Unveiling the principle of microRNA-mediated redundancy in cellular pathway regulation

Abstract

Understanding the multifaceted nature of microRNA (miRNA) function in mammalian cells is still a challenge. Commonly accepted principles of cooperativity and multiplicity of miRNA function imply that individual mRNAs can be targeted by several miRNAs whereas a single miRNA may concomitantly regulate a subset of different genes. However, there is a paucity of information whether multiple miRNAs regulate critical cellular events and thereby acting redundantly. To gain insight into this notion, we conducted an unbiased high-content miRNA screen by individually introducing 1139 miRNA mimics into Chinese hamster ovary (CHO) cells. We discovered that 66% of all miRNAs significantly impacted on proliferation, protein expression, apoptosis and necrosis. In summary, we provide evidence for a substantial degree of redundancy among miRNAs to maintain cellular homeostasis.

Keywords: 3'-untranslated region; 3'UTR; AF647; AlexaFluor→647; CH; CHO; CO2; Chinese hamster; Chinese hamster ovary; Cricetulus griseus; MIR; PI; RNA interference; RNAi; SEAP; base pair; bp; cDNA; carbon dioxide; cgr; chinese hamster ovary; mRNA; mature microRNA; miR; miRNA; microRNA; nc; non-coding; pathway regulation; pre-miR; precursor microRNA; propidium iodide; puro; puromycin; qRT-PCR; quantitative reverse-transcriptase real-time PCR; redundancy; rounds per minute; rpm; screen; secreted alkaline phosphatase; siRNA; small-interfering RNA.

Figure 1:

Confirmation of functional small ncRNA transfection on all miRNA screen plates (n=73) in the primary miRNA screen. Normalized mean values of (A) viable cell density and (B) SEAP protein expression level of siDeath and siSEAP siRNA transfected CHO-SEAP cells, respectively. Sample values were normalized to the respective NT siRNA transfected on-plate control cells. Data are presented as mean ± s.e.m. and for statistical analysis an unpaired, 2-tailed t-test was applied (*** p <0.001). (C) Overview on how many miRNAs significantly affected crucial cellular processes such as protein expression, cell proliferation, necrosis and apoptosis in CHO-SEAP cells. Data are presented as percentages of the entire miRNA library comprising 1139 murine miRNA mimics and the amount of miRNAs increasing (green) or decreasing (red) observed phenotypes are indicated. miRNAs were considered as impactful if the change in phenotype was statistically significant against the negative control cells.

Figure 2:

Influence of single miRNAs on different cell phenotypes in CHO-SEAP cells. (A) Effects of individual pro- and anti-productive miRNAs on SEAP protein level (black) and viable cell concentration (gray) 72 h following transfection in the primary miRNA screen. (B) Effects of individual pro- and anti-proliferative miRNAs on viable cell concentration (black) and apoptosis (gray) 72 h following transfection in the primary miRNA screen. (C) Effects of individual pro-necrotic miRNAs on necrotic (black) and apoptotic (white) cell death as well as viable cell concentration (gray) 72 h following transfection in the primary miRNA screen. (D) Effects of individual pro-apoptotic miRNAs on apoptosis (black) and viable cell concentration (gray) 72 h following transfection in the primary miRNA screen. Triplicate sample values of each miRNA were normalized to the mean value of the miR-NT transfected control cells. Error bars represent the standard deviation (SD) of 3 independent transfections. Statistics: One-way ANOVA (* p <0.05; ** p<0.01; *** p<0.001). (E) VENN diagram of bioinformatically predicted target genes for miR-17–5p, miR-20b-5p, miR-30a-5p, miR-214–3p and miR-16–5p in humans involved in translational control. The five different prediction algorithms miRanda, TargetScan, DIANA-mT, miRDB or miRWalk were used and overlaps indicate shared target genes.

Figure 3:

Vast numbers of miRNAs redundantly regulate multiple cellular pathways in CHO-SEAP cells. (A) Phenotypic distribution of all impactful miRNAs significantly affecting cell proliferation, protein expression, apoptosis and necrosis. Subdivisions indicate the total number of miRNAs regulating only a single (red), 2 (green, violet and blue) or multiple (orange) cellular processes. (B) Influence of anti-apoptotic (black bars) miRNAs on cell-specific SEAP productivity (red bars). (C) Influence of anti-apoptotic miRNAs on cell proliferation (green bars). (D) Influence of anti-apoptotic miRNAs on necrotic cell death (blue bars). (E) Influence of anti-apoptotic miRNAs on cell proliferation and cell-specific SEAP productivity. Data are presented as mean ± SD for 3 independent transfections. Statistics: One-way ANOVA (* p <0.05; ** p<0.01; *** p<0.001).

Figure 4:

(A) Impactful murine miRNAs from the primary miRNA screen were found to be either expressed in CHO cells (green), not expressed but present as mature miRNA sequence on the CHO genome (red), or did not have any equivalent in CHO cells (gray). (B) Schematic overview on the main principles of miRNA-mediated regulation of cellular pathways in mammals. A single miRNA can regulate various effector genes of a particular pathway since miRNAs are capable of post-transcriptionally regulating hundreds of different mRNAs due to the imperfect nature of target recognition. This principle is known as multiplicity of miRNA function. Furthermore, as most mRNAs have been found to possess multiple binding sites for miRNAs, individual mRNAs can be regulated by different miRNAs in parallel which known as cooperative miRNA function. Finally, vast miRNAs are capable of regulating multiple cellular processes by targeting various effector genes of different pathways thereby ensuring cellular homeostasis. This phenomenon is called redundancy in miRNA function.