MMP-14 Exhibits Greater Expression, Content and Activity Compared to MMP-15 in Human Renal Carcinoma.

Membrane-type metalloproteinases (including MMP-14 and MMP-15) are enzymes involved in the degradation of extracellular matrix components. In cancer, they are involved in processes such as cellular invasion, angiogenesis and metastasis. Therefore, the aim of this study was to evaluate the expression, content and activity of MMP-14 and MMP-15 in human renal cell carcinoma. Samples of healthy kidney tissue (n = 20) and tissue from clear-cell kidney cancer (n = 20) were examined. The presence and contents of the MMPs were assessed using Western blot and ELISA techniques, respectively. Their activity-both actual and specific-was evaluated using fluorimetric analysis. Both control and cancer human kidney tissues contain MMP-14 and MMP-15 enzymes in the form of high-molecular-weight complexes. Moreover, these enzymes occur in both active and latent forms. Their content in cancer tissues is very similar, but with a noteworthy decrease in content with an increase in the kidney cancer grade for both membrane-type metalloproteinases. Even more notable is the highest content of the investigated enzymes represented by MMP-14 in the control tissues. Considering the actual and specific activity outcomes, MMP-14 dominates over MMP-15 in all of the investigated tissues. Nevertheless, we also noted a significant enhancement of the activity of both metalloproteinases with an increase in the grade of renal cancer. The expression and activity of both enzymes were detected in all examined renal cancer tissues. However, our findings suggest that transmembrane metalloproteinase 14 (MMP-14) plays a much more significant and essential role than MMP-15 in the studied renal carcinoma tissues. Therefore, it seems that MMP-14 could be a promising target in the diagnosis, prognosis and therapy of renal cell carcinoma.

An MMP-based risk score strongly distinguishes prognosis in hepatocellular carcinoma after resection.

Aim: To first explore the prognostic value of MMP11 and MMP15 in hepatocellular carcinoma. Methods: MMP11/MMP15 expression was immunohistochemically detected and correlated with clinicopathologic variables and survival and confirmed in publicly available databases. An MMP-based risk score (MMPRS) was established. Results: Tumoral MMP11/MMP15 expression was higher and univariately associated with crucial clinicopathologic parameters, overall survival and disease-free survival in all patients and/or many subsets. Multivariately, MMP11/MMP15 expression remained significant. Their overexpression and prognostic value were confirmed in the Ualcan and Kaplan-Meier plotter databases. Critically, the novel MMPRS integrating MMP11, MMP15 and tumor-node-metastasis stage identified subgroups with the best and worst prognoses, with much higher predictive power. Conclusion: MMP11 and MMP15 served as prognosticators in hepatocellular carcinoma. MMPRS might work more accurately.

MMP11 and MMP15, involved in cancer dissemination, were found to have important biological functions in several cancers. However, their prognostic value in hepatocellular carcinoma (HCC) remains unknown. In the present study, it was found that MMP11 and MMP15 were overexpressed and predictive of the outcome of HCC. Moreover, the novel MMP-based risk score integrating MMP11, MMP15 and tumor­node­metastasis stage had much higher prognostic power. MMP11, MMP15 and especially the MMP-based risk score were identified as promising indicators of prognosis in HCC.

Differential Expression and Localization of ADAMTS Proteinases in Proliferative Diabetic Retinopathy.

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR.

Inhibition of medaka ovulation by gap junction blockers due to its disrupting effect on the transcriptional process of LH-induced Mmp15 expression.

Using medaka, we found that in vitro follicle ovulation, but not germinal vesicle breakdown, was inhibited by three gap junction blockers, carbenoxolone, mefloquine, and flufenamic acid. The blockers specifically inhibited follicular expression of matrix metalloproteinase-15 mRNA and the protein (mmp15/Mmp15), a protease indispensable for medaka ovulation, indicating that gap junctional communication may be required for successful ovulation and mmp15/Mmp15 expression. Further experiments using carbenoxolone as the representative of the gap junction blockers showed that expression of nuclear progestin receptor (Pgr), a transcription factor required for mmp15 expression, was not affected by carbenoxolone treatment, but the formation of phosphorylated Pgr was considerably suppressed. Carbenoxolone treatment caused a decrease in the Pgr binding to the promoter region of mmp15. mRNA expression of cyclin-dependent protein kinase-9 (cdk9) and cyclin I (ccni), whose translation products are demonstrated to be involved in Pgr phosphorylation in the medaka ovulating follicles, was suppressed by carbenoxolone treatment. Transcripts of connexin 34.5 (cx34.5) and connexin 35.4 (cx35.4) were dominantly expressed in the follicle cells of ovulating follicles. The results indicate that gap junctional communication plays an important role in medaka ovulation.

Silencing PROK2 Inhibits Invasion of Human Cervical Cancer Cells by Targeting MMP15 Expression.

Cervical cancer is the second most frequent type of gynecologic cancer worldwide. Prokineticin 2 (PROK2) is reported to be involved in tumor progression in some malignant tumors. However, the role of PROK2 in the development of cervical cancer remains unknown. Our results indicate that PROK2 is overexpressed in the human cervical cancer. Cervical cancer patients with high PROK2 expression have a shorter overall survival rate (OS) and disease-free survival rate (DFS). PROK2 acts as a potential biomarker for predicting OS and DFS of cervical cancer patients. We further show that PROK2 is important factor for oncogenic migration and invasion in human cervical cancer cells. Knockdown PROK2 significantly inhibited cell migration, invasion, and MMP15 protein expression in HeLa cells. High expression of MMP15 is confirmed in the human cervical cancer, is significantly associated with the shorter overall survival rate (OS) and is correlated with PROK2 expression. Overexpression of PROK2 using PROK2 plasmid significantly reverses the function of knockdown PROK2, and further upregulates MMP15 expression, migration and invasion of human cervical cancer cells. In conclusion, our findings are the first to demonstrate the role of PROK2 as a novel and potential biomarker for clinical use, and reveal the oncogenic functions of PROK2 as therapeutic target for cervical cancer.

Fluctuation of Rac1 activity is associated with the phenotypic and transcriptional heterogeneity of glioma cells.

Phenotypic heterogeneity of cancer cells is caused not only by genetic and epigenetic alterations but also by stochastic variation of intracellular signaling molecules. Using cells that stably express Förster resonance energy transfer (FRET) biosensors, we show here a correlation between a temporal fluctuation in the activity of Rac1 and the invasive properties of C6 glioma cells. By using long-term time-lapse imaging, we found that Rac1 activity in C6 glioma cells fluctuated over a timescale that was substantially longer than that of the replication cycle. Because the relative level of Rac1 activity in each cell was unaffected by a suspension-adhesion procedure, we were able to sort C6 glioma cells according to the levels of Rac1 activity, yielding Rac1(high) and Rac1(low) cells. The Rac1(high) cells invaded more efficiently than did Rac1(low) cells in a Matrigel invasion assay. We assessed the transcriptional profiles of Rac1(high) and Rac1(low) cells and performed gene ontology analysis. Among the 14 genes that were most associated with the term 'membrane' (membrane-related genes) in Rac1(high) cells, we identified four genes that were associated with glioma invasion and Rac1 activity by using siRNA knockdown experiments. Among the transcription factors upregulated in Rac1(high) cells, Egr2 was found to positively regulate expression of the four membrane-related invasion-associated genes. The identified signaling network might cause the fluctuations in Rac1 activity and the heterogeneity in the invasive capacity of glioma cells.

m1A demethylase Alkbh3 regulates neurogenesis through m1A demethylation of Mmp15 mRNA.

BACKGROUND: N1-Methyladenosine (m1A) is an abundant modification of transcripts regulating mRNA structure and translation efficiency. However, the characteristics and biological functions of mRNA m1A modification in adult hippocampal neurogenesis remain enigmatic. RESULTS: We found that m1A demethylase Alkbh3 was dramatically enriched in neurons and neuronal genesis. Functionally, depletion of Alkbh3 in neural stem cells (NSCs) significantly decreased m1A modification, neuronal differentiation and proliferation coupling with increasing gliogenesis, whereas overexpressing Alkbh3 facilitated neuronal differentiation and proliferation. Mechanistically, the m1A demethylation of Mmp15 mRNA by Alkbh3 improved its RNA stability and translational efficacy, which promoted neurogenesis. Therapeutically, the silencing of Alkbh3 reduced hippocampal neurogenesis and impaired spatial memory in the adult mice. CONCLUSIONS: We reveal a novel function of m1A demethylation on Mmp15 mRNA in Alkbh3-mediated neurogenesis, which shed light on advancing Alkbh3 regulation of neurogenesis as a novel neurotherapeutic strategy.

Magnolin targeting of the JNK/Sp1/MMP15 signaling axis suppresses cervical cancer microenvironment and metastasis via microbiota modulation.

Magnolin (MGL), a compound derived from the magnolia plant, has inhibitory effects on tumor cell invasion and growth. His study aims to explore the antitumor effect and underlying molecular mechanism of MGL against human cervical cancer. We found that MGL inhibited the proliferation, migration, and invasiveness of cervical cancer cells in vitro and in vivo. The underlying mechanism was shown to involve MGL-induced inhibition of JNK/Sp1-mediated MMP15 transcription and translation. Overexpression of JNK/Sp1 resulted in significant restoration of MMP15 expression and the migration and invasion capabilities of MGL-treated cervical cancer cells. MGL modulated the cervical cancer microenvironment by inhibiting cell metastasis via targeting IL-10/IL-10 receptor B (IL-10RB) expression, thereby attenuating JNK/Sp1-mediated MMP15 expression. Analysis of the gut microbiota of mice fed MGL revealed a significant augmentation in Lachnospiraceae bacteria, known for their production of sodium butyrate. In vivo experiments also demonstrated synergistic inhibition of cervical cancer cell metastasis by MGL and sodium butyrate co-administration. Our study provides pioneering evidence of a novel mechanism by which MGL inhibits tumor growth and metastasis through the IL-10/IL-10RB targeting of the JNK/Sp1/MMP15 axis in human cervical cancer cells.

Asiatic acid from centella asiatica exert anti-invasive ability in human renal cancer cells by modulation of ERK/p38MAPK-mediated MMP15 expression.

BACKGROUND: Asiatic acid (AA) is a naturally pentacyclic triterpenoids extracted from traditional medicine Centella asiatica l. that has demonstrated possesses potential health benefits and antitumor ability. However, the precise anticancer effects and mechanisms by which AA impact RCC cells remains unclear. METHODS: Cell proliferation and cell cycle distribution were detected by MTT, colony formation assay and PI stain by flow cytometry, respectively. Cell mobility and invasiveness were determined by in vitro migration and invasion assay. The secretory MMP15 was detected by ELISA assay. Quantitative RT-PCR, siRNA, and immunoblot were used to determine gene expression/regulation and protein expression, respectively. Antimetastatic effect of AA were performed to lung nodule numbers in vivo metastasis mice model. MMP15, pERK1/2 and p-p38MAPK expressions were determined by immunohistochemistry. RESULTS: Our findings indicated cell proliferation and cell cycle distribution of RCC cells were not significantly influenced by AA treatment. AA suppressed cell migration, invasion and significantly down-regulated mRNA and protein expression of MMP-15 (Matrix Metallopeptidase-15). Activation of ERK1/2 and p38MAPK were inhibited with AA, whereas combined AA with siRNA-ERK or siRNA-p38MAPK markedly reduced the metastatic effect and decreased MMP-15 expression in 786-O and A498 cells. Finally, AA significantly reduced the lung metastasis formation and metastasis-related proteins of human 786-O cells in vivo metastasis mice model. CONCLUSION: AA inhibits the metastatic properties of RCC cells via inhibition of the p-ERK/p-p38MAPK axis and the subsequent down-regulation of MMP-15 in vitro and in vivo. Further study of AA as a potential anti-metastatic agent for RCC is warranted.

[Intervention effects of miR-125b-5p on cognitive dysfunction induced by traumatic brain injury in rats and its mechanisms].

OBJECTIVE: To investigate the effects and molecular mechanisms of miR-125b-5p on cognitive dysfunction caused by traumatic brain injury (TBI). METHODS: The rats were randomly divided into control group, TBI group (model group), NC Agomir group (false negative group) and miR-125b-5p agomir group (high expression group), with 5 rats in each group. The false negative group and the high expression group were injected with NC agomir and miR-125b-5p agomir, respectively. The brain injury model was established by modified Feeney method except control group. Animal behavioral experiments were utilized for evaluation of the motor coordination, learning and memory and the degree of nerve damage in rats; and enzyme-linked immunosorbent assays (ELISA) and Western blot (WB) were used for determination of the expression levels of inflammatory factors and nerve-related factors in the hippocampus of rats in each group respectively. Finally, combined with bioinformatics, downstream target genes of miR-125b-5p were predicted and verified by reverse transcription polymerase chain reaction (RT-PCR) and WB. RESULTS: Compared with control group, mir-125b-5p expression level, motor coordination ability, learning and memory ability, brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) expression levels of rats in model group and false negative group were decreased significantly, the MNSS score, the expressions of interleukins (IL-1ß, IL 6), tumor necrosis factor-α(TNF-α) and glial fibrillary acid protein(GFAF) were increased significantly (P＜0.01);However, compared with model group and false negative group, the above situation of rats in high expression group was opposite (P＜0.01). Bioassay showed that MMP-15 was the downstream target gene of miR-125b-5p. Compared with the control group, the expression of MMP-15 in model group and false negative group was increased significantly (P＜0.01);Compared with model group and false negative group, the expression of MMP-15 in high expression group was decreased significantly (P＜0.01) . CONCLUSION: miR-125b-5p can improve cognitive dysfunction induced by TBI in rats, which may be related to regulating the expression level of MMP-15, thereby inhibiting the neuroinflammatory response after TBI and promoting neuronal regeneration.

Tiki proteins are substrates of membrane-type matrix metalloproteinases.

Tiki proteins represent a new family of Wnt-specific proteases that inhibit Wnt signalling by cleaving and inactivating Wnt proteins. Tiki proteins are glycosylphosphatidylinositol (GPI)-anchored proteases and function in both Wnt-producing and Wnt-responsive cells. However, how Tiki proteins are regulated remains elusive. In this study, we demonstrate that matrix metalloproteinase 15 (MMP15) interacts with TIKI2 and degrades TIKI2 on the cell surface. Functionally, MMP15 relieves the inhibitory effect of TIKI2 on Wnt signalling in Wnt-responsive cells. We further show that Tiki proteins are substrates of MMP14, MMP15 and MMP16 but not MMP3 or MMP13. Our study provides insights into the potential regulation of Tiki family proteins by other proteases.

LINC00963 facilitates acute myeloid leukemia development by modulating miR-608/MMP-15.

Despite continuous improvements of AML therapy, the prognosis of AML patients remains unsatisfactory. Recently, lncRNAs have been reported to participate in the development of AML. Our data demonstrated that MMP15 and LINC00963 were upregulated and miR-608 was decreased in AML cells (THP-1, HL-60, HEL and MOLM-13) compared to HS-5 cells. RT-qPCR results showed that LINC00963 levels were higher in the serum and bone marrow of AML cases than in controls. Moreover, overexpression of LINC00963 promoted AML cell growth and EMT progression in both THP-1 and HL-60 cells. Furthermore, miR-608 levels were downregulated in the serum and bone marrow of AML cases compared with controls, and Pearson's correlation analysis indicated that LINC00963 was negatively correlated with miR-608 in the serum and bone marrow of AML samples. In addition, we demonstrated that LINC00963 sponged miR-608 expression and that MMP-15 was a target of miR-608 in AML cells. Finally, rescue experiments indicated that ectopic expression of LINC00963 accelerated cell growth and EMT development by modulating MMP-15. These data demonstrated that LINC00963 acted as an oncogene and may be a potential target for AML treatment.

Silencing LINC00482 inhibits tumor-associated inflammation and angiogenesis through down-regulation of MMP-15 via FOXA1 in bladder cancer.

Multiple studies have previously demonstrated that long intergenic non-coding RNAs (lincRNAs) play an important role in the development of bladder cancer. However, little is known regarding the underlying molecular mechanisms of LINC00482 functions in bladder cancer. The current study aimed to elucidate the role of LINC00482 in the progression of bladder cancer. The initial step was to detect the expressions of LINC00482 and MMP15 in bladder cancer cells and tissue. According to the results from the RT-qPCR, LINC00482 and MMP15 were both highly expressed in bladder cancer cells and tissue. The relationship among LINC00482, FOXA1 and MMP15 was studied via dual-luciferase reporter assay. LINC00482 was positively correlated with MMP15. LINC00482 promoted MMP15 expression by recruiting FOXA1. Using the gain- and loss-of-function approaches, silencing of LINC00482 resulted in the downregulation of VEGF and NF-κB protein levels, decreased expression of inflammatory factors, and inhibited angiogenesis. Silencing of LINC00482 also suppressed tumor-associated inflammation and angiogenesis in vivo, which was found to be reversed by the overexpression of MMP15. The present study demonstrated that LINC00482 induced the expression of MMP15 by interacting with FOXA1, thereby contributing to the inflammation and angiogenesis in bladder cancer.

The oncoprotein HBXIP facilitates metastasis of hepatocellular carcinoma cells by activation of MMP15 expression.

Background: Due to the high recurrence and metastasis rate, the clinical outcomes of patients with hepatocellular carcinoma (HCC) are still unsatisfactory. Hepatitis B virus X-interacting protein (HBXIP) has been reported to play crucial roles in carcinogenesis. Purpose: We aimed to reveal the functional significance and underlying mechanism of HBXIP in HCC metastasis. Methods: Cell transwell assay, in vivo metastasis model, real-time PCR, western blot analysis, luciferase reporter and chromatin immunoprecipitation assays were applied. Results: Here, we detected the HBXIP expression level and determined its clinical significance in HCC. We found that HBXIP was significantly upregulated in HCC tissues, and correlated with vascular invasion, tumor metastasis and worse prognosis of HCC patients. HBXIP enhanced cell migration and invasion in vitro, and promoted the metastasis of HCC in vivo. Furthermore, we confirmed that HBXIP increased MMP15 expression through association with proto-oncogene c-myc. Depletion of c-myc abolished HBXIP-mediated MMP-15 upregulation. We also observed a positive correlation between HBXIP and MMP15 expression in HCC tissues. Conclusion: Our results establish a novel function for HBXIP-MMP15 regulation in HCC metastasis and suggest its candidacy as a new prognostic biomarker and therapeutic target for HCC metastasis.

Generation of mmp15b Zebrafish Mutant to Investigate Liver Diseases.

Upon gene inactivation in animal models, the zebrafish (Danio rerio) has become a useful model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. Mainly, CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR-associated protein 9) is a technology that enables geneticists to edit parts of the genome. In this study, we utilized this technology to generate an mmp15b mutant by using zebrafish as an animal model. MMP15 is the membrane-type MMP (MT-MMP) which is a recently identified matrix metalloproteinase (MMP) capable of degrading all kinds of extracellular matrix proteins as well as numerous bioactive molecules. Although the newly-established mmp15b zebrafish mutant didn't exhibit morphological phenotypes in the developing embryos, it might be further utilized to understand the role of MMP15 in liver-related diseases, such as liver fibrosis, and associated pathogeneses in humans.

Involvement of the nuclear progestin receptor in LH-induced expression of membrane type 2-matrix metalloproteinase required for follicle rupture during ovulation in the medaka, Oryzias latipes.

Hormonal regulation of the expression of Mmp15, a proteolytic enzyme indispensable for ovulation in the teleost medaka, was investigated. In an in vitro culture system using preovulatory follicles, Mmp15 expression and ovulation were induced in the presence of recombinant luteinizing hormone (rLh). Both rLh-induced Mmp15 expression and ovulation were 17α, 20ß-dihydroxy-4-pregnen-3-one-dependent, suggesting the involvement of a nuclear progestin receptor (Pgr). In vitro follicle ovulation and Mmp15 expression were reduced by treatment with the Pgr antagonist RU-486. Like Pgr, the transcription factor CCAAT/enhancer-binding protein ß (Cebpb) was induced by rLh. ChIP analyses indicated that Pgr and Cebpb bound to the mmp15 promoter region. These results indicate that the rLh-induced expression of Mmp15 is mediated by Pgr and Cebpb. A differential timing of expression of Pgr and Cebpb in the preovulatory follicles appears to explain the considerably long time-lag from the pgr gene activation to mmp15 gene expression.