edu (M.M.M.);
tsherman@southalabama.edu (T.D.S.)
2
Department of Pharmacology, USA College of Medicine, Mobile, AL 36688-0002, USA;
dh1001@jagmail.southalabama.edu
* Correspondence: borchert@southalabama.edu; Tel.: +1-251-460-7310
Received: 6 April 2018; Accepted: 21 April 2018; Published: 25 April 2018
Abstract: RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly
being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their
processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target
mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased
in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous
reports have now identified an array of miRNAs that directly contribute to various malignancies
although the majority of their targets remain largely undefined. Here we propose that modulating
the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated
participant in pathology. In order to more accurately characterize the relationship between these
two regulatory processes, this study examined RNA editing events within mRNA sequences of
two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits
could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell
lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were
located in 3 ′ untranslated regions (UTRs). When these locations were screened against the list of
currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant
alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be
misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to
directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these
two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and
physiological responses, we hypothesized that the differential RNA editing of DFFA in these two
cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western
blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but
not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7,
but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be
an underappreciated function of ADAR and may help further elucidate the role of RNA editing in
tumor pathogenicity.
Keywords: ADAR; breast; cancer; inosine; microRNA; microRNA targeting; RNA editing
Processes 2018, 6, 42; doi:10.3390/pr6050042
www.mdpi.com/journal/processes
130