MicroRNAs tend to synergistically control expression of genes encoding extensively-expressed proteins in humans

Introduction

MicroRNAs (miRNAs) belong to a super-family of ∼22 nucleotides single-stranded non-coding RNA molecules, which are extensively implicated in pathophysiological activities (Bartel, 2009; Shukla, Singh & Barik, 2011). After a long biogenesis process, functionalities of miRNAs rely on their interaction with argonaute proteins and co-assembling into the RNA-induced silencing complex (RISC). Despite being energy-consuming, such a unique gene regulation pattern may be significant to human cells owing to their macro roles in biological regulation (Adlakha & Seth, 2017; Park et al., 2016; Srinivasan & Das, 2015).

One miRNA usually physically interacts with hundreds of target genes (Zhou & Yang, 2012), suggesting the advantage of high-throughput and integrative gene regulation (Liu et al., 2016). However, it was also proposed that an inherent deficiency should be not ignored regarding the operation of a ‘one to many’ system like this. Targeting hundreds of genes undoubtedly dissipates the efficacy of the miRNA-RISC machine due to the abundance dilution effect of target genes (Arvey et al., 2010). Undoubtedly, comprehensive and effective regulation covering all target genes needs high miRNA output with sufficient RISC machines in which mature miRNA is embedded. Such a strategy may be uneconomical for widespread adoption due to excessive occupancy of cellular energy and material. Actually, the completed human miRNA expression profiles have shown that only a very small portion of miRNAs can afford this way, such as miR-1 that is strongly expressed in heart and miR-122a that is highly expressed in liver (Ritchie, Flamant & Rasko, 2010). Just recently, it has been revealed that ∼10–15% of human miRNAs are tissue-specific, replying limited cell load capacity for miRNAs (Ludwig et al., 2016).

Indirect and non-physical interactions exist among miRNAs, which forms the basis of the overall effect of miRNA-mediated gene regulation. Several possible patterns of miRNA–miRNA interactions (MMIs) were uncovered by far. As one miRNA can physically interact with hundreds of target genes, this causes with a great probability that different miRNAs may competitively bind the 3′-untranslated region of the same gene. Competition for binding is the main and fundamental pattern of miRNA-miRNA interactions (MMIs) in the miRNA world (Jens & Rajewsky, 2015). Expect the competition pattern, different miRNAs may show cooperativity due to being co-regulated by the same transcription factor and targeting genes with functional interconnections (Na & Kim, 2013; Shi et al., 2013; Guo et al., 2014). Restricted MMI is another pattern that two miRNAs have completely or partially complementary structures and constitute an endogenous sense and antisense miRNA pair (Guo et al., 2014). Great expression difference of the two miRNAs in a pair was a major feature of this pattern.

Based on our previous knowledge of miRNA regulation (Yuan et al., 2015; Zhu et al., 2011; Zhu et al., 2013), we proposed that miRNAs might adopt an alternative pattern of MMI that promotes a manner of more economical and efficient gene regulation in response to real-time adjustment of cellular signals. It less depends on miRNA abundance and more rely on synergistic miRNA-miRNA collaboration for fine-tuning protein output (Skommer et al., 2014). On the whole, synergistic gene regulation may optimize the regulatory efficacy of miRNAs (Xu et al., 2011). Importantly, it is less expensive in cellular energy consumption compared to extraordinarily high expression of miRNAs. In order to validate this hypothesis, we performed a quantitative assessment of miRNA synergism by calculating miRNA synergy score (Zhu et al., 2013). Benefited from the increasingly clear human protein expression atlas (Uhlén et al., 2015), this method enables us to explore potential principles in miRNA regulation at the dimension of human organs. Additionally, in vitro experiment was performed to show the potential benefit of miRNA synergy. Such efforts are intended to shed novel insights into the biological significance of miRNA-miRNA collaboration and provide implications for better understanding the existence of miRNAs in humans.

Materials and Methods

Results

Comparison of UEPs and DEPs in organ expression, network topology and GO terms

After retrieving the protein expression data from the HPA database, we calculated the CV value for each of the 6,328 proteins regarding its HPA-defined abundance ranks in human cells. The majority of the proteins were located with the CV range from 40% to 120% (Fig. 1A). This distribution characteristic was more clearly observed after log2 transformation of CVs. In total, we identified 1,340 UEPs and 1,115 DEPs. Unlike DEPs, UEPs tend to be more uniformly expressed in human organs, have more interacting neighbors, and constitute closer associations with each other in clusters (Figs. 1B–1D). Comparably, DEPs are not ubiquitously expressed, and more marginalized in the human PPI network and execute functions in isolation. More than these, GO analysis disclosed that UEPs tend to be intensively involved in gene transcription and related biological processes, be located in the nucleus or nearby, and play roles in the housekeeping functions such as being transcription regulators (Table S3). This result indicates the crucial role of UEPs in the maintenance of basic cell activities. In contrast, DEPs are more implicated in organ-specific biological processes and perform molecular functions at the plasma membrane and cytoplasmic organelles.

MiRNAs’ regulation tendency on mRNAs encoding UEPs or DEPs is independent of miRNA expression

We investigated the difference of UEPs and DEPs from the miRNA regulation aspect. UEP-encoding mRNAs instead of those encoding DEPs are averagely under more dense control of miRNAs in the whole genome context (Fig. 2A). Even when MTIs are restricted into one organ such as CVS, preference of miRNAs for UEP-encoding mRNAs is also obvious to observe (Fig. 2B). The highest skewness value highlighted miR-133a of its definite trend in regulating DEP-encoding mRNAs in CVS (Fig. 2C). Both miR-1 and 133a are cardiac-specific miRNAs. However, we found that they showed different tendencies on gene regulation (Fig. 2D). This result suggests that regulating organ-specific genes may be not just a necessary condition for the organ-specific functions of miRNAs, such as miR-1 (Yang et al., 2007).

MiRNAs regulate UEP-encoding mRNAs in a synergistic pattern

MiRNAs may act synergistically with each other (Xu et al., 2011). With the evolution of the complexity of a biological system, synergism is undoubtedly more advantageous than isolated regulation in terms of management strategy (Corning, 1995; Stelling et al., 2004). Altered expression of only a few numbers of miRNAs caused systemic changes via disrupting synergistic associations between miRNAs (Figs. 3A–3C). If all conceivable miRNA pairs are supposed to be synergistic 75.5% of synergistic miRNA interactions will be affected only when half of all the miRNAs undergo altered expression (Fig. 3D). In particular, to those the hubs in the miRNA–miRNA synergistic network, adaptation of this management strategy may be more energy-saving due to plenty of synergistic partners (Fig. 3E). Further result indicates that miRNAs with small skewness values comprise most of the synergistic miRNA–miRNA associations in human organs (Fig. 4). This finding implies that the molecular behaviors of UEPs, rather than those of DEPs, are under the surveillance of a dense synergistic miRNA regulation network. In this invisible network, several miRNAs should be paid more attention owing to their more powerful and extensive collaboration with other miRNAs (Fig. 5). They are miR-1, miR-21, miR-141, miR-155, miR-181b, miR-200a, and miR-206. Compared with other miRNAs, miR-21 had a significant impact on the whole cell state of the three cell lines at a mono-transfection concentration of 25 nM (Fig. 6). MiR-133a tends to regulate DEP-encoding mRNAs implying its relatively isolated action, our in vitro experiment verified its failing to influence the whole cell state of the three cell lines.

Discussion

The new discovery of miRNAs and other kinds of non-coding RNAs greatly consolidates the dominant position of genes in regulating cellular activities (Cech & Steitz, 2014). Genes are not only carriers and messengers of genetic information, but also direct supervisors on whether such information is accurately translated into phenotypes. Despite many efforts it is not still clear about the regulation layer that is constituted by thousands of miRNAs by now (Boettger & Braun, 2012; Stroynowska-Czerwinska, Fiszer & Krzyzosiak, 2014). In this study, system-level insight was attempted to shed into the overall effect of miRNAs from the perspective of miRNA–miRNA collaboration.

It was confirmed that abundant expression of target genes produces the abundance dilution effect for miRNA-mediated regulation (Arvey et al., 2010). Denzler and his colleagues (2014) further proved that the overall abundance of target genes is generally higher than that of the corresponding miRNA. This causes insensitive influence of competitive endogenous RNA (ceRNA)-mediated derepression on miRNA activity (Denzler et al., 2016). The above findings imply that miRNAs may do not perform functions in isolation and collaboration of different miRNAs is a means of effective regulation of gene expression.

Functional studies have confirmed that miRNAs might act synergistically, strongly suggesting collaborative gene regulation (Huang et al., 2016; Raut et al., 2016; Xue et al., 2016). The above evidence is properly consistent with the conclusion inferred by the systems biology analyses performed before (Xu et al., 2011; Zhu et al., 2013). Extensive miRNA–miRNA synergism may exist in the miRNA layer. Precisely because of this, slight adjustment of miRNAs’ abundance can cause an apparent variation in the state of cells. Synergistic miRNAs action allows cells to adjust their live-or-death status more flexibly and rigorously to adapt to internal and external alternation. Due to acting synergistically with other miRNAs, low concentration transfection of miR-21 showed an obvious impact on the viability of all of the studied cell lines. Our result is in agreement with the established knowledge that miR-21 is a well-known miRNA that targets tumor suppressor genes and was shown experimentally in many studies to control apoptosis (Buscaglia & Li, 2011). This finding also implies that altered miRNA expression may not only independently affect the translation of its own target gene products but also produces a radiative impact on other miRNAs that have synergistic associations with it. Put briefly, miRNA synergy amplifies the effects of miRNA expression alteration. As cell viability assay is only one way of measuring effects of miRNAs on the cell state and it cannot be excluded that the other examined miRNAs with high potential to regulate UEP-encoding mRNAs can affect the cell state in another way. However, further studies are definitely needed to investigate the miRNAs through other experimental methods.

Functional execution of miRNAs requires high cellular energy consumption as a prerequisite (Shukla, Singh & Barik, 2011). Besides miRNA regulatory crosstalk (Jens & Rajewsky, 2015), miRNA synergy may represent an alternative pattern of energetically optimal miRNA-mediated regulation of post-transcriptional gene expression. However, we supposed that this obvious advantage brought by miRNA synergy might rely heavily on the complexity of the biological system. As miRNA synergy score calculation disclosed, only dense PPIs between target gene products could lead to meaningful miRNA synergy (Zhu et al., 2013). This explains well that one can expect strong synergistic potential of the cardic-specific miRNA miR-1 instead of miR-133a. Our findings indicate that miR-1 has a tendency to regulate UEP-encoding mRNAs whereas miR-133a tends to regulate DEP-encoding mRNAs. A deeper insight is that only complex life forms such as human can afford the coexistence of hundreds of miRNAs in a cell owing to the intricate PPI network that is an essential condition for miRNA synergy to occur.

Besides, our results further point out that miRNA synergism may be associated with the organ expression of target gene products. UEPs that have broad and balanced expression in human organs are subject to over-supervision by synergistic miRNA regulation. Broad and balanced expression of UEPs is a reasonable reflection of their important biological roles in cellular activities such as significant participation in gene transcription. Uniformed expression of large number of proteins throughout human organs implies that different cells share a similar basic state, which ensures that the communication between cells is relatively equal in the physiological environment. Strengthened synergistic miRNA regulation on UEP-encoding mRNAs suggests that miRNA synergism should contribute to the maintaining of their uniformed expression throughout human organs. Due to allowing cells more finely tune the protein abundance of UEPs, miRNA synergism leads to more economical adaptation of cells to the intracellular and extracellular environments.

The complexity of the biological system is described by the following characteristics such as robustness, redundancy, and crosstalk (Jia et al., 2009). Biological robustness lies in that altered expression of a single gene may be insufficient to effectively affect the overall system (Kitano, 2004). Although this guarantees the relative stability of a biological system, it makes cells difficult to adapt to the external environment changes in a timely manner. Due to the existence of functional linkages between system components, cells cannot merely change one component but keep others unchanged when faced with altered conditions. For instance, as cells encounter excessive oxidative stress, adjustment of the overall cellular signals could be observed (Chandra, Samali & Orrenius, 2000; Kiffin, Bandyopadhyay & Cuervo, 2006; Reuter et al., 2010). Robustness and adaptability are mutually contradictory. Biological system requires an integrated gene/protein management strategy for coordinating diverse biological signals. By targeting hundreds of mRNA messengers, single miRNA is in a position to accomplish integrated gene regulation (Backes et al., 2017). Compared to mRNA-mediated protein expression control, indirect regulation by miRNAs is high-throughput (Baek et al., 2008). This may be the biological significance of miRNAs’ presence in human biology. More importantly, the unique synergistic action mechanism of miRNAs provides richer control skills for cells and better overcomes the huge energy consumption in biosynthesis of miRNAs and assembly of RISC protein complex.

Undoubtedly, more in-depth exploration of constituent factors for effective miRNA target site (Agarwal et al., 2015) would greatly promote our understanding of synergistic interactions between miRNAs. Differential 3′-untranslated region isoforms may lead to inconsistent target gene profiles of a miRNA in different types of cells (Nam et al., 2014). Combined with the fact that ∼10–15% of human miRNAs are tissue-specific (Ludwig et al., 2016), this finding implies that synergistic collaboration between miRNAs may be cell-type-specific or tissue-specific. However, more experimental researches are definitely to explore this.

In conclusion, no longer depending on self-expression lets miRNA synergism maximize the effectiveness of fine-tuning protein output. For complex biological systems, miRNA synergism seems to be a very energy-economical solution by which cells can better deal with the contradictory relationship between system robustness and system adaptability. Our findings provide a new understanding of the biological significance of miRNAs in the organ scale.

Supplemental Information