Role of microRNA-mediated MMP regulation in the treatment and diagnosis of malignant tumors

Abstract

Matrix metalloproteinases (MMPs) play important roles in tumor cell proliferation and apoptosis and contribute to tumor growth, angiogenesis, migration, and invasion primarily via extracellular matrix (ECM) degradation and/or the activation of pre-pro-growth factors. Recently, there has been considerable interest in the posttranscriptional regulation of MMPs via microRNAs (miRs). In this review, we highlight the complicated interactive network comprised of different MMPs and their regulating microRNAs, as well as the ways in which these interactions influence cancer development, including tumor angiogenesis, growth, invasion, and metastasis. Based on the conclusive roles that microRNAs play in the regulation of MMPs during cancer progression, we discuss the potential use of microRNA-mediated MMP regulation in the diagnosis and treatment of tumors from the clinical perspective. In particular, microRNA-mediated MMP regulation may lead to the development of promising new MMP inhibitors that target MMPs more selectively, and this approach may also target multiple molecules in a network, leading to the efficient regulation of distinct biological processes relevant to malignant tumors. A thorough understanding of the mechanisms underlying microRNA-mediated MMP regulation during tumor progression will help to provide new insights into the diagnosis and treatment of malignant tumors.

Introduction

The matrix metalloproteinase (MMP) family is comprised of structurally related, zinc-dependent endopeptidases that degrade various components of the extracellular matrix (ECM) and basement membrane.¹ MMPs play important roles in multiple physiological and pathological processes, including tissue remodeling, embryonic development, mammary involution, bone resorption, and wound healing.^2,3 Due to their capacity to degrade the ECM and promote the migration of endothelial cells, MMPs are also involved in tumor growth, angiogenesis, invasion and metastasis.^4-6 The expression levels of MMPs are increased in almost every type of human cancer and are correlated with different tumor stages. Hence, MMP levels are currently used for cancer diagnoses, and MMPs have been targeted in the development of anti-tumor therapies that aim to increase overall survival in patients with several types of solid tumors.^7,8

The proteolytic activity of MMPs is strictly controlled at the levels of transcription, zymogen activation, and endogenous inhibition. Furthermore, an emerging concept in MMP regulation is their intra/extracellular localization, as both secreted and membrane-bound MMPs can be localized to various intracellular sites. The specific regulatory mechanisms governing MMP activity at these levels have been summarized in the literature;^9-13 indeed, a greater understanding of the regulatory mechanisms that control MMP activity may provide new avenues for the diagnosis and therapeutic intervention of malignant tumors, and many diagnostic reagents and drugs are designed to target these key regulatory points.

Recently, there has been considerable interest in the posttranscriptional regulation of MMPs via microRNAs (miRs). MicroRNAs are a family of small, non-coding RNA molecules ranging from approximately 17 to 25 nucleotides in length, which function in posttranscriptional gene regulation via translational repression or the degradation of their mRNA targets. Bioinformatic analyses have predicted potential microRNA-binding sites in the 3′-untranslated region (UTR) of several MMPs,¹⁴ and recent studies have demonstrated that microRNAs participate in MMP regulation at the posttranscriptional level and ultimately influence the translation and expression of MMP genes.^15,16 Furthermore, microRNAs are involved in the regulation of key cellular processes, such as proliferation, differentiation, and apoptosis, which implies that microRNAs may play important roles in cancer progression.^17-19 Accordingly, MMP-mediated microRNA regulation may play an important role in cancer development, and elucidating the function of MMP-mediated microRNA regulation in malignant tumors may further clarify the posttranscriptional regulatory mechanisms governing MMP gene expression. In addition, this work may reveal novel biomarkers that could enhance early detection, define therapeutic responses, and complement current therapeutics.

MMPs are found in all species, and 24 different MMPs have been isolated in humans. Based on their domain organization, sequence similarities, and the specificity of their substrates, MMPs can be classified into four different groups: gelatinases, matrilysins, archetypal, and furin-activated MMPs.^20,21 The study of whether functionally related MMPs, which are often allocated into the same group, share overlapping posttranscriptional regulatory control mechanisms or differ greatly in their composition of microRNA binding sites is of particular interest. In this review, we illustrate the complicated interactive network comprised of different types of MMPs and the microRNAs that regulate them, and we also highlight the manner in which these interactions influence cancer progression, including tumor angiogenesis, growth, invasion, and metastasis. Based on the conclusive role that microRNA-mediated MMP regulation plays in cancer progression, we discuss the potential use of miRNA-mediated MMP regulation in the diagnosis and treatment of tumors.

MicroRNA-Mediated Regulation of Archetypal MMPs

Within this category, and according to their substrate specificities, typical MMPs are classified into one of three subgroups: collagenases, stromelysins, and other archetypal MMPs.

Collagenases

The collagenase subgroup is composed of three enzymes: MMP-1, MMP-8, and MMP-13 (also known as collagenases-1, -2, and -3, respectively). Collagenases are the principal secreted endopeptidases and are capable of cleaving the collagenous ECM. More specifically, collagenases cleave the collagen triple helix into characteristic 3/4 and 1/4 fragments at specific sites on the α-chains (at Gly775 and Leu:Ile776).²² As a result, collagenases can regulate cell growth and survival and may play important roles in many pathological situations, particularly during tumor progression and metastasis. Several microRNAs are responsible for the inhibition or promotion of tumor invasion and metastasis, which are mediated, at least in part, via the posttranscriptional regulation of collagenase gene expression.

In the collagenase subgroup, the function of MMP-1 during tumor metastasis has received much attention. During breast tumor metastasis to the bone, derepression of MMP-1 expression was shown to promote tumor metastasis and was associated with the ectopic expression of miR-224, which inhibits RKIP gene expression by directly targeting its 3′-UTR.²³ Similarly, during breast tumor metastasis to the brain, MMP-1 was more highly expressed in LvBr2 cells (brain-trophic metastatic MDA-MB-435-LvBr2 cells, which were established by injecting MDA-MB-435 cells into the left ventricles of immunodeficient mice) as compared with parental MDA-MB-435 cells. In contrast, MMP-1 expression in LvBr2 cells was decreased when miR-146a was overexpressed via the suppression of Akt activation.²⁴ In addition to breast cancer metastasis, microRNA-mediated MMP-1 regulation is also involved in colon cancer and oral tongue squamous cell carcinoma (OTSCC) metastasis. In colon cancer cells, both the mRNA and protein levels of MMP-1 were decreased in miR-34a-transfected cells via Fra-1 inhibition, as Fra-1 directly induces MMP-1 promoter activities.²⁵ In OTSCC cells, miR-222 was shown to regulate MMP1 expression through both a direct cis-regulatory mechanism (targeting MMP1 at the mRNA level) and an indirect trans-regulatory mechanism (indirectly controlling MMP1 gene expression by targeting SOD2).²⁶ Thus, miR-mediated MMP-1 regulation contributes to the metastasis of several malignant tumors and may serve as a novel therapeutic target for patients who are at risk for metastatic disease.

The distribution of MMP-13 is restricted in normal tissues, and MMP-13 can degrade components of the basement membrane to promote tumor invasion and progression. Both of these features make this enzyme an interesting pharmaceutical target in malignant tumors. Although an extensive overview of MMP-13 expression in malignant tumors was published more than 10 years ago, the posttranscriptional regulatory mechanisms governing MMP-13 expression were not reported until recently. In cutaneous squamous cell carcinoma (CSCC) cells, miR-125b was observed to downregulate MMP-13 expression by binding to the 3′-UTR of its mRNA, thereby expanding our knowledge of the MMP-13 regulatory network.²⁷ This novel regulatory mechanism may also contribute to MMP-13 upregulation in other types of cancers with decreased miR-125b levels, such as breast cancer,²⁸ oral SCC,²⁹ and bladder cancer.³⁰ In addition, Osaki et al. reported that MMP-13 was the direct target of miR-143 in osteosarcoma.³¹ These authors observed that miR-143 was downregulated to the greatest extent of any miRNA in lung osteosarcoma metastatic lesions and significantly promoted their invasiveness and that MMP-13 was the protein most commonly downregulated by miR-143. These results indicate that miR-143 downregulation is correlated with human osteosarcoma lung metastasis by promoting cellular invasion, which is likely mediated via MMP-13 upregulation.³¹ Taken together, these findings suggest that miRNAs targeting MMP-13 could be developed as new molecular targets for malignant tumors.

The final member of the human collagenase family is MMP-8, which is mainly secreted by neutrophils during inflammatory reactions to promote neutrophil migration through basement membranes and connective tissue. MMP-8 expression has also been detected in certain types of malignant tumors, including invasive squamous cell carcinomas,³² ovarian cancers,³³ and skin carcinomas.³⁴ However, the posttranscriptional regulatory mechanisms by which MMP-8 is controlled remain under investigation, and few studies have examined microRNA-mediated MMP-8 regulation.

Stromelysins and other archetypal MMPs

Stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) share the same structural design as collagenases but degrade different components of the ECM in the absence of native collagen.²⁰ Both stromelysin-1 and stromelysin-2 are expressed by fibroblasts and epithelial cells and are secreted into the extracellular space, where they play important roles in immunity, mammary gland development, and wound healing.³⁵ In addition, stromelysin-1 also processes several bioactive substrates, including stromal cell-derived factor-1, E-cadherin, and pro-interleukin-1 β (IL-1β), resulting in the promotion of tumor invasion.²² A recent study revealed that one of the molecular mechanisms governing the role of MMP-3 in the invasion of malignant gliomas may involve microRNA-mediated regulation. In this study, a luciferase activity assay confirmed that miR-152 attenuated MMP-3 protein expression by directly binding to the MMP-3 transcript. Furthermore, miR-152 significantly decreased cell invasiveness, and anti-miR-152 inhibitors counteracted the miR-152-mediated inhibition of invasion, although miR-152 did not reduce tube formation in cultured endothelial cells.³⁶

With the exception of miR-152, to the best of our knowledge, no other evidence supports the involvement of microRNAs in stromelysin regulation or the regulation of other archetypal MMPs (e.g., MMP-12, -19, -20, and -27). However, miR-452 is predicted to target the transcript encoding MMP-12 in human alveolar macrophages.³⁷ Further studies will be required to fully understand the posttranscriptional mechanisms that control the gene expression of stromelysins and other archetypal MMPs.

MicroRNA-Mediated Gelatinase Regulation

Gelatinases are a class of MMPs that have been historically defined according to their affinities for denatured collagen. This class includes MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B),² both of which play key roles in the remodeling of the collagenous ECM by degrading not only a broad spectrum of ECM molecules, including collagen types I, IV, V, VII, X, and IX, elastin, fibronectin, aggrecan, vitronectin, and laminin²² but also many non-ECM molecules, such as pro-TNF-α,³⁵ transforming growth factor (TGF)-β,³⁸ pro-IL-1β, pro-IL-8, and monocyte chemoattractant protein (MCP)-3.³⁹ Thus, gelatinases are deeply involved in various aspects of tumor progression, including tumor cell growth, apoptosis, migration, invasion and metastasis, and have long been considered to be some of the most important anti-tumor targets.

MicroRNA-mediated MMP-2 and MMP-9 regulation

To identify the role of microRNAs in tumorigenesis, Han et al. silenced Dicer, an essential component of the microRNA processing machinery, and found that the impairment of microRNA processing enhanced the expression of MMP-2 and MMP-9, leading to the enhanced proliferative activity and invasive ability of tumor cell lines. These results suggested that MMP-2 and MMP-9 may be controlled by microRNAs.⁴⁰

The detailed underlying mechanism for microRNA-mediated MMP-2 and MMP-9 regulation was then further examined by a number of research groups that studied the functions of several microRNAs, including miR-125b, miR-143, miR-196b, miR-206, miR-21, miR-340, miR-451, miR-9, and miR-7, in various cancers. For instance, miR-125b was shown to alter the expression of both gelatinases to decrease glioblastoma⁴¹ and hepatocellular carcinoma (HCC) cell invasion.⁴² Similarly, miR-21 promoted cell migration and invasion by upregulating MMP-2 and MMP-9 expression in HCC⁴³ and significantly increased proliferation and prevented apoptosis by enhancing gelatinase expression in pancreatic cancer.⁴⁴ However, MMP-2 and MMP-9 expression in pancreatic cancer was shown to be modulated by miR-143 via a regulatory mechanism that differed from that of miR-21. In Panc-1 cells, miR-143 expression significantly decreased MMP-2 and MMP-9 expression at the protein level and inhibited migration, invasion, liver metastasis, and xenograft tumor growth in vivo.⁴⁵ In addition to the previously outlined regulation of miR-125b, both gelatinases were also regulated by miR-451⁴⁶ and miR-7 in gliobastoma,⁴⁷ resulting in altered cell proliferation, apoptosis, and invasion. The impact of miR-451 on cell invasion and survival, as well as the repression function of miR-7 in glioblastoma,⁴⁷ is likely to be regulated by both MMP-2 and MMP-9,⁴⁶ which supports the tumor suppressor role of microRNAs in gelatinase regulation. In breast cancer, two other microRNAs have been shown to indirectly regulate MMP-2 and MMP-9 expression.^48,49 First, miR-206 was found to downregulate MMP-2 and MMP-9 expression at the protein level and inhibit the invasion and migration of MDA-MB-231 cells in vitro.⁴⁸ In addition, Wu et al. found that miR-340 directly targeted the c-Met gene and consequently inhibited MMP-2 and MMP-9 expression, thereby suppressing tumor cell migration and invasion.⁴⁹ In addition, miR-9 and miR-196b were shown to regulate gelatinase expression in uveal melanoma⁵⁰ and gastric cancer,⁵¹ respectively. In uveal melanoma cells, miR-9 negatively modulated MMP-2 and MMP-9 expression indirectly by directly targeting the 3′-UTR of their upstream target, NFκB1.⁵⁰ In gastric cancer, MMP-2 and MMP-9 were regulated by miR-196b in the same manner. Although gelatinases were upregulated in miR-196b-treated cells, miR-196b was not predicted to directly target the 3′-UTR of MMP-2 and MMP-9 by TargetScan predictions. Therefore, miR-196b-mediated promotion of cell migration may be the result of other genes that control gelatinases.⁵¹

Finally, multiple microRNAs have been shown to regulate gelatinases during tumor invasion and metastasis via different signaling pathways. In particular, tissue-specific effects of microRNAs on gelatinases were observed for miR-125b and miR-21.^41-44 However, with the exception of miR-451 in glioblastoma, microRNAs regulate gelatinase expression indirectly, and the detailed regulatory mechanisms therefore remain unclear and should be elucidated in future studies.

MicroRNA-mediated MMP-2 regulation

Although both gelatinases play similar roles in tumor progression, subtle differences exist between MMP-2 and MMP-9 due to differences in their expression profiles. The constitutive expression of MMP-2 has been reported in endothelial cells, fibroblasts, keratinocytes and chondrocytes, whereas MMP-9 is expressed in alveolar macrophages, trophoblasts, osteoclasts and polymorphonuclear leukocytes. In addition, both MMP-2 and MMP-9 may be regulated by tissue-specific microRNAs.

MMP-2 was first confirmed to be a target of miR-29b in prostate cancer cells, leading to the identification of miR-29b as a novel target with potential implications for invasion and metastasis.⁵² Fang et al. demonstrated that MMP-2 was a direct target of miR-29b in HCC and that miR-29b exerted its anti-angiogenic function, at least in part, by suppressing MMP-2 expression in tumor cells.⁵³ These results were also demonstrated in breast cancer⁵⁴ and colorectal cancer.⁵⁵

Apart from miR-29b directly targeting MMP-2, MMP-2 is indirectly regulated by several other microRNAs in various cancers. For instance, inhibitors of both miR-10b and miR-21 synergistically suppress MMP-2 protein expression in U87MG cells (human glioblastoma cells).⁵⁶ Similarly, miR-21 is involved in the cell migration and tumorigenicity of laryngeal squamous cell carcinoma (LSCC) via the regulation of MMP-2 expression.⁵⁷ Very recently, MMP-2 expression was reported to be inhibited by miR-139 via the suppression of IGF-IR/MEK/ERK signaling in colorectal cancer.⁵⁸ Similarly, MMP-2 expression was inhibited by miR-101 in lung cancer⁵⁹ and by miR-146a in castration-resistant prostate cancer (CRPC), although the molecular mechanisms remain unclear. Conversely, MMP-2 was upregulated following the ectopic expression of miR-26a, dramatically enhancing lung cancer cell migration and invasion.⁶⁰

MicroRNA-mediated MMP-9 regulation

Studies of microRNA-mediated MMP-9 regulation have mainly focused on glioblastomas and liver cancers. Yan et al. established the MMP-9-specific microRNA expression profile by performing microRNA microarrays in 60 glioblastoma multiforme samples and identified the MMP-9 specific microRNA miR-491-5p, which suppressed glioma cell invasion by directly targeting MMP-9. This observation may provide potential targets for the development of anti-invasion therapies in glioblastoma.⁶¹ In addition, miR-125b⁶² and miR-218^63,64 were also reported to regulate MMP-9 expression in glioblastoma. In liver cancer cells, MMP-9 expression was decreased in SK-HEP-1 cells that had been transfected with pre-miR-338-3p, while MMP-9 expression was increased in SK-HEP-1 cells that had been transfected with anti-miR-338-3p. However, these authors did not detect any changes in MMP-2 expression after the transfection of pre-miR-338-3p or anti-miR-338-3p.⁶⁵ Similarly, a close relationship between the expression of miR-224 and MMP-9 protein was observed in HepG2 cells.⁶⁶ In addition, Gao et al. recently reported that MMP-9, but not MMP-2, was significantly decreased in miR-145-expressing cells, although it was not a direct target of miR-145. This result suggests that miR-145 suppresses tumor metastasis by inhibiting N-cadherin protein translation and then indirectly downregulates its downstream effector, MMP-9.⁶⁷

Taken together, these results show that MMP-2 and MMP-9 have attracted the greatest attention due to their extensive roles in the invasion and metastasis of a variety of tumors. Furthermore, the roles of MMP-2 and MMP-9 are dependent, at least in part, on the control of serial microRNAs, and both gelatinases were shown to be regulated via both distinct and overlapping molecular microRNA-mediated mechanisms.

MicroRNA-Mediated Regulation of Furin-Activated MMPs

This group of MMPs contains a specific furin recognition motif that is located between the propeptide and the catalytic domain, enabling furin-dependent activation of the proenzyme for subsequent secretion.²⁰ The furin-activated MMPs encompass three subgroups, including membrane type MMPs (MT-MMPs), type II transmembrane MMPs, and secreted MMPs.

Secreted MMPs, including MMP-11, -21, and -28, are processed intracellularly by furin-like proteases and are secreted as active forms. Clinical investigations have revealed that MMP-11 overexpression is correlated with lower survival rates in patients with breast, head, neck or colon cancers.¹⁰ In addition, MMP-21 was shown to be upregulated in several human tumors, including colorectal cancer, breast cancer, prostate cancer, pancreatic cancer, Merkel cell carcinoma, and HCC, and has generally been associated with poor prognoses.¹⁰ However, to the best of our knowledge, the molecular mechanisms underlying the pathological activity of MMP-21 have not yet been described. Similarly, MMP-28 (epilysin) was found to be expressed in several tumors and was associated with cellular proliferation.⁶⁸ Type II transmembrane MMPs (such as MMP-23A and MMP-23B) are unique among the members of the matrixin family because they lack a signal peptide and instead possess a novel cysteine array motif and an immunoglobulin domain. Expression analyses have demonstrated that MMP-23 is mainly produced by reproductive tissues, such as the ovaries and testes.⁶⁹ However, the in vivo functions of this protease in tumorigenesis remain undetermined.

Among the furin-activated MMPs, membrane-type MMPs are considered to be the most relevant to the processes of tumorigenesis and tumor development. Membrane-type MMPs (MMP-14, -15, -16, -17, -24, and -25) contain membrane-anchoring domains that localize them to the cell surface, and this feature makes these enzymes optimal pericellular proteolytic machines able to control the local environment surrounding both normal and tumor cells. Although all of the members of this subgroup have been demonstrated to be associated with angiogenesis, cellular invasion, and tumor metastasis and have been linked to poor prognoses in several tumors,^70-74 the ability of microRNAs to regulate membrane-type MMPs has only been reported for MMP-14 and MMP-16.

MicroRNA-mediated MMP-14 regulation

MMP-14, also referred to as membrane type-1 matrix metalloproteinase (MT1-MMP), is mainly believed to be important for the cleavage of components of the ECM and other cell surface proteins, as well as the activation of proMMP-2. MMP-14 is involved in different stages of tumor progression, ranging from initial tumor development, growth, and angiogenesis to invasion, metastasis, and growth at secondary sites. Clinical studies have revealed that MMP-14 overexpression is associated with poor prognoses in patients with various malignant tumors.^75-78 However, the mechanisms underlying MMP-14 expression in tumors remain largely uncharacterized. Of note, MMP-14 expression has been reported to be associated with microRNAs in pancreatic cancer, neuroblastoma, and glioma.

In pancreatic cancers, the miR-200 family, let-7, and miR-155/miR-216b were shown to regulate MMP-14 expression both in vivo and in vitro.^79-81 Moreover, miR-200 expression was drastically downregulated in aggressive MIAPaCa-2 cells, and the re-expression of miR-200c led to decreased MMP-14 expression. In contrast, following miR-200c knockdown, a marked increase in MMP-14 expression was observed, which resulted in the acquisition of an epithelial–mesenchymal transition (EMT) phenotype and tumor cell aggressiveness.⁷⁹ Furthermore, MMP-14 expression was significantly correlated with let-7 levels in pancreatic cancer specimens. Interestingly, these authors evaluated the role of MMP-14 in the regulation of let-7 expression by generating Panc-1 cells expressing either the full-length or tail-less (ΔC) MMP-14 protein. The results revealed that MMP-14 was overexpressed at the protein level in PDAC cells grown in collagen, whereas the let-7 levels were significantly repressed in these cells as compared with control PDAC cells, indicating that MMP-14 represses let-7 expression in the collagen milieu.⁸⁰ In addition, Ali et al. demonstrated that MMP-14 expression was increased in both the KC (K-Ras^G12D and Pdx1-Cre) and KCI (K-Ras^G12D, Pdx1-Cre, and Ink4a/Arf) transgenic mouse models and that MMP-14 expression could be attenuated in cells that had been transfected with pre-miR-216a. In contrast, inactivation of miR-155 led to decreased MMP-14 expression in RInk-1 cells (which were derived from KCI animals) that had been transfected with miR-155-ASO (antisense oligonucleotides).⁸¹ In conclusion, these results suggested that the targeted inactivation of miR-155, or re-expression of miR-216a/miR-200c/let-7, could potentially be utilized as a therapeutic approach for pancreatic cancer. Therefore, it is tempting to speculate that MMP-14 inactivation via either the targeted re-expression of microRNAs or the downregulation of microRNAs may be useful for the treatment of pancreatic cancer.

MMP-14 was shown to be overexpressed in neuroblastomas⁸² and gliomas. In particular, MMP-14 was shown to be indirectly regulated by miR-10b in neuroblastomas, whereas MMP-14 was directly regulated by miR-9 in gliomas. Sun et al. found that miR-10b induced glioma cell invasion by modulating MMP-14 expression and directly targeting the HOXD10 gene.⁸³ In addition, it is worth noting that miR-9 was the first microRNA shown to directly target MMP-14, although several microRNAs have been shown to be involved in MMP-14 regulation. Furthermore, overexpression or knockdown of miR-9 inversely affected MMP-14 expression and regulated the luciferase activity of the MMP-14 3′-UTR luciferase reporter system and inhibited or stimulated the migration, invasion and angiogenesis of neuroblastoma cells, respectively.⁸⁴

MicroRNA-mediated MMP-16 regulation

Membrane type-3 matrix metalloproteinase (MT3-MMP), also known as MMP-16, is overexpressed in many significantly invasive cancers and is associated with poor patient prognoses.^85,86 These observations may be due, in part, to the deregulated expression of microRNAs, which regulate the expression of MMP-16, thereby contributing to tumor invasion and metastasis. An in silico analysis using available bioinformatic resources revealed that miR-146a/b were candidate microRNAs that could bind to the 3′-UTR of MMP-16. Confirmation of MMP-16 regulation by miR-146a was obtained via reporter gene assays using the pmiR REPORT vector, which contained the 3′-UTR of MMP-16 and the miR-146a binding site. Significant decreases in luciferase activity were observed in Caco-2 cells, which indicated that MMP-16 serves as a direct target of miR-146a in colon cancer.⁸⁷ Likewise, miR-146b was shown to be significantly dysregulated in human glioblastoma tissue using a microRNA microarray. Furthermore, MMP-16 is one of the direct downstream functional targets of miR-146b, which may partially explain the characteristic migration and invasion of glioma cells.⁸⁸ Taken together, these findings indicate that miR-146 a/b may act as metastasis-inhibiting microRNAs during cancer and may be potential therapeutic targets for MMP-16 inhibition.

MicroRNAs Regulate Different Groups of MMPs Concurrently

MMPs from the same groups, based on their similar structures and domain organization, may be co-regulated under certain conditions and share similar posttranscriptional regulatory mechanisms. Functionally related MMPs likely differ greatly in the composition of the microRNA binding sites in their respective 3′-UTRs. Nevertheless, different groups of MMPs may be co-regulated concurrently by the same microRNAs. For instance, Fra-1 was identified as a new target of miR-34a and was suggested to directly induce MMP-1 and MMP-9 promoter activities in cancer cells.⁸⁹ In addition, inhibition of Fra-1 by miR-34a led to MMP-1 and MMP-9 downregulation in colon cancer cells.²⁵ In this sense, functionally related MMPs from different groups, such as gelatinases (MMP-9) or collagenases (MMP-1), may be co-regulated via overlapping signaling pathways. Liu et al. showed an inverse correlation between the levels of miR-218 and MMP mRNAs (MMP-2, -7, -9; MMP-2 and -9 belong to gelatinase group, and MMP-7 belongs to the matrilysin group) in 60 glioblastoma multiforme tissues, although miR-218 overexpression was only found to reduce MMP-9 expression and not MMP-2 or -7 expression.⁶⁴

In addition to the experimental results obtained by individual research groups, the regulation of distinct MMPs (belonging to different groups) by single microRNAs in tumor cells has been studied and reviewed. For example, miR-125b was shown to indirectly regulate gelatinases in gliomas^41,62 and liver cancers,⁴² although it was shown to directly target and regulate MMP-13 in CSCCs.²⁷ Similarly, miR-143 was found to indirectly regulate gelatinases in pancreatic carcinomas. However, miR-143 directly targeted and regulated MMP-13 in osteosarcomas, and miR-146a regulated MMP-2 in prostatic carcinoma,⁹⁰ MMP-16 in colon carcinoma⁸⁷ and MMP-1 in breast cancer.²⁴ Nevertheless, to the best of our knowledge, no single microRNA has been shown to regulate distinct MMPs belonging to different groups in the same type of tumor.

MicroRNAs Regulate Endogenous MMP Inhibition

Many natural inhibitors of MMPs exist that regulate MMP activity. Therefore, microRNAs that regulate the endogenous inhibition of MMPs also play a role in controlling MMP expression. The most common inhibitors are tissue inhibitors of metalloproteinases (TIMPs), α2-macroglobulin (α₂M), reversion-inducing cysteine-rich protein with kazal motifs (RECK), and endostatin.²¹

TIMPs are a group of endogenous proteins capable of regulating MMP proteolytic activity in the pericellular space. Four TIMPs (TIMP-1, -2, -3, and -4) have been identified based on differences in their abilities to inhibit various MMPs.^91-93 Another inhibitor of MMPs is α₂M, a non-specific protease inhibitor, which is commonly expressed in the human plasma and serum. However, RECK, a GPI-anchored glycoprotein, specifically inhibits the activities of MMP-2, -9, and -14. The fourth known natural inhibitor of MMPs is endostatin. Endostatin is a cleavage product of type XVIII collagen and is able to inhibit proMMP-2/MMP-2 activation in vitro. Although α₂M and endostatin provide a negative feedback loop for MMP-activity, the posttranscriptional regulatory mechanisms underlying their activity remain uncharacterized and require further elucidation. In contrast, accumulating evidence supports the microRNA-mediated regulation of both TIMPs and RECK.

MicroRNA-mediated TIMP regulation

The 4 human TIMPs are generally considered to be broad-spectrum inhibitors of all MMPs found in humans, although some differences in specificity exist. For example, TIMP-1 is more restricted in its inhibitory range than the other three TIMPs, as it demonstrates a relatively low affinity for membrane-type MMPs, such as MMP-14, MMP-16, and MMP-24, as well as for MMP-19.⁹⁴ In addition, some relatively subtle differences exist between the affinities of different TIMPs for other MMPs. For example, TIMP-2 and -3 are weaker inhibitors of MMP-3 and MMP-7 than TIMP-1, which is in contrast to their affinities for other MMPs. TIMPs are multifunctional proteins that have numerous biological activities, including the modulation of cell proliferation, migration and invasion, angiogenesis, apoptosis, and synaptic plasticity. These activities may partially arise from metalloproteinase inhibition, as the regulation of ECM catabolism can influence cellular behavior, but many of these activities have been shown to be independent of MMP inhibition. The extensive ability of TIMPs to modulate tumorigenesis has attracted attention to their regulatory mechanisms.

TIMP-3 was identified as the target of at least three microRNAs, including miR-191 in endometrial cancer and miR-103/miR-181b in HCC.^95-97 Wang et al. investigated the molecular mechanism underlying the role of miR-181b in promoting HCC cell proliferation, migration, invasion, and tumor growth in nude mice and found that miR-181b functioned by decreasing the expression of its target, TIMP-3.⁹⁶ Similarly, He et al. found that miR-191 likely induced cells to transition into mesenchymal-like cells by directly targeting TIMP-3 and inhibiting TIMP-3 protein expression.⁹⁷ Furthermore, miR-580, 588, or 190 overexpression resulted in the downregulation of TIMP-3 and functioned to repress the angiogenic phenotype in gliomas,⁹⁸ and additional studies have shown that miR-21, miR-221, and -222 can also regulate TIMP-3 expression.^99,100

MicroRNAs regulate RECK

RECK, a metastasis suppressor gene that can negatively regulate MMP-2, -9, and -14 activities and inhibit tumor invasion and metastasis, is a membrane-anchored glycoprotein that has been detected in a variety of normal human tissues.¹⁰¹ However, reduced RECK expression has been observed in various types of tumor tissues and is frequently associated with poor prognoses.¹⁰² Thus, RECK upregulation may represent a valuable therapeutic option for improving patient prognosis and blocking tumor progression, and microRNA antagonists previously shown to upregulate RECK may serve as potential candidates for cancer therapy.

Gabriely et al. found that transfection of glioma cells with miR-21-ASO led to elevated levels of RECK and TIMP-3, which reduced MMP activity and decreased the migratory and invasive abilities of glioma cells.¹⁰⁰ RECK was later identified to be a target of miR-21 in gastric cancer.¹⁰³ At present, a number of findings have suggested that miR-21 may be important for the progression of multiple cancers, including prostate cancers,¹⁰⁴ liver cancers,^105,106 gliomas,¹⁰⁷ osteosarcomas,¹⁰⁸ and pancreatic adenocarcinomas,¹⁰⁹ due to its direct regulation of RECK.

In addition to the previously proposed miR-21-mediated regulation of RECK in various cancers, miR-7 was also shown to regulate RECK, and both microRNAs were confirmed to regulate RECK in oral cancers. Increased levels of miR-7 and miR-21 were shown to posttranscriptionally repress RECK in oral squamous cell carcinomas (OSCC) via direct microRNA-mediated regulation, and environmental changes may further modulate RECK mRNA levels via alterations in miR-7 and miR-21 levels.¹¹⁰ Recently, miR-222 was shown to be another novel regulator of RECK in H. pylori-associated gastric cancers, functioning to promote cell proliferation and colony formation in vitro. In addition, RECK is also controlled by miR-15b/16, miR-372/373, and miR-342 and can mediate multiple signaling pathways under different microenvironmental conditions.^111,112 Although these microRNAs can simultaneously target RECK, different amounts of specific microRNAs are required to achieve target regulation.

Conclusions and Perspectives

As uncontrolled MMP activity has been linked to cancer progression, MMPs are attractive targets for the development of specific inhibitors that may have clinical applications. However, all of the clinical trials that have been conducted using MMP inhibitors in advanced cancer patients have failed, with the exception of Metastat (which has entered Phase II trials). Although there may be many factors that have contributed to this failure of MMP inhibitors in the clinic, we identified two possible reasons in this review. First, these MMPs share generally similar active site structures, have overlapping specificities, and play numerous key roles in important biological processes other than tumor development, which makes it difficult to design MMP inhibitors that are highly selective and have low side effect profiles. Second, all of the clinical trials that have been conducted to date involved patients with terminal-stage cancers, where several overlapping pathways may come into play.¹¹³

Therefore, in order to improve the applicability of MMPs for tumor therapy, new MMP inhibitors should be able to both regulate individual MMPs selectively and control a network of interconnected molecules in which the central node is represented by an individual MMP. In this respect, miRNA-mediated MMP regulation may lead to the development of MMP inhibitors because miRNAs can target MMPs more selectively without the interference of the structural similarities of MMP catalytic domains. Moreover, miRNAs can target multiple molecules, frequently in the context of a network, which makes them extremely efficient at regulating distinct biological processes that are relevant to malignant tumors.

In this review, we concluded that different microRNAs function to improve or inhibit tumor growth, angiogenesis and metastasis by regulating different types of MMPs either directly or indirectly (Table 1).

Table 1. Functional and pathological implications of microRNA-mediated MMP regulation.

MMPs	Group	MicroRNA		Cancer type	Effect of microRNA on tumor development
		Directly target MMP	Indirectly target MMP
MMP-1	Collagenases	miR-22226	miR-224,23 miR-146a,24 miR-34a25	breast cancer,23,24 colon cancer,25 OTSCC26	Invasion and migration
MMP-2/MMP-9	Gelatinases		Dicer,40 miR-125b,41,42 miR-21,43,44 miR-143,45 miR-451,46 miR-7,47 miR-206,48 miR-340,49 miR-9,50 miR-196b51	GMB,41,46,47 liver cancer,42,43 pancreatic cancer,44,45 breast cancer,40,48,49 uveal melanoma,50 gastric cancer51	Invasion and migration; proliferation and apoptosis44,46,47
MMP-2	Gelatinases	miR-29b52-55	miR-21,56,57 miR-139,58 miR-101,59 miR-146a,90 miR-26a60	liver cancer,53 breast cancer,54 colon cancer,55,58 GMB,56 LSCC,57 lung cancer,59,60 prostatic cancer52,90	Invasion and migration; angiogensis53
MMP-9	Gelatinases	miR-491-5p61	miR-125b,62 miR-145,67 miR-218,63,64 miR-224,66 miR-373,31 miR-338-3p65	GMB,62,61,63,64 gastric cancer,67 liver cancer66,65	Invasion and migration
MMP-3	Stromelysins	miR-15236		GMB36	Invasion
MMP-13	Collagenases	miR-125b,27 miR-14331		CSCC,27 osteosarcoma31	Invasion and migration
MMP-14	Transmembrane	miR-10b,83 miR-984	miR-200,79 let-7,80 miR-155,81 miR-216b81	pancreatic cancer,79,80,81 GMB,83 neuroblastoma84	Invasion and migration; angiogensis84
MMP-16	Transmembrane	miR-146a,87 miR-146b88		colon cancer,87 GMB88	Invasion and migration
RECK	Inhibitor	miR-21,100,103-110 miR-7,110 miR-222112	miR-342113	prostatic cancer,104 liver cancer,105,106 GMB,107,100 osteosarcoma,108 pancreatic cancer,109 gastric cancer,103,112 OSCC,110 colon cancer113	Invasion and migration; proliferation and apoptosis109,112,113
TIMP-3	Inhibitor	miR-103,69 miR-181b,96 miR-19197	miR-580/588/19098	endometrial cancer,69 liver cancer,96,97 GMB98	invasion and migration; angiogensis98

Abbreviations: OTSCC, oral tongue squamous cell carcinoma; GMB, glioblastoma; LSCC, laryngeal squamous cell carcinoma; OSCC, oral squamous cell carcinomas; CSCC, cutaneous squamous cell carcinoma.

Cell migration and invasion occur as normal events in a number of physiological processes, but uncontrolled migration and invasion lead to metastasis, which causes up to 90% of human cancer deaths. All MMPs are involved in cell migration and invasion and are frequently controlled by various microRNAs. In particular, MMP-2 and -9 are the most commonly studied MMPs in the processes of migration, invasion, and metastasis. Although more than a dozen microRNAs were shown to suppress tumor migration and invasion by controlling MMP-2 and MMP-9 (either concurrently or alone), only miR-29b was found to directly target MMP-2, and only miR-491-5p was found to directly target MMP-9.^53,61 Regarding furin-activated MMPs, miR-9 was shown to target MMP-14 to inhibit the invasion, migration, and metastasis of neuroblastoma cells. Furthermore, miR-146a/b inhibits the migration and invasion of glioma and Caco-2 cells by targeting MMP-16.^87,88 Among archetypal MMPs, miR-152 reduces glioma cell invasion by targeting MMP-3,³⁶ and miR-222 reduces cell invasion by targeting MMP-1 in OTSCC.²⁶ miR-125b and miR-143 play similar roles, as miR-125b directly targets MMP-13 in CSCC, and miR-143 directly targets MMP-13 in osteosarcomas.^27,31 In contrast, miR-21 acts as an oncomir and promotes metastasis by targeting RECK in prostate cancer. Accordingly, MMPs and their inhibitors promote or inhibit the migration and invasion of multiple cancers by regulating microRNAs either directly or indirectly. In parallel, miR-21, miR-451, miR-7, and miR-222 were shown to either promote or inhibit cell proliferation, apoptos,is and tumor growth via the direct or indirect regulation of MMPs.^{44,46,47,109,112,114}

Fewer studies have examined the ability of microRNAs to target MMPs and interfere with tumor angiogenesis, although MMPs are known to participate in the tumor angiogenic process, mainly by degrading the ECM and releasing and/or activating pro-angiogenic growth factors, resulting in the enhanced local formation of new vessels. In this review, only miR-29b and miR-9 were reported to suppress tumor angiogenesis by inhibiting MMP-2 and MMP-14, respectively.^53,84 However, microRNA-mediated MMP regulation may not always have positive effects on tumor angiogenesis. For example, miR-152 did not reduce tube formation in cultured endothelial cells, although it did promote cell invasion by directly targeting MMP-3.³⁶ Furthermore, although TIMP-3 has been demonstrated to inhibit angiogenesis,⁹⁴ miR580/588/190 targeted TIMP-3 directly and functioned to inhibit hypoxia-induced angiogenesis in glioblastoma.⁹⁸ Therefore, the specific function of microRNAs in MMP regulation should be studied on an individual basis, and no absolute rule applies for all MMPs. Overall, the interactions between miRNAs and the MMP network demonstrate that miRNAs play essential roles in the activation of MMP function and indicate that the interplay and crosstalk between these molecules promote tumor angiogenesis.

In conclusion, the total number and composition of microRNAs that regulate each MMP vary widely, and the same microRNA can affect different types of MMPs, likely due to tissue-specific effects. Multiple regulatory pathways are involved in microRNA-mediated MMP regulation, and many of these remain unknown. Furthermore, many of the MMPs that are involved in the process of tumorigenesis, including MMP-7, -8, -10, -12, -15, -17, -21, -22, -23, -24, -26, and -27, have not yet been shown to be regulated by microRNAs. Based on the growing relevance of microRNAs in the regulation of tumorigenesis and tumor development, it is tempting to speculate that further work in this field will reveal that additional microRNAs can target specific MMPs, thus contributing to their functional regulation in the different physiological and pathological contexts in which they are implicated. A thorough understanding of the miRNA-mediated regulatory mechanisms governing tumor progression should help to generate additional strategies for, and provide new insights into, the diagnosis and treatment of malignant tumors.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.