Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design.


Introduction
Cellular paths are controlled through a variety of mechanisms, since extracellular and intracellular signals are carried out by proteins, nucleotides, carbohydrates, ions like Ca 2+ , etc. A novel mechanism of cell path regulation is yielded by microRNAs, small RNA molecules that actively inhibit expression of all kind of proteins. Each miRNA is able to regulate a great number of proteins, which means these little molecules have great impact on cellular fate through cell pathway regulation. Reviewing how miRNA families are expressed in clusters and how they regulated cell path signaling will increase the knowledge of cervical cancer progression, giving advantages for therapeutic, diagnostic, and prognostic methodology design. Several works are enriching this knowledge by the generation of experimental evidence of miRNAs' participation in regulation of cell paths. Therefore, coupling the information available we propose cell paths affected through miRNAs in cervical cancer. The advantages generated by gain or loss of function in miRNA expression on cancer development [1,2] could be due to the effect of one or more members of the family or cluster. This work shows several misregulated miRNAs that belong to miRNA families and clusters (Table 1), suggesting a complex system of regulation affecting genes and cellular processes.

miRNAs Families Altered in Cervical Cancer
A gene family is a group of genes with a common phylogenetic origin and possible functional homology [3]. The members of miRNA families may be grouped in clusters expressed from a same transcript. However, it is important to note that not all miRNAs from a cluster are expressed at the same time even though the cluster is generated from a single transcript. Short clusters of miRNAs usually include two or three miRNAs and the larger are from four onwards [4]. The distance between miRNAs in a cluster could be from 1 to 10 Kb [5][6][7]. Additionally, most of the clusters have evolutionary conservation, implying an important biological function. It should be noted that paralogous miRNAs are located on different chromosomes; therefore, they should be expected to have differential regulation and expression [8]. Members of different miRNA families have evolved to target a diverse set of transcripts [9,10] regulating several cellular signaling pathways. It this work we point out the pathways regulated by families of miRNAs, grouped in 20 clusters, involved in cervical cancer.

Regulation of PI3K-AKT and MAPK Axis by miR-133b from the Cluster miR-206~133b
The cluster miR-133a-2~1-1 is formed by miR-1-1 and miR-133a-2 [11], localized on chromosome 20, having 10,536 nt of distance between them. In this sense miR-1-2 and miR-133a-1 could be expressed as a cluster since the distance between them is 3219 nt long and they are localized on chromosome 18. In a similar way, miR-133b and miR-206 are localized on chromosome 6 and the distance between them is 4607 nt long [6]. Interestingly, miR-1 and miR-133 from the clusters miR-133a-2~1-1 and miR1-2~133a-1 have opposite expression in cervical cancer: whereas miR-1 is downregulated, miR-133 is upregulated [12]. MiR-133b from the cluster miR-206~133b over-expression activates AKT1, ERK1, and ERK2 phosphorylation, inducing cell proliferation and colony formation in cervical cell lines by the degradation/inhibition of mRNAs and proteins of mammalian sterile 20-like kinase 2 (MST2), cell division control protein 42 homolog (CDC42), and Ras homolog gene family member A (RhoA) [13] (Figure 1). Proliferation and apoptosis are regulated through Raf-1 and MST2 activation. Raf-1 is activated by Ras inducing MEK1, ERK1, and ERK2 phosphorylation [14], while MST2 triggers apoptosis through PUMA and caspases activation. MST2-Raf-1 interaction inhibits MST2 activation while MST2-RASSF1A interaction activates MST2. RASSF1A and Raf-1 compete for binding sites within MST2 [14,15]. Apoptosis stimulus induces MST2-RASSF1A interaction, activating MST2. AKT phosphorylates MST2, inducing Raf-1 interaction and thereby releasing RASSF1A from the complex inducing MST2 inhibition [15]. Furthermore, RhoA and CDC42 inhibit ERK1/2 activation as well as AKT signaling through PTEN activation [16][17][18]; however, it should be noted that RhoA could activate the cell signaling PI3K/AKT and ERK1/2 by the activation of Plexin-B1 [19,20], and probably by CDC42 too (Figure 1). It seems that a cell-specific pathway axis activation or inactivation response is dependent on intensity and time [18]. The AKT and MAPK cell signaling pathways have an intricate and complex regulation to induce cancer that in part could be explained by miRNA family members' misregulation. MiR-133a and miR-133b differ at a single 3′ terminal nt and have similar expression levels in cervical cancer [12]; therefore, it is possible that there is a similar type of regulation on AKT and MAPK signaling ( Figure 1).
Xu et al. [22] showed that miR-19a/b from the cluster miR17~92 downregulates cullin 5 (CUL5) at the mRNA and protein level ( Figure 2). CUL5 is a scaffold protein in E3 ubiquitin ligase complexes, which targets substrates for ubiquitin-dependent proteasome-mediated degradation [23]. The cullin-ring ligases form a bridge with the substrate-binding cytokine-inducible suppressors of cytokine signaling (SOCS) adaptor proteins and the E2 ubiquitin-conjugating enzyme [24]. Notch transcriptional signaling substitutes SOCS for ankyrin repeat and SOCS box-containing (Asb2) from the cullin-based complex constituted by S-phase kinase-associated protein 1 (skp1), skp2, CUL5, CUL1, E47, and E2 ( Figure 2), inducing ubiquitination of its targets, which could be involved in differentiation and proliferation [25,26]. MAPK signaling is needed for E47 degradation [27] (Figure 2). A dual effect of these cell-signaling pathways is possible in cancer development: on one hand a turn-on of systems that favor tumor properties by phosphorylation, and on the other hand inducing ubiquitination-degradation of tumor suppressor genes by regulation of specific proteins of the ubiquitination-degradation system.
The clusters miR17~92 and miR106a~363 are involved in the positive regulation of the PI3K-AKT signaling pathway. Mature miR-92, which could be formed from the cluster miR17~92 and/or miR106a~363, downregulates PTEN in cervical carcinoma cells, thus turning on PI3K-AKT signaling [32] ( Figure 2). One of the most important points in the carcinogenesis process is the transduction of cell signaling, favoring the hallmarks of cancer. Interestingly, PI3K signaling is activated through Notch pathway a common oncogene in cervical cancer [33,34]. The members of the clusters miR17~92, miR106a~363, and miR106b~25 could potentially regulate CUL5, TNKS2, and PTEN because they have a seed sequence that hybridizes with their mRNA (microRNA.org, MIRDB, Target Scan). The clusters miR17~92, miR106a~363, and miR106b~25 function as onco-miRs, activating several cellular signaling pathways that induce cervical cancer development.

Regulation of uPA-PLG-MMP and JAG-NOTCH Axis by Members of miR-23b~27b~24-1 Cluster
The families miR-23, miR-24, and miR-27 are organized in two clusters ( Table 1). The cluster miR-23a~27a~24-2 is localized on chromosome 19 while cluster miR-23b~27b~24-1 is on chromosome 9 [4,6,35]. In cervical carcinoma cells it was shown that miR-23b diminished uPA expression via 3′-UTR mRNA binding [36] (Figure 3). This protein is a serine protease that degrades plasminogen to plasmin, thus activating metalloproteinases and inducing extracellular matrix (ECM) degradation. Recently it was shown that an increase of Notch receptors' expression triggers the Notch pathway by uPA activation [37]. Additionally, JAG-Notch activation stimulates uPA transcription [38,39], inducing a mutual signaling activation exacerbated by miR-23b absence favoring cancer progression (Figure 3). The clusters miR-23a~27a~24-2 and 23b~27b~24-1 have a high similarity; however, it appears that the members of these clusters have opposite profile expressions: whereas miR-27a is upregulated, miR-23b and miR-27b are downregulated in cervical cancer [12]. MiR-23a, miR-27a, and miR-27b from the two clusters have sequences that may potentially regulate uPA (microRNA.org, MIRDB, and Target Scan). It remains to be addressed whether miRNAs of these clusters regulated the same targets to elucidate the participation of these clusters in cervical cancer's advance.

Figure 3.
Regulation of JAG1-NOTCH1-uPA, Cyclin D-CDK4/6-p21-p27, and PI3K-PDK1-AKT1 cell signaling by members of the miR-23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR17~92, miR183~96~182, and miR-302s clusters. MiR-34a, a member of the miR-34 family, downregulates JAG1 and NOCTH1 expression. JAG1 is activated by uPA inducing ECM reorganization via plasminogen-plasmin-MMP, inducing the hallmarks of cancer. Protein expression of uPA is decreased by miR-23b, a member of the cluster 23b~27b~24-1. The complex cyclin D-CDK4/6 phosphorylates RB inducing the liberation of E2F, favoring c-Myc, c-Fos and COX-2 transcription provoking cellular division. Cellular division progression is regulated by the inhibition of cyclin D via the miR-302s cluster and CDK4/6 through p18Ink4c, p21, and p27. The PI3K-PDK1-AKT1 axis decreases p21 and p27 protein expression, delivering CDK4/6 to be complexed with cyclin D. This signaling pathway is regulated by the downregulation of AKT due to the miR-302s cluster increasing p21 and p27 proteins, thereby inhibiting the complex cyclin D-CDK4/6. CDK4/6 is inhibited by p18Ink4c, which in turn is downregulated by miR-34a, inducing apoptosis and causing proliferation to diminish. Another point of control is given by the miR-29a~29b-1 cluster hindering CDK4/6, which provokes inhibition of the complex cyclin D-CDK4/6. YY-1, a target of the cluster miR-29a~29b-1, induces transcription of c-Myc in the absence of this cluster common in cervical cancer. Cell cycle continuity is dependent on the levels and the complex formed of cyclin D-CDK4/6 and cyclin E-CDK4/6. FOXO1 could directly hinder CDK4/6, thus impeding the formation and activation of the complex cyclin D-CDK4/6 and cyclin E-CDK4/6 and indirectly via p27. Cyclin D is indirectly upregulated by miR-182, one member of the cluster miR183~96~182, through FOXO1 downregulation conducing to proliferation, migration, invasion, and apoptosis induction. These cellular processes are enhanced via FOXO1 phosphorylation by PI3K-PDK1-AKT, which is recognized by skp2, a subunit of the skip1/cul1/F-box ubiquitin protein complex, targeting it to degradation via proteasomes. MiR-92 diminishes the expression of PTEN, triggering PI3K-PDK1-AKT signaling, which is conducive to FOXO1 reduction. In bold are the miRNAs with effect on genes with validated experimental data.

Regulation of HSP47, YY-1 and CDK6 Axis by miR-29a~29b-1 and miR-29b-2~29c Clusters
The members of miR-29 family are divided into two clusters (Table 1). In the first one, miR-29a and miR-29b-1 are localized on chromosome 7, whereas in the second miR-29b-2 and miR-29c reside on chromosome 1 [6]. Interestingly the members of the family miR-29 have differential expression patterns in cervical cancer: while miR-29a and miR-29c are downregulated, the expression of miR-29b is not altered. The restoration of miR-29a inhibits mRNA and protein of Heat-shock protein 47 (HSP47) via 3′-UTR. This protein belongs to serpin, a superfamily of serine protease inhibitors and a molecular chaperone involved in the maturation of collagen molecules [40]. The overexpression of HSP47 could be crucial in the metastasis processes because structural and architectural changes are needed in the extracellular matrix for carcinogenesis. In a similar way, changes in the normal cell cycle are required in cancer. The proteins YY-1 and CDK6 are upregulated in CIN 1 and cervical cancer, respectively, by miR-29a and miR-29b, showing a fine-tuning regulation in the cervical carcinoma progress [12,41] (Figure 3). Given the similarity between miR-29a, b, and c, the members of the two clusters could regulate YY-1 and CDK6. It should be noted that the members of the miR-29 family have different effects on the cell cycle, indicating target specificity [42]. YY-1 regulates several genes related to cancer like HER2, COX-2, c-Myc, and c-Fox [43]. In addition, CDK-6, in complex with Cyclin D, phosphorylates and inhibits the interaction of pRB with E2F1, inducing target transcription and proliferation [44] (Figure 3).

Regulation of P18Ink4c and JAG-NOTCH-uPA Axis by miR-34a
P18Ink4c is a CDK4/6 inhibitor that is increased in CIN 2 and carcinoma but not in the normal cervix. De novo infection of human keratinocyte-derived raft tissue by oncogenic HPV increased p18Ink4c expression, suggesting it is one of the first oncogenes activated upon HPV infection. It is well known that oncogenic HPV reduces p53 expression as well as their effectors, like miR-34a, a member of the family miR-34. The validated targets of miR-34a participate in cervical cancer progression, as is the case with p18Ink4c [45], Jagged, and Notch1 affecting cell cycle and Notch signaling directly and indirectly through uPA, respectively [46] (Figure 3). miR-34 family members have a sequence similarity of more than 80%, so it is possible to have targets in common between miR-34a, miR-34b, and miR-34c, which are clustered on chromosome 11 and separated only by 418 nt [6]. Actually, Notch and Jag1 are potential targets of the cluster miR-34b~34c (microRNA.org, Target Scan, and MIRDB). The expression of this cluster could potentiate the downregulation of these proteins affecting Notch signaling, thus balancing proliferation/apoptosis signals.

FOXO1-Cullin-Rings Axis Are Regulated by the Cluster miR183~96~182
The cluster miR183~96~182 is localized on chromosome 7 [47]. MiR-182 and miR-183 are upregulated [48] while miR-96 is downregulated in cervical cancer [49]. The inhibition of miR-182 diminishes tumor growth and its overexpression inhibits apoptosis and increases the S phase cell cycle by downregulating the FOXO1 protein in cervical carcinoma [48]. FOXO1 is an inhibitor of cyclin/CDK complex like cyclin D1 and D2-CDK4 and cyclin E-CDK2 (Figure 3). FOXO1 phosphorylation by PI3K-AKT is recognized by skp2, a subunit of the skip1/cul1/F-box protein ubiquitin complex, targeting it to degradation via proteasomes [50] (Figure 3). The function and relation of miR-182 and FOXO1 seems primordial in carcinogenesis because this axis regulates several complexes that direct the cell cycle's advance [51]. Axis by miR-let-7c~99a, miR-125a~let-7e~99b, and miR-100~let-7a-2 Clusters miR-99 and miR-125 families are evolutionarily related and are clustered with members of the family let-7. Interestingly, miR-125a, let-7e, and miR-99b are localized on chromosome 19, separated only by 468 and 642 nt, respectively. Let-7c and miR-99a are clustered on chromosome 21, separated by 739 nt, as well as miR-100 and let-7a-2, which are grouped on chromosome 11 with 5707 nt of distance between them. MiR-99a and miR-125b-2 are localized on chromosome 21, separated by 52,068 nt; likewise miR-100 and miR-125b-1 are on chromosome 11 with 52,385 nt of distance between them. Whether these miRNAs are expressed as a cluster is not known; however, they regulate similar genes [6] impacting on common cell signaling.
miR-100 and miR-125b have similar profile expression, pointing to an anti-oncogenic function. miR-100 of the cluster miR-100~let-7a-2 and miR-125b-1 are downregulated in the early steps of cervical cancer progression [12]. In this sense, miR-100 inhibits PLK1 [58], a protein that participates in G2/M phase check-point regulation to block cell progression induced by DNA damage; however, some tumor cells override this check-point [59]. Upon UV-DNA damage, the kinases ATM and ATR are activated, inducing Chk1/2 phosphorylation and activation and thereby triggering CDC25 phosphatase inhibition, resulting in CDK1 phosphorylation and G2/M arrest. Additionally, ATM/ATR inactivates Bora, leading to cascade inhibition of Aurora A, PLK1, and CDC25 (Figure 1). The overexpression or constant activity of PLK1 is conducive to MDM2 activation and concomitant p53 degradation via ubiquitination. Likewise, CDC25 activation and CDK1 dephosphorylation favor G2/M progression [60]. Interestingly, miR-100 regulates these cell-signaling pathways, making it a new player in the G2/M checkpoint. The profile expression of the cluster miR100~125b-1 and the similarity between them point to the fact that miR-125b might participate in this signaling, too. Other important targets of miR-100 are phosphatase (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like) (CTDSPL), enzyme N-Myristoyltransferase 1 (NMT1), Transmembrane Protein 30A (TMEM30A), and chromatin remodeler SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 5 (SMARCA5), HOXA1, and mTOR. MiR-125b inhibits the PI3K/AKT pathway through downregulation of mRNA and protein PIK3CD via 3′-UTR binding, which is conducive to protein kinase A (AKT) and mTOR phosphorylation, inducing tumor growth volume inhibition [61] (Figure 1). Downregulation of the targets of the families and clusters of miR-99 and miR-125 hinders proliferation and migration, highlighting the importance of function loss in these families in cervical carcinogenesis progression. The imbalance of signaling pathways gives advantages toward development of cancer.

Author Contributions
Luis Steven Servín-González and Angelica Judith Granados-López analyzed the literature and wrote the paper; Jesús Adrián López analyzed the literature, organized the data, and created the original idea.

Conflicts of Interest
The authors declare no conflict of interest.