MiR-181a-5p Inhibits Cancer Cell Migration And Angiogenesis Via Downregulation Of Matrix Metalloproteinase-14

Background and AimsMetastasis, the dissemination of cancer cells from the primary tumor to a distant organ, accounts for 90% of human cancer related deaths. Pathologically, metastasis is a multistep process consisting of a series of discrete biologic processes. One of the key biologic steps is cancer cell invasion, which is involved in almost every stage of the metastatic cascade from initiation of metastasis to establishment of a secondary tumor.Matrix metalloproteinases (MMPs) are a family of 25 highly homologous Zn++-dependent endopeptidases. The primary functions of MMPs are traditionally considered to be the degradation of cell adhesion molecules and removal of extracellular matrix (ECM) to permit cancer cell invasion. MMPs have been recently recognized as cell migration enhancers, independent of their proteolytic activity. Matrix metalloproteinase-14 (MMP-14) is a membrane inserted MMP that has been found to play critical roles in cancer invasion and metastasis by cleaving EC8M and basement membrane proteins, activating proMMP-2 and-13, inducing the activation of growth factors, and enhancing cell migration. Phenotypically, MMP-14 plays a critical role in converting epithelial cells to migratory mesenchymal-like cells (epithelial-to-mesenchymal transition, EMT), which is considered an important mechanism for the initial step in the metastatic process. However, the regulatory mechanism of MMP-14 expression during cancer progression remains to be characterized.It has been reported that MMP-14 expression is upregulated by transcription factors such as Spl and Egr-1. MMP-14 has also been demonstrated to be transcriptionally regulated by hypoxia-inducible factor-2a. Not only can MMP-14 expression be positively regulated by these transcription factors, it can also be negatively regulated by microRNAs (miRNAs, miR), such as miR-9 and-133a.miRNAs or miR are small, highly conserved noncoding RNAs that have been reported to participate in the metastatic process by negatively or positively regulating gene expression of metastasis associated genes through posttranscriptional repression, mRNA degradation, or promoter activation. Although miRNAs can positively affect gene expression by binding to the promoter region of a regulated gene, the majority of miRNAs are reported to negatively regulate target gene expression by repressing translation or inducing sequence-specific degradation of target mRNAs through interaction with the 3’-untranslated regions (3’UTR) of target mRNAs.miRNAs are transcribed as approximately 70 nucleotide precursors and subsequently processed by the RNase-Ⅲ type enzyme Dicer to give an approximately 22 nucleotide mature product. More than 1,500 miRNA genes have been identified in the human genome that collectively control an estimated 30% of all human genes. Each miRNA appears to regulate the expression of tens to hundreds of genes to efficiently coordinate multiple cellular pathways. Interestingly, many miRNAs exist as a multi-member family, indicating their functional redundancy.miR-181a-5p belongs to the miR-181s family, which includes four highly conserved mature miRNAs:miR-181a, b, c, and d. They are derived independently from six precursors located on three different chromosomes. This new class of genes has recently been shown to play a central role in malignant transformation. In contrast, miR-181s are downregulated in many tumors and thus appear to function as tumor suppressor genes.In this study, we identified miR-181a-5p as a negative regulator of MMP-14 by directly targeting the 3’UTR of MMP-14 mRNA, resulting in decreased cell migration, invasion, and angiogenesis. Our data highlight a functional role of miR-181a-5p in cancer dissemination and uncover a potential prognostic biomarker and molecular target for the prevention of cancer metastasis.Materials and MethodsIn section one, we demonstrated that MMP-14 is upregulated in various human cancers and the expression level of MMP-14 is correlated with the invasive status of human cancer by using data mining, laser capture microdissection, real time PCR and immunohistochemistry approaches.In section two, miR-181s was identified as potential regulators of MMP-14. We confirmed that the observed downregulation of MMP-14 by miR-181a-5p depends directly on a single cognate recognition site in the 3’UTR of MMP-14 mRNA via bioinformatics analysis, DNA construction, lentivirus system, real time PCR, western blot and dual-luciferase reporter assay.In section three, we explored the effect of functional MMP-14 by miR-181a-5p, using DNA construction, lentivirus system, gelatin zymography, western blot, Transwell chamber migration assay, dot-based 2D migration assay, dot-based 3D invasion assay, macrodissection, real time PCR, cell surface biotinylation assay, chicken chorioallantoic membrane invasion and angiogenesis assay to demonstrate that miR-181a-5p is a critical regulator for MMP-14 expression and can affect MMP-14-mediated cancer cell migration, invasion, and angiogenesis.Results1. MMP-14 is upregulated in various human cancersUpregulated MMP-14 expression in cancer cells has been shown to promote tumor growth and metastasis. Although MMP-14 has been reported to be upregulated in various human cancers, there is no systematic analysis of MMP-14 expression in different cancers from different organs. By mining DNA microarray databases at Oncomine (Cancer Profiling Database), expression of MMP-14 in human cancer specimens was assessed. When assigning a P< 0.01 limit, a myriad of cancers displayed upregulation of MMP-14 as compared with respective nonmalignant control tissues.To validate the data mining results, human breast cancer specimens were examined for the expression of MMP-14 by examining mRNA and protein levels. A laser capture microdissection (LCM) technique was used to harvest breast carcinoma cells and tumor-adjacent normal epithelial cells as we previously used followed by real-time RT-PCR for the mRNA of MMP-14. Our data show that MMP-14 is selectively expressed in human breast ductal carcinoma in situ (DCIS) and in invasive ductal carcinoma, but not in normal epithelial cells. Expression of MMP-14 in invasive ductal carcinoma is elevated as compared with DCIS, although there is no statistical significance among them. To confirm this observation, IHC was performed in the corresponding human breast cancer specimens using an anti-MMP-14 antibody. In agreement with the real-time RT-PCR data, MMP-14 protein was detected only in human breast cancer cells but not in adjacent normal epithelial cells.To further confirm our data mining results showing that the upregulation of MMP-14 is a general phenomenon in cancer, human colon cancer specimens were examined by IHC using the anti-MMP-14 antibody. We observed that MMP-14 is minimally expressed in normal colonic mucosal cells, but increased intensity of staining is found in cancer cells at the invasive front. Thus, our study indicates that MMP-14 is upregulated in human cancers and the expression level of MMP-14 is correlated with the invasive status of human cancer.2. Computational prediction of miR-181s as potential regulators of MMP-14MMP-14 is upregulated in human cancers; however, the regulatory mechanism of MMP-14 remains to be fully characterized. When MMP-14-GFP chimeric cDNA (MMP-14-GFP) containing the 3’UTR of MMP-14 (MMP-14-GFP/3’UTR) was transfected into minimally invasive human breast cancer MCF-7 and highly invasive MDA-MB-231 cells, we noticed that the expression of MMP-14-GFP in MCF-7 cells is relatively weak as compared with that in MDA-MB-231 cells. This difference, however, was not seen in these cells when transfected with the MMP-14-GFP chimera that does not contain the 3’UTR. This observation is reproduced in prostate cancer cell lines of LNCaP (minimally invasive) and DU145 and PC3 (highly invasive), suggesting that the 3’UTR of MMP-14 mRNA may contain a regulatory sequence(s) that is responsible for differential expression of MMP-14 in cancer lines.It is known that the 3’UTR often contains miRNA response elements (MRE), which are sequences to which miRNAs bind. To identify putative MREs within the 3’UTR of MMP-14 mRNA, we used TargetScan to search for potential MREs within the 3’UTR. This analysis revealed multiple MREs that potentially interact with miR-22,-24,-26,-133,-150, and-181s. To increase the probability of postulated MREs within the 3’UTR, another miRNA prediction algorithm, miRanda, was used. By analyzing predicted MREs from both TargetScan and miRanda, only miR-133 and-181s were found to overlap between the two computational analyses. The MRE of miR-181 within the 3’UTR between 291 nt and 297 nt received the highest prediction score for binding to miR-181s, and hence miR-181s were further characterized.3. Interference of ectopically expressed MMP-14 by miR-181 a-5pBecause miR-181 isoforms contain identical seed sequence to the MMP-14 3’UTR, miR-181a-2 along with the 140-bp flanking regions at both sides of miR-181a-2 was amplified from the chromosomal DNA and the resultant DNA was cloned into an MDH1-PGK-GFP 2.0 retroviral vector that contains a GFP reporter (Addgene). On the basis of recent miRNA nomenclature guidelines (http://www.mirbase.org), we designated the mature sequence of hsa-miR-181a-2 in the vector as miR-181 a-5p/GFP. Using a similar strategy, miR-128 that was not predicted to bind the MMP-14 3’UTR was also cloned in MDH1-PGK-GFP 2.0 retroviral vector as a control (miR-128/GFP). Ectopic expression of miR-181a-5p was examined by transient transfection of the cDNAs encoding miR-181a-5p/GFP as well as control vectors into COS-1 cells, which do not express detectable endogenous MMP-14. Mature miR-181a-5p and-128 were detected in the transfected COS-1 cells by a real-time RT-PCR approach with 5-and 7-fold increases of miR-181 a-5p and-128, respectively, as compared with vector control, indicating that mature miR-181 a-5p and-128 are efficiently produced in the transfected cells.We then examined whether miR-181a-5p can affect ectopically expressed MMP-14 in COS-1 cells by cotransfecting MMP-14/3’UTR with miR-control, miR-181a-5p/GFP, and miR-128/GFP, respectively. To increase the expression efficiency for both miRNA and MMP-14/3’UTR, COS-1 cells were first made to stably express each miRNA and was followed by transient transfection of MMP-14/3’UTR cDNA and vector control. Ectopic expression of miR-181a-5p, but not miR-control and miR-128, led to reduced MMP-14mRNAand protein levels, suggesting that MMP-14 mRNA is downregulated by miR-181a-5p.To further determine whether the loss of MMP-14 expression in both mRNA and protein levels by miR-181a-5p is due to the existence of the MMP-14 3’UTR, we used a previously generated plasmid DNA encoding only the open reading frame of MMP-14, and therefore lacks the 3’UTR of MMP-14. Western blotting revealed that there was no distinct difference in ectopic MMP-14 expression between the cells that overexpressed either miR-181a-5p or miR-control, confirming the existence of a miR-181 response element within the 3’UTR.To further validate the role of miR-181a-5p in regulation of MMP-14 expression, we generated U6 promoter-driven sponge to downregulate miR-181a-5p. When the sponges were stably introduced into MCF-7 cells that express a high level of endogenous miR-181a-5p, we found that the sponges efficiently reduced the miR-181 a-5p level in the cells as compared with the control.To determine whether reduced endogenous miR-181a-5p by the sponges leads to enhanced MMP-14 expression, endogenous and ectopically expressed MMP-14 were examined. It has been reported that hypoxia induces MMP-14 expression. When MCF-7 cells stably expressing the sponges were cultured under hypoxia (1%O2) for 48 hours, endogenous MMP-14 increased in cells expressing miR-181 a-5p sponges as compared with control sponges. To further validate the role of the miR-181a-5p in regulating MMP-14 expression, the effect of miR-181 a-5p on ectopic MMP-14 was examined. MCF-7 cells stably expressing the sponges were transfected with MMP-14-GFP/3’UTR and subsequently analyzed for the MMP-14-GFP mRNA and protein expressions. Ectopic expression of miR-181a-5p sponge, but not the miR-control sponge, led to upregulated MMP-14-GFP in both mRNA and protein levels, further suggesting that MMP-14 is negatively regulated by miR-181a-5p.4. Direct interaction of miR-181a-5p in the MMP-14 3’UTRTo determine whether miR-181a-5p targets the MMP-14 3’UTR directly, we used a luciferase reporter gene fused to a sequence of the 3’UTR of MMP-14 that contains the predicted miR-181a-5p response element (Luc/3’UTR). As a control, the predicted miR-181a-5p response element (7 nt) was converted to the complementary sequence to eliminate potential miR-181a-5p binding by using a site-direct mutagenesis approach (Luc/3’UTRmu). Expression of miR-181a-5p significantly reduced luciferase activity of Luc/3’UTR, whereas it had no effect on cells expressing miR-control or Luc/3’UTRmu. When Luc/3’UTR reporter gene was transfected into MCF-7 cells stably expressing miR-181a-5p sponges, the luciferase activity significantly increased as compared with miR-control sponge infected cells, whereas luciferase activity from Luc/3’UTRmu was not affected by miR-181a-5p sponge. Hence, the observed downregulation of MMP-14 by miR-181a-5p depends directly on a single cognate recognition site in the 3’UTR of MMP-14 mRNA.Using an imaging-based assay, we observed that fluorescence intensity in COS-1 cells coexpressing MMP-14-GFP/3’UTR with miR-181a-5p (lacking GFP) significantly decreased as compared with control. Conversely, fluorescence intensity of MMP-14-GFP/3’UTR in MCF-7 cells stably expressing the miR-181a-5p sponge significantly increased as compared with the control sponge.5. Effect of functional MMP-14 by miR-181a-5pWe previously reported that functional MMP-14 enhances proteolytic activity and cell migratory ability. MMP-2, a secretory MMP that promotes cancer invasion and metastasis, has been used as an indicator for functional MMP-14. We then examined whether reduced MMP-14 expression by miR-181a-5p results in decreased MMP-2 activation and cell migration. Coexpression of MMP-14/3’UTR with miR-181a-5p/GFP in COS-1 cells resulted in reduced proMMP-2 activation as demonstrated by decreased intermediate (intMMP-2) and fully activated (actMMP-2) MMP-2, along with increased latent MMP-2 (proMMP-2) as compared with cells expressing miR-control and miR-128. This decrease correlates with reduced MMP-14 expression. Because TIMP-2 is a natural inhibitor of MMP-14, we examined whether the decreased activity of MMP-14 by miR-181a-5p is due to upregulated TIMP-2. Our Western blotting data rules out that possibility because miR-181a-5p does not enhance TIMP-2 expression in MDA-MB-231 cells.Consistent with MMP-2 activation, ectopic expression of miR-181a-5p/GFP along with MMP-14/3’UTR in COS-1 cells led to a decrease in cell migration. Importantly, miR-128 was unable to inhibit migration in the COS-1 cells expressing MMP-14/3’UTR, suggesting that miR-181a-5p is a key factor in control of MMP-14 expression, whose downregulation results in decreased proteolytic potential and cell migratory ability.6. Inverse correlation of miR-181a-5p and MMP-14 in human cancer cell lines and human cancer specimensIt has been reported that upregulated MMP-14 is often observed in invasive human cancers and correlates with aggressiveness of human cancer cell lines. To investigate the correlation between the endogenousmiR-181a-5p level and MMP-14 expression in human cancer cell lines, we surveyed human cancer cell lines using real-time RT-PCR and Western blotting. As expected, MMP-14 expresses at a relatively high level in aggressive cancer lines, including HT1080, MDA-MB-231, and breast cancer stem SK-3rd cells. Interestingly, miR-181a-5p expression is significantly lower in these cells as compared with less aggressive cancer cell lines, such as MCF-7 and SK-BR3 lines. To determine clinical significance, we examined the correlation between miR-181a-5p and MMP-14 in human cancer specimens. Because MMP-14 is minimally expressed in normal primary colonic mucosal cells, but an increased intensity of staining is found in cancer cells located at the invasive front, we harvested tumor cells at the invasive front of human colon cancer specimens as well as tumor-adjacent normal epithelial cells using a macrodissection technique followed by real-time RT-PCR for miR-181a-5p. Consistent with the observation in cell lines, miR-181a-5p is downregulated in invasive tumor cells as compared with tumor-adjacent normal epithelial cells. These data suggest that expression of MMP-14 in human invasive cancer is affected by miR-181a-5p levels. To further delineate the causal relationship between miR-181a-5p and MMP-14 expression in cancer progression, we used MDAMB-231 cells because this cell line is highly invasive and expresses a high level of MMP-14 as compared with other cancer cell lines. When MDA-MB-231 cells were transiently transfected with miR-181a-5p/GFP or miR-control, the endogenous MMP-14 expression at both mRNA and protein level was significantly reduced. This correlation was also reproduced in HT1080 cells that express a high level of endogenous MMP-14.Specific downregulation of MMP-14 expression has been shown to significantly inhibit cancer cell migration and invasion, even though other MMPs continued to be expressed. Therefore, we asked whether miR-181a-5p can affect cancer cell migration. Expression of miR-181a-5p in MDAMB-231 cells resulted in a significant decrease in cell migration as examined by a Transwell chamber migration assay. The ability of miR-181a-5p to reduce cell migration was further confirmed in HT1080 cells ectopically expressing miR-181a-5p.Because cell migration is a critical determination of cancer invasion, a three-dimensional (3D) invasion assay was used to determine whether overexpression of miR-181a-5p in MDA-MB-231 cells and HT1080 cells could inhibit cell invasion. As expected, when overexpressing miR-181a-5p, cell invasive ability was dramatically decreased in both cell lines.Because MMP-14 is a transmembrane protease, we next asked whether reduced cancer cell migration and invasion by miR-181a-5p were due to the loss of cell surface MMP-14. By cell surface biotinylation assay, we found that cell surface MMP-14 was markedly reduced in HT1080 cells ectopically expressing high levels of miR-181a-5p.7. Overexpression of miR-181a-5p attenuates in vivo invasion and angiogenesisMMP-14 is linked to enhanced cancer invasion. To directly examine whether miR-181a-5p is capable of inhibition of MMP-14-mediated cancer cell invasion through basement membrane in vivo, the chick chorioallantoic membrane (CAM) invasion assay was used. The CAM consists of the chorionic epithelium and underlying allantoic membrane that is primarily made of type IV collagen, which simulates the basement membrane of human epithelium. Invasion of cancer cells through the epithelium and basement membrane of the upper CAM into connective tissue was examined by hematoxylin and eosin staining. miR-control MDA-MB-231 cells that were loaded onto the CAM invaded into the connective tissues through the breached basement membrane. In contrast, MDA-MB-231 cells stably expressing miR-181a-5p failed to cross through the basement membrane. In addition, more new blood vessels underneath the CAM can be found in miR-control cells as compared with miR-181a-5p cells. This reduction in invasion, along with the decrease in angiogenesis, suggests that miR-181a-5p is effective at interfering with the in vivo invasive ability of MDA-MB-231 cells, which normally express high levels of MMP-14.To further characterize the antiangiogenic activity of miR-181a-5p through downregulation of MMP-14 expression, HT1080 cells were used. Because minimal miR-181a-5p is present in HT1080 cells, we stably expressed miR-181a-5p in HT1080 cells and applied the cells over chorioallantoic membranes via sponges as previously described. miR-181a-5p, but not miR-control, statistically impaired new blood vessel formation induced by HT1080 cells.Taken together, we, for the first time, demonstrate that miR-181a-5p is a critical regulator for MMP-14 expression and can affect MMP-14-mediated cancer cell migration, invasion, and angiogenesis.ConclusionsIn this study, we show that miR-181a-5p is downregulated in aggressive human breast and colon cancers where its levels correlate inversely with MMP-14 expression. In clinical specimens, enhanced expression of MMP-14 was observed in cancer cells located at the invasive front of tumors where miR-181a-5p was downregulated relative to adjacent normal cells. Bioinformatics analyses defined a potential miR-181a-5p response element within the 3’-untranslated region of MMP-14 that was validated in reporter gene experiments. Ectopic miR-181a-5p reduced MMP-14 expression, whereas miR-181a-5p attenuation elevated MMP-14 expression. In support of a critical relationship between these two genes, miR-181a-5p-mediated reduction of MMP-14 levels was sufficient to decrease cancer cell migration, invasion, and activation of pro-MMP-2. Furthermore, this reduction in MMP-14 levels was sufficient to reduce in vivo invasion and angiogenesis in chick chorioallantoic membrane assays.Taken together, our results establish the regulation of MMP-14 in cancers by miR-181a-5p through a posttranscriptional mechanism, and they further suggest strategies to elevate miR-181a-5p to prevent cancer metastasis.