Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner

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.

Keywords: dsRNA sensing; miR-155; microRNA; microRNA mimics; siRNA.

FIGURE 1.

Several miRNA mimics trigger interferon response and reduce the growth of MCF-7 cultures. (A) Culture confluence of MCF-7 cells (normalized to initial confluence) was measured using an IncuCyte at 48 h post-transfection with increasing concentrations of miRNA mimics (5, 10, 25, 50, and 100 nM). Negative control mimic was tested only at 5 and 100 nM. Results are sorted by the lengths of the tested miRNA. Error bars, SEM of nine scans per well (n = 2). (B,C) Heatmap of unsupervised clustering (B) and LogOdds/FoldChange volcano density plots (darkness of blue shade proportional to density of probe data points) (C) of 1005 microarray-based differentially expressed genes (FDR < 0.01) in MCF-7 cells transfected in triplicate with 10 nM miR-155 or miR-199a5p mimic compared to negative control mimic; top 25 commonly induced genes are listed in heatmap inset. (D) Culture confluence of MCF-7 cells was monitored using IncuCyte (normalized to initial confluence) over 48 h following transfection of MCF-7 cells with microRNA mimics at 10 nM; SEM of nine scans per well (n = 2). Colors as in A. (E) RT-qPCR analysis of total RNA from MCF-7 cells matched to D, collected at 24 h post-transfection. Single gene data is normalized to the geometrical mean of the housekeepers DGUOK and eEF1A1. Error bars. SD of three technical replicates per sample (n = 2).

FIGURE 2.

The effect of miR-155 mimic on MCF-7 can be eliminated by antisense inhibitors. Normalized culture confluence (as in Fig. 1) was monitored for 48 h following transfection of MCF-7 cells with combinations of negative control mimic, negative control inhibitor, miR-155 mimic, or miR-155 inhibitor as indicated. (Left) Pretreatment with inhibitors 4 h prior to mimic transfection (ratio 20:1); (middle) cotransfection of premixed inhibitor and mimic (ratio 5:1); (right) transfection of inhibitor and mimic, prepared separately (ratio 5:1). Error bars, SEM of nine scans per well (n = 2).

FIGURE 3.

The effect of miR-155 mimic in MCF-7 is length-dependent. (A) Schematic illustration of custom lengthened negative control and shortened miR-145 and miR-155 mimics. Not to scale. (B) Normalized confluence readings collected post-transfection of MCF-7 cells with 10 nM of mimic variants (as in Fig. 1). Error bars, SEM of nine scans per well (n = 2). (C) Western blot analysis of MCF-7 cells matched to B, collected 24 h post-transfection; protein samples were sequentially probed for the dsRNA-response sensors RIG-I and MDA5, interferon-induced phosphorylated STAT1-Tyr701 and total STAT1, apoptosis marker PARP (F: full-length; Cl: cleaved), and actin as a loading control. (D) Western blot analysis of dsRNA-response elements (as in C) in MCF-7 cells collected 24 h post-treatment with apoptosis inducing actinomycin D (10 nM), cyclohexamide (100 nM), or DMSO alone. (E) Volcano density plots (as in Fig. 1) of microarray-based differentially expressed genes of MCF-7 cells transfected the indicated mimic variants. The y-axes are on a common scale.

FIGURE 4.

The effect of miR-155 mimic in MCF-7 depends on the seed of the passenger strand. (A) Schematic illustration of standard negative control and miR-155 mimics and custom shortened and/or mutated variants of miR-155 (21-mer wild-type, mutant 1 and mutant 2). Anti-seed, passenger strand seed sequence. Not to scale. (B) Normalized confluence readings collected post-transfection of MCF-7 cells with 10 nM of mimic variants (as in Fig. 1). Error bars: SEM of nine scans per well (n = 2). (C) Western blot analysis of MCF-7 cells matched to B, collected 24 h post-transfection; protein samples were sequentially probed as in Figure 3. Lanes of negative control and miR-155 wt mimics are repeated from Figure 3 for comparison. (D) Volcano density plot (as in Fig. 1) of microarray-based differentially expressed genes of MCF-7 cells transfected the indicated mimic variants. The y-axes are on a common scale.

FIGURE 5.

Variant-specific changes in gene expression. The heatmap depicts unsupervised clustering of the union of the top 50 most deregulated Illumina probes in each of the six contrasts (155wt_21mer versus neg_21mer; 155mut1_21mer versus neg_21mer; 155mut2_21mer versus neg_21mer; 155wt_23mer versus neg_21mer; neg_23mer versus neg_21mer; 155wt_23mer versus neg_23mer). Mimics are color-coded as in Figure 4.

FIGURE 6.

Sensitivity to miRNA mimic length is cell type–specific. (A) Culture confluence (normalized to initial confluence) of the indicated six breast cancer cell lines was measured using an IncuCyte at 48 h post-transfection of 10 nM mimic variants (as in Fig. 1). Error bars, SEM of nine scans; D'Agostino and Pearson normality test to identify parametric data; unpaired student t-test: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (B) Light microscopy images taken 48 h post transfection of dsRNA Poly (I:C) or matched untreated cultures of the same six cell lines. Cell lines are ordered from most to least sensitive to Poly (I:C) transfection.