Abstract
MicroRNAs are short (17–26) noncoding RNAs driving or modulating physiological and pathological cellular events. Overexpression of miR-155 is pathogenic in B-cell malignancy but was also reported in a number of solid tumors—in particular, in breast cancer, where its role remains unclear and often contradictory. Using representative cell line models, we sought to determine whether the discrepant miR-155 effects in breast cancer could be explained by the heterogeneity of the disease. The growth of six breast cancer cell lines transfected with several miRNA mimics was analyzed. We found MCF-7 cell growth to be inhibited by miR-155 and miR-145 mimics, both 23-nt long, but not by a number of shorter mimics, including a universal commercial negative control. Microarray and Western blot analyses revealed induction of apoptosis, associated with interferon-β after activation of the double-stranded RNA sensor pathway. 3′ Trimming of the miRNA mimics to 21 nt substantially reduced their growth-inhibitory potency. Mutating the canonical seed of the miR-155 mimic had no effect on the induced inhibition, which was abolished by mutating the miRNA seed of the artificial passenger strand. A panel of breast cancer cell lines showed a wide range of sensitivities to 23-mer mimics, broadly consistent with the sensitivity of the cell lines to Poly (I:C). We demonstrate two sources for nonspecific in vitro effects by miRNA mimics: duplex length and the artificial passenger strand. We highlight the danger of a universal 21-mer negative control and the importance of using matched seed mutants for reliable interpretation of phenotypes.
Highlights
MicroRNAs are a class of noncoding RNAs, generally conserved across the higher eukaryotes and typically ranging between 17 and 26 nt in length (Kozomara and Griffiths-Jones 2014)
Alongside miR-155, we tested a commercial universal negative control, oncogenic miR-21, and five known tumor-suppressor miRNAs natively arising from predominantly two primary transcripts
Transfecting up to 100 nM of the nontargeting negative control mimic had no observable effects on MCF-7 confluence
Summary
MicroRNAs (miRNAs) are a class of noncoding RNAs, generally conserved across the higher eukaryotes and typically ranging between 17 and 26 nt in length (miRBase21) (Kozomara and Griffiths-Jones 2014). (for review, see Ha and Kim 2014), miRNA genes are transcribed by RNA polymerase II to yield primary miRNAs (pri-miRNAs) that go through an initial nuclear maturation stage, mediated by Drosha and its coactivator DGCR8, and resulting in imperfectly base-paired stem– loop precursors (pre-miRNAs) of ∼70 nt. After their export into the cytoplasm, a further maturation step is executed by Dicer, assisted by TRBP, PACT, and Ago in a poised complex, by cleaving the precursor’s loop and generating a short imperfect double-stranded RNA (dsRNA) stem (miRNA duplex) (Wilson et al 2015). Ago orchestrates the last maturation step, preferentially incorporating one of the duplex RNA strands into the RISC, responsible for translational repression and RNA degradation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
