SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

As the “master” microRNA that is induced by hypoxia, miR-210 is involved in multiple processes in the hypoxia pathway. However, whether miR-210 mediates hypoxia-induced tumor cell metastasis still remains unclear. Here, we demonstrate that miR-210 is frequently up-regulated in hepatocellular carcinoma (HCC) samples and promotes the migration and invasion of HCC cells. Furthermore, miR-210 can be induced by hypoxia in HCC cells and mediates hypoxia-induced HCC cell metastasis. We identify vacuole membrane protein 1 (VMP1) as the direct and functional downstream target of miR-210; in addition, we show that its expression is negatively correlated with the expression of miR-210 in HCC. Intriguingly, VMP1 is reduced by hypoxia, and down-regulation of VMP1 by miR-210 mediates hypoxia-induced HCC cell metastasis. Conclusion: These findings extend our understanding of the function of miR-210 in the hypoxia pathway, and this newly identified hypoxia/miR-210/VMP1 pathway should facilitate the development of novel therapeutics against hypoxic tumor cells. (HEPATOLOGY 2011)

The rapid growth of solid tumor cells requires extra nutrients and oxygen from the environ ment. Although tumors often induce angiogenesis, the abnormal organization of the microvasculature of tumors cannot provide enough oxygen to meet the needs of tumor cells, which ultimately results in hypoxia inside the tumors. Hypoxic tumors are often aggressive, likely to metastasize and resistant to chemotherapy and radiation.1 Investigation of hypoxia-induced signaling pathways will help to develop new tools for the treatment of hypoxic tumors. Among the many factors regulated by hypoxia, hypoxia-induced factor (HIF) plays a central role in the ability of tumor cells to sense and respond to hypoxia.2 HIF is a transcriptional factor involved in many aspects of cancer, including cell proliferation, angiogenesis, invasion, and metastasis, by regulating a number of downstream protein-coding genes, including vascular endothelial growth factor (VEGF) and CXCR4 (cysteine-X-cysteine receptor 4).

Recently, in addition to regulating protein-coding genes, HIF has been found to modulate the transcription of many microRNAs (miRNAs). Among them, miR-210 is the most robustly and consistently up-regulated miRNA in response to hypoxic stress. Moreover, miR-210 acts as a “versatile” molecule and affects many aspects of the cellular response to hypoxia. In the initiation of cancer, miR-210 is induced by HIF1α and can be regarded as a tumor hypoxia marker in vivo.3 By regulating several targets, miR-210 can initially inhibit the proliferation of tumor xenografts. Moreover, miR-210 also regulates cell cycle progression under hypoxia.4 In addition to the inhibitory effect on tumor growth, miR-210 can decrease mitochondrial function and up-regulate glycolysis of tumor cells under hypoxia, which helps cells adapt to hypoxic stress and survive.5, 6 Usually, hypoxia inside tumors can stimulate angiogenesis to sustain tumor growth. In hypoxic conditions, miR-210 also induces the formation of human umbilical vein endothelial cells (HUVEC) into capillary-like structures and promotes the migration of the endothelial cells by targeting the receptor tyrosine kinase ligand, ephrin-A3 (EFNA3).7 Collectively, these results demonstrate that miR-210 can prepare tumor cells for hypoxic stress and may induce angiogenesis to support the growth of the surviving tumor cells. Increasing evidence shows that metastasis is another important hallmark of hypoxic tumors.8 Hypoxia can directly modulate the gene expression program that can drive the metastatic spread of tumor cells by inducing HIF. Although miR-210, the “master” miRNA induced by hypoxia, is involved in multiple processes in the hypoxia response pathway, the functional role of miR-210 in the migration and invasion of tumor cells under hypoxia and the implications of this have yet to be elucidated. Evidence has shown that miR-210 is overexpressed in tumor samples and associated with the aggressiveness of tumors.9 Bioinformatics analysis indicates that miR-210 is coupled to the hypoxia/VEGF signal pathway in tumors.9 This coupling implies that miR-210 may have a role in the hypoxia-induced spread of tumor cells.

In this study, we have demonstrated that miR-210 is often up-regulated in hepatocellular carcinoma (HCC) and can drive the metastatic spread of HCC cells. In addition, we found that miR-210 is induced by hypoxic conditions in HCC cells and can mediate hypoxia-induced HCC cell migration and invasion by directly targeting vacuole membrane protein 1 (VMP1). Our results indicate that miR-210 up-regulation under hypoxia not only helps tumor cells to adjust to hypoxic stress but also confers an aggressive phenotype to hypoxic tumor cells.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell Culture and Tumor Specimens.

HEK (human embryonic kidney) 293T, SMMC-7721, and Huh-7 cells were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37°C. To induce hypoxia, cells were incubated in an atmosphere of 2% O2, 5% CO2, and 93% N2 at 37°C. Forty-eight pairs of human primary HCC and matched adjacent noncancerous liver tissues were collected from the Qi Dong Liver Cancer Institute/Hospital, Qi Dong, China. Clinicopathological features including sex, age, virus infection status, alpha-fetoprotein level, cirrhosis, and intrahepatic metastasis and tumor stages are listed in Supporting Table 1. All human specimens were obtained with informed consent and approved by the Ethical Review Committee of the World Health Organization of the Collaborating Center for Research in Human Production, which has been authorized by the Shanghai Municipal Government. All tissue samples were freshly frozen at −80°C and carefully examined by pathologists.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction.

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was synthesized using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time polymerase chain reaction (PCR) analyses were performed with SYBR Premix Ex Taq (TaKaRa). The primers used are listed in Supporting Table 2. For the detection of mature miR-210, RNA was reverse-transcribed by a specific reverse-transcription primer (Applied Biosystems, Foster City, CA). The expression of miR-210 was quantified by quantitative reverse transcription polymerase chain reaction (RT-PCR) using TaqMan miRNA assays (Applied Biosystems).

Vector Constructs.

The pre–miR-210 sequence was amplified and cloned into the pMD-18T vector (Invitrogen), and after digestion with BamHI and EcoRI, it was subcloned into the pWPXL vector (a gift from Didier Trono, École Polytechnique Fédérale de Lausanne). The open reading frame (ORF) of VMP1 was amplified and cloned into the pWPXL vector. The wild-type and mutant 3′-untranslated regions (UTRs) of VMP1 were cloned downstream of a firefly luciferase cassette driven by a cytomegalovirus promoter in a pCDNA3.0 vector. The primers used are shown in Supporting Table 2.

Lentivirus Production and Transduction.

Virus particles were harvested 48 hours after pWPXL–miR-210 transfection with the packaging plasmid psPAX2 and vesicular stomatitis virus glycoprotein G envelope plasmid pMD2.G (a gift from Didier Trono) into HEK293T cells by Lipofectamine 2000 reagent (Invitrogen). Huh-7 and SMMC-7721 cells were infected with recombinant lentivirus transducing units plus 6 μg/mL polybrene (Sigma, St. Louis, MO).

Oligonucleotide Transfection.

The miR-210 mimics were synthesized by Genepharma (Shanghai, China). The miR-210 inhibitor and small interfering RNAs against VMP1 were synthesized by Ribobio (Guangzhou, China). The sequences used are shown in Supporting Table 2. Cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).

In Vitro Migration and Invasion Assays.

For the migration assays, 5 × 104 cells in serum-free medium were placed into the top chamber of each insert (BD Biosciences, Franklin Lakes, NJ). For the invasion assays, 105 cells in serum-free medium were placed into the top chamber of each insert coated with 150 μg Matrigel (BD Biosciences). After several hours of incubation at 37°C, cells adhering to the lower membrane were stained with 0.1% crystal violet in 20% methanol, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan).

Cell Proliferation and Colony Formation Assays.

Cell proliferation was measured by the Cell Counting Kit-8 (CCK8; Dojindo, Shanghai, China) according to the manufacturer's instructions. For colony formation assays, 1000 cells were plated into each well of a six-well plate and incubated at 37°C for 2 weeks. Cells were stained with 0.1% crystal violet in 20% methanol and counted using an inverted microscope.

Tumor Formation in Nude Mice.

Huh-7 cells (2 × 106) stably expressing the vector or miR-210 were subcutaneously injected into 4- to 6-week-old nude mice. After 4 weeks, mice were sacrificed and the tumors were weighed. Mice were manipulated and housed according to protocols approved by the Shanghai Medical Experimental Animal Care Commission.

Luciferase Assay.

HEK293T cells were plated into 96-well plates and transfected with 100 ng pWPXL-miR-210 or pWPXL, 50 ng luciferase reporter, and 5 ng Renilla luciferase. Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter system (Promega, Madison, WI).

Western Blot.

Proteins were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat milk and incubated with mouse anti-VMP1 polyclonal antibody (Abcam, Southampton, UK) or mouse anti–β-actin monoclonal antibody (Sigma). The protein complex was detected with enhanced chemiluminescence reagents (Pierce, Rockford, IL).

Statistical Analysis.

The results are presented as means and standard error of the mean. The data were subjected to the Student t test (two-tailed; P < 0.05 was considered significant) unless otherwise specified (paired t test, Pearson's correlation).

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

miR-210 Is Frequently Up-Regulated in HCC and Induced by Hypoxia in HCC Cells.

To determine the expression of miR-210 in HCC, the mature miR-210 was detected in 48 pairs of HCC and corresponding noncancerous liver samples using quantitative RT-PCR. The results showed that the expression of miR-210 was significantly up-regulated in HCC when compared to the noncancerous liver samples (P < 0.0001, Fig. 1A). The overall expression level of miR-210 increased 5.2-fold in 29 HCC samples (60.42%), was unchanged in 10 samples and was down-regulated in nine samples (Fig. 1B), indicating that up-regulation of miR-210 is a frequent event in HCC. Given that miR-210 is induced by hypoxia in many cell types, we next sought to determine whether miR-210 could be induced by hypoxia in HCC cells. miR-210 was up-regulated by hypoxia in two HCC cell lines, SMMC-7721 and Huh-7 (Fig. 1C,D). Moreover, the longer the exposure time in hypoxia, the greater the induction of miR-210 expression, suggesting that miR-210 can indeed be regulated by hypoxia in HCC cells.

thumbnail image

Figure 1. miR-210 is often up-regulated in HCC and is induced by hypoxia in HCC cells. (A,B) Expression of mature miR-210 was determined by TaqMan real-time PCR in 48 pairs of HCC and adjacent noncancerous liver tissue (NT). U6 (U6 small nuclear RNA) was used as an internal control. (C,D) miR-210 was induced under hypoxia, with the most robust induction seen after 48 hours in 2% O2 in (C) SMMC-7721 and (D) Huh-7 cells. Western blot of HIF1α is shown to indicate the hypoxic condition.

Download figure to PowerPoint

miR-210 Increases Migration and Invasion of HCC Cells and Mediates Hypoxia-Induced Migration and Invasion of HCC Cells.

To investigate the biological function of miR-210 in HCC, we first determined the expression of miR-210 in various HCC cells and constructed two HCC stable transfectants overexpressing miR-210 (Supporting Fig. 1). Because previous studies indicated that miR-210 can inhibit the proliferation of tumor cells in vitro and in vivo,3 we determined the effect of miR-210 on the proliferation of HCC cells. As shown in Supporting Fig. 2, miR-210 slightly suppressed the growth of Huh-7 cells (Supporting Fig. 2A). However, miR-210 significantly inhibited the colony-forming ability and tumor growth of Huh-7 cells (Supporting Fig. 2B,C). These results are consistent with a previous study.3

To test the hypothesis whether miR-210 might have a role in hypoxia-induced metastasis, we first determined whether miR-210 can affect the migration and invasion of HCC cells. In transwell experiments with or without Matrigel, the cells transiently transfected with the miR-210 mimic showed higher migration and invasion potential than the control cells (Fig. 2A,B). In addition, SMMC-7721 and Huh-7 cells that stably overexpress miR-210 showed significant increases in migration and invasion compared to the control cells (Fig. 2C,D). To further support the findings, we performed the transwell assays after transfecting SMMC-7721 and Huh-7 cells with miR-210 inhibitors to decrease the expression of endogenous miR-210. The results demonstrated that the migration and invasion abilities of SMMC-7721 and Huh-7 cells were significantly impaired when the endogenous miR-210 was decreased (Fig. 2E,F). Taken together, these results indicate that although miR-210 inhibits the growth of HCC cells, it promotes the migration and invasion of HCC cells.

thumbnail image

Figure 2. miR-210 promotes HCC cell migration and invasion. (A,B) Transwell migration (A) and invasion (B) assays of SMMC-7721 and Huh-7 cells were performed after transfection with miR-210 mimics or negative control (NC). (C,D) Transwell migration (C) and invasion (D) assays of SMMC-7721 and Huh-7 cells were performed after transduction with miR-210 or vector lentivirus. (E,F) Transwell migration (E) and invasion (F) assays of SMMC-7721 and Huh-7 cells were performed after transfection with miR-210 inhibitor or negative control. In all panels, the results are representative of at least three independent experiments. Statistical analysis was performed with the Student t test.

Download figure to PowerPoint

Given that miR-210 can promote the migration and invasion of HCC cells, we hypothesized that miR-210 might have a role in hypoxia-induced migration and invasion. To test this hypothesis, we first determined the migration and invasion ability of HCC cells under hypoxic conditions. Hypoxia significantly increased the migration and invasion potentials of SMMC-7721 and Huh-7 cells (Fig. 3), consistent with previous reports.10 After transfection with the miR-210 inhibitor, the migration and invasion abilities of SMMC-7721 and Huh-7 cells induced by hypoxia were dramatically diminished (Fig. 3). In conclusion, these results suggest that miR-210 is an important mediator for hypoxia-induced migration and invasion by HCC cells.

thumbnail image

Figure 3. miR-210 mediates hypoxia-induced HCC cell migration and invasion. (A,B) Transwell migration and invasion assays of SMMC-7721 cells were performed after transfection with miR-210 inhibitor or negative control (NC) in normoxic or hypoxic conditions. (C,D) Transwell migration and invasion assays of Huh-7 cells were performed after transfection with miR-210 inhibitor or negative control in normoxic or hypoxic conditions. In all panels, the results are representative of at least three independent experiments. Statistical analysis was performed with the Student t test.

Download figure to PowerPoint

VMP1 Is the Direct Downstream Target of miR-210 in HCC.

To explore the mechanism by which miR-210 promotes the migration and invasion of HCC cells, we performed microarray analyses to search for genes regulated by miR-210. When the results were compared with those from the TargetScan prediction program, a total of 26 candidate genes (Fig. 4A and Supporting Table 3) were identified. After the preliminary screen, we identified VMP1 and CPEB2 (cytoplasmic polyadenylation element binding protein 2) as possible targets of miR-210. In addition, VMP1 expression appeared to be possibly regulated by hypoxia. These results suggested that VMP1 was a direct downstream target of miR-210 in HCC cells. We constructed a vector containing the 3′-UTR of VMP1 with the binding site of miR-210 directly downstream of the luciferase reporter gene (Fig. 4B). The vector was cotransfected with miR-210 or control into HEK293T cells, and the relative luciferase activity was determined. The results showed that when compared to the control, the relative luciferase activity was significantly decreased by miR-210, whereas the luciferase activity was not altered by the vector containing the mutant 3′-UTR (Fig. 4C). These results indicate that miR-210 can directly bind to the 3′-UTR of VMP1. Furthermore, we showed that miR-210 can suppress the messenger RNA (mRNA) and protein levels of VMP1 in HCC cells (Fig. 4D,E) and that there is dose-dependence between the expression of VMP1 and miR-210 (Supporting Fig. 3). Taken together, these results indicate that miR-210 can down-regulate the expression of VMP1 in HCC cells by directly binding to its 3′-UTR, which illustrates that VMP1 may act as a downstream target of miR-210 in HCC cells.

thumbnail image

Figure 4. miR-210 down-regulates VMP1 expression by directly targeting its 3′-UTR. (A) Schema of the candidate genes identified by two independent approaches. The left circle represents a bioinformatics prediction of putative target genes using TargetScan. The right circle indicates mRNA microarray analysis that identified genes down-regulated by miR-210. (B) Sketch of the construction of wild-type or mutant VMP1 3′-UTR vectors. The mutant binding sequences are italicized. (C) Relative luciferase activity assays of luciferase reporters with wild-type or mutant VMP1 3′-UTR were performed after cotransfection with pWPXL–miR-210 plasmids or vector control. The Renilla luciferase vector was cotransfected as an internal control. (D) The protein levels of VMP1 were determined by western blot assays in SMMC-7721 and Huh-7 cells after infection with miR-210 or control lentivirus, miR-210 inhibitor, or negative control (NC). β-Actin served as an internal control. (E) The expression levels of VMP1 mRNA were determined in SMMC-7721 and Huh-7 cells after infection with miR-210 or control lentivirus by quantitative real-time PCR assays. β-Actin served as an internal control.

Download figure to PowerPoint

VMP1 Is Frequently Down-Regulated and Inversely Associated with the Expression of miR-210 in HCC.

VMP1 has been reported to be involved in cell autophagy11 and is down-regulated in metastatic breast cancer.12 However, the expression of VMP1 in HCC was unknown. The protein level of VMP1 was decreased in HCC samples compared to the noncancerous liver tissues (Fig. 5A). In 48 HCC samples, VMP1 showed strong staining (score of 3 or 4) in 18 samples (37.5%) and weak or no staining (score of 0, 1, or 2) in 30 samples (62.5%) (Fig. 5B). In contrast, VMP1 showed strong staining (score of 3 or 4) in all 48 noncancerous liver tissues. Intriguingly, the expression of VMP1 in the normal liver was strong in all 10 samples (score of 3 or 4). Furthermore, the expression level of VMP1 was inversely correlated with miR-210 expression levels in HCC samples (Fig. 5C). These observations indicate that VMP1 is frequently down-regulated in HCC samples, and the reduction may be caused by the overexpression of miR-210.

thumbnail image

Figure 5. VMP1 is frequently down-regulated and is negatively correlated with miR-210 expression in HCC. (A) Immunohistochemical staining of VMP1 is shown in HCC, adjacent noncancerous tissue (Non.), and normal liver tissue (Normal). (B) The statistical analyses of cases grouped according to the scoring. Statistical significance was assessed using the χ2 test. HCC, n = 48; adjacent noncancerous tissue, n = 48; Normal liver tissues, n = 10. (C) Correlations between the expression levels of miR-210 and VMP1. Box plots indicate the relative expression of miR-210. The expression levels of VMP1 were ranked according to the results of immunohistochemical staining. Low scores were 0, 1, or 2; high scores were 3 or 4. Statistical analysis was performed using the Student t test.

Download figure to PowerPoint

VMP1 Hinders the Migration and Invasion of HCC Cells, and Restoration of VMP1 Abrogates the Migration and Invasion of HCC Cells Induced by miR-210.

To further explore the biological function of VMP1 in HCC, siRNAs targeting VMP1 were designed and transfected into SMMC-7721 and Huh-7 cells. The results showed that siRNA targeting VMP1 can significantly decrease the expression of VMP1 in the two cell lines (Fig. 6A and Supporting Fig. 4). As a result, the migration and invasion abilities of the two cell lines were significantly increased by the VMP1 siRNA but not by the control siRNA (Fig. 6A). To further support these findings, we also introduced the VMP1 ORF into the SMMC-7721 and Huh-7 cells and found that VMP1 overexpression can significantly hinder the migration and invasion of the two cell lines (Fig. 6B). Together, the effect of overexpression of VMP1 on HCC cells was the opposite of that of miR-210, and reduction of VMP1 phenocopied the effect of miR-210 on HCC cells, suggesting that VMP1 may be a functional target of miR-210 in HCC.

thumbnail image

Figure 6. VMP1 functions as a metastasis suppressor and can block miR-210–induced HCC cell migration and invasion. (A) Transwell migration and invasion assays of SMMC-7721 and Huh-7 cells were performed after transfection with small interfering RNA (siRNA) against VMP1 or with negative control (NC). VMP1 protein levels were detected by western blot analysis. (B) Transwell migration and invasion assays of SMMC-7721 and Huh-7 cells were performed after transduction by VMP1 (ORF without the 3′-UTR) or with the control lentivirus. VMP1 protein levels were detected by western blot analysis. (C,D) Transwell migration and invasion assays of SMMC-7721–vector or SMMC-7721–miR-210 cells (C) and Huh-7–vector and Huh-7–miR-210 cells (D) were performed after transduction with VMP1 or vector control lentivirus. VMP1 protein was detected by western blot analysis. In all panels, results are representative of at least three independent experiments. Statistical analysis was performed using the Student t test.

Download figure to PowerPoint

If VMP1 is indeed the functional target of miR-210 in HCC, then restoration of VMP1 in miR-210 overexpressed HCC cells should abrogate the effects of miR-210. To test this hypothesis, we introduced the ORF of VMP1 into HCC cells overexpressing miR-210. Introduction of VMP1 into HCC cells overexpressing miR-210 restored the protein level of VMP1 in SMMC-7721 and Huh-7 cells (Fig. 6C,D). After the restoration of VMP1, the migration and invasion of SMMC-7721 and Huh-7 cells induced by miR-210 were significantly decreased (Fig. 6C,D). These results indicated that VMP1 is a bona fide functional target of miR-210 in HCC cells.

VMP1 Is Repressed by Hypoxia, and Down-Regulation of VMP1 by miR-210 Mediates Hypoxia-Induced HCC Metastasis.

To further determine whether VMP1 is involved in miR-210–induced HCC cell metastasis under hypoxia, we first detected the expression level of VMP1 under hypoxic conditions. The results showed that expression of VMP1 in HCC cells was decreased under hypoxia when compared to its expression under normoxia (Fig. 7A, upper panel). When an miR-210 inhibitor was transfected into Huh-7 cells, the decrease in VMP1 induced by hypoxia was abrogated (Fig. 7A, lower panel), which indicated that the reduction in VMP1 was mediated by the up-regulation of miR-210 under hypoxic conditions. Transwell assays indicated that overexpression of VMP1 impaired the hypoxia-induced migration and invasion (Fig. 7B). To further determine the function of VMP1 in miR-210–induced HCC metastasis under hypoxia, siVMP1 and control siRNA were cotransfected with miR-210 inhibitor into Huh-7 cells. In transwell experiments, the impairment of miR-210 inhibitor on the hypoxia-induced HCC cell migration and invasion was partially relieved by siVMP1, but not by control siRNA (Fig. 7C). Moreover, reintroducing VMP1 into miR-210 overexpressing cells abolished miR-210–induced migration and invasion under hypoxia (Fig. 7D). Taken together, these results indicate that VMP1 was repressed by hypoxia, and down-regulation of VMP1 by miR-210 mediated the hypoxia-induced HCC metastasis.

thumbnail image

Figure 7. VMP1 down-regulation mediates miR-210–driving HCC cell migration and invasion under hypoxia. (A) The protein levels of VMP1 in Huh-7 cells. The cells were transfected with miR-210 inhibitor or negative control and then exposed to hypoxia at 24 hours after transfection. Cells were collected and subjected to western blot analysis at specific time points as indicated. β-Actin served as an internal control. (B) Transwell migration and invasion assays of Huh-7–vector and Huh-7–VMP1 cells were performed under normoxic or hypoxic conditions. The protein levels of VMP1 were determined by western blot assays. (C) Transwell migration and invasion assays of Huh-7 cells were performed after transfection with miR-210 inhibitor, VMP1 siRNA (siVMP1), or negative control (Hypoxia) in normoxic or hypoxic conditions. The protein levels of VMP1 were determined by western blot assays. (D) Transwell migration and invasion assays of Huh-7 cells were performed after transduction with miR-210, VMP1, or control lentivirus (Hypoxia) in normoxic or hypoxic conditions. The protein levels of VMP1 were determined by western blot assays. In panels (A) through (D), results are representative of at least three independent experiments. Statistical analysis was performed using the Student t test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we found that miR-210 was up-regulated in 60.42% of HCC samples and induced by hypoxia in HCC cells. Actually, the overexpression of miR-210 in cancers is a common event. It has been shown that miR-210 is up-regulated in breast cancer,9, 13, 14 lung cancer,15 oral carcinoma,16 pancreatic cancer,17 melanoma,18 head and neck cancer,3 and renal cancer.19 Moreover, the overexpression of miR-210 is associated with poor prognosis and inversely correlated with patient survival in some cancers.3, 13, 14, 17, 19 Together with our results, these findings demonstrate that miR-210 may play an important role in the development and progression of human cancer.

The exploration of miR-210 function indicated that miR-210 plays roles in many cellular processes of physiological and malignant conditions. miR-210 is induced during the differentiation of erythroid cells,20 and it can also modulate adipogenesis and repress the WNT signaling pathway in adipogenesis.21 As the most consistently and robustly up-regulated miRNA induced by hypoxia,22 miR-210 regulates multiple processes in the hypoxic pathway. It may regulate the angiogenic response to hypoxia by suppressing the expression of EFNA3.7 Consistent with this result, the expression of miR-210 is up-regulated in breast cancer and is correlated with the hypoxia/VEGF signaling pathway.13 In epithelial ovarian cancer, however, miR-210 is frequently deleted and inhibits cell cycle progression by targeting E2F3.4 In head and neck cancer, miR-210 acts as a marker of tumor hypoxia23 and suppresses the proliferation of tumor cells during cancer initiation.3

In the present study, we also found that miR-210 inhibited the growth of HCC cells in vitro and in vivo. Intriguingly, we found that miR-210 markedly promoted the migration and invasion of HCC cells. To the best of our knowledge, this is the first report showing that miR-210 enhances the migration and invasion of tumor cells. Furthermore, as a master miRNA induced by hypoxia, miR-210 was found to mediate hypoxia-induced migration and invasion. Our results imply that miR-210 not only modulates tumor cell growth but also regulates the migration and invasion of tumor cells under hypoxia, which extends the repertoire of the miR-210 function. The overexpression of miR-210 in tumors may not simply act as a tumor hypoxia marker or adjust cells to hypoxia stress, but also have a biological role in the malignancy and metastasis of tumors, which may explain why miR-210 is correlated with poor prognosis of cancer patients.

Investigation of the mechanism by which miR-210 regulates the migration and invasiveness of HCC cells revealed that VMP1 was the functional target of miR-210. VMP1 was first found to be activated in acute pancreatitis.24, 25 VMP1 promotes the formation of cellular vacuoles and apoptosis of acinar cells during chronic pancreatitis.25, 26 In addition, VMP1 is involved in the autophagy of mammalian cells.27, 28 In pancreatic cancer, VMP1 is induced by gemcitabine, and it promotes the apoptosis of pancreatic cancer cells.29 VMP1 is reduced in kidney cancer metastases and metastatic breast cancer cell lines.12 Down-regulation of VMP1 by siRNA significantly increases the invasiveness of noninvasive kidney cancer cells. In accordance with this study, we found that VMP1 is also reduced in HCC samples and acts to suppress metastasis in HCC. Furthermore, we found that the expression of VMP1 is inversely correlated with the expression of miR-210 in HCC, which demonstrates that down-regulation of VMP1 is, at least in part, caused by the up-regulation of miR-210 in HCC. Importantly, we found that VMP1 is a bona fide functional target of miR-210 and that the down-regulation of VMP1 by miR-210–mediated hypoxia-induced tumor cell spreading, indicating that hypoxia-miR-210–VMP1 is a new signaling pathway that is involved in hypoxia-induced cancer metastasis. However, the mechanism by which VMP1 inhibits the migration and invasion of tumor cells is still unknown. Previous research demonstrated that VMP1 colocalizes with the tight junction protein, zona occludens-1 (ZO-1).12 However, whether miR-210 regulation of HCC metastasis is mediated via the regulation of VMP1 and ZO-1, which may further affect the tight junctions and thus the metastatic spread of HCC cells, remains unknown and warrants further investigation.

In conclusion, we found that the “master” microRNA, miR-210, in hypoxia is frequently increased in HCC and is involved in hypoxia-induced HCC metastasis. VMP1, a putative metastasis suppressor in HCC, is identified as a direct and functional target of miR-210. Moreover, its down-regulation by miR-210 mediates hypoxia-induced HCC migration and invasion. These findings facilitate our understanding of the mechanism of tumor cell metastasis under hypoxic conditions, and this newly identified hypoxia/miR-210/VMP1 pathway may contribute to the development of new therapeutics against hypoxic tumors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are most grateful for the pWPXL, psPAX2, and pMD2.G lentivirus plasmids that were provided by Professor Didier Trono from the School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 2004; 4: 437-447.
  • 2
    Chan DA, Sutphin PD, Yen SE, Giaccia AJ. Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha. Mol Cell Biol 2005; 25: 6415-6426.
  • 3
    Huang X, Ding L, Bennewith KL, Tong RT, Welford SM, Ang KK, et al. Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation. Mol Cell 2009; 35: 856-867.
  • 4
    Giannakakis A, Sandaltzopoulos R, Greshock J, Liang S, Huang J, Hasegawa K, et al. miR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer. Cancer Biol Ther 2008; 7: 255-264.
  • 5
    Chen Z, Li Y, Zhang H, Huang P, Luthra R. Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression. Oncogene 2010; 29: 4362-4368.
  • 6
    Chan SY, Zhang YY, Hemann C, Mahoney CE, Zweier JL, Loscalzo J. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab 2009; 10: 273-284.
  • 7
    Fasanaro P, D'Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 2008; 283: 15878-15883.
  • 8
    Chan DA, Giaccia AJ. Hypoxia, gene expression, and metastasis. Cancer Metastasis Rev 2007; 26: 333-339.
  • 9
    Foekens JA, Sieuwerts AM, Smid M, Look MP, de Weerd V, Boersma AW, et al. Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer. Proc Natl Acad Sci U S A 2008; 105: 13021-13026.
  • 10
    Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 2006; 441: 437-443.
  • 11
    Tian Y, Li Z, Hu W, Ren H, Tian E, Zhao Y, et al. C. elegans screen identifies autophagy genes specific to multicellular organisms. Cell 2010; 141: 1042-1055.
  • 12
    Sauermann M, Sahin O, Sultmann H, Hahne F, Blaszkiewicz S, Majety M, et al. Reduced expression of vacuole membrane protein 1 affects the invasion capacity of tumor cells. Oncogene 2008; 27: 1320-1326.
  • 13
    Camps C, Buffa FM, Colella S, Moore J, Sotiriou C, Sheldon H, et al. hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer. Clin Cancer Res 2008; 14: 1340-1348.
  • 14
    Radojicic J, Zaravinos A, Vrekoussis T, Kafousi M, Spandidos DA, Stathopoulos EN. MicroRNA expression analysis in triple-negative (ER, PR and Her2/neu) breast cancer. Cell Cycle 2011; 10: 507-517.
  • 15
    Puissegur MP, Mazure NM, Bertero T, Pradelli L, Grosso S, Robbe-Sermesant K, et al. miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity. Cell Death Differ 2011; 18: 465-478.
  • 16
    Scapoli L, Palmieri A, Lo Muzio L, Pezzetti F, Rubini C, Girardi A, et al. MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression. Int J Immunopathol Pharmacol 2010; 23: 1229-1234.
  • 17
    Greither T, Grochola LF, Udelnow A, Lautenschlager C, Wurl P, Taubert H. Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int J Cancer 2010; 126: 73-80.
  • 18
    Satzger I, Mattern A, Kuettler U, Weinspach D, Voelker B, Kapp A, et al. MicroRNA-15b represents an independent prognostic parameter and is correlated with tumor cell proliferation and apoptosis in malignant melanoma. Int J Cancer 2010; 126: 2553-2562.
  • 19
    Neal CS, Michael MZ, Rawlings LH, Van der Hoek MB, Gleadle JM. The VHL-dependent regulation of microRNAs in renal cancer. BMC Med 2010; 8: 64.
  • 20
    Bianchi N, Zuccato C, Lampronti I, Borgatti M, Gambari R. Expression of miR-210 during erythroid differentiation and induction of gamma-globin gene expression. BMB Rep 2009; 42: 493-499.
  • 21
    Qin L, Chen Y, Niu Y, Chen W, Wang Q, Xiao S, et al. A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/beta-catenin signaling pathway. BMC Genomics 2010; 11: 320.
  • 22
    Huang X, Le QT, Giaccia AJ. MiR-210–micromanager of the hypoxia pathway. Trends Mol Med 2010; 16: 230-237.
  • 23
    Gee HE, Camps C, Buffa FM, Patiar S, Winter SC, Betts G, et al. hsa-mir-210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer. Cancer 2010; 116: 2148-2158.
  • 24
    Dusetti NJ, Jiang Y, Vaccaro MI, Tomasini R, Azizi Samir A, Calvo EL, et al. Cloning and expression of the rat vacuole membrane protein 1 (VMP1), a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation. Biochem Biophys Res Commun 2002; 290: 641-649.
  • 25
    Vaccaro MI, Grasso D, Ropolo A, Iovanna JL, Cerquetti MC. VMP1 expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis. Pancreatology 2003; 3: 69-74.
  • 26
    Jiang PH, Motoo Y, Vaccaro MI, Iovanna JL, Okada G, Sawabu N. Expression of vacuole membrane protein 1 (VMP1) in spontaneous chronic pancreatitis in the WBN/Kob rat. Pancreas 2004; 29: 225-230.
  • 27
    Vaccaro MI, Ropolo A, Grasso D, Iovanna JL. A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy. Autophagy 2008; 4: 388-390.
  • 28
    Ropolo A, Grasso D, Pardo R, Sacchetti ML, Archange C, Lo Ré A, et al. The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells. J Biol Chem 2007; 282: 37124-37133.
  • 29
    Pardo R, Lo Ré A, Archange C, Ropolo A, Papademetrio DL, Gonzalez CD, et al. Gemcitabine induces the VMP1-mediated autophagy pathway to promote apoptotic death in human pancreatic cancer cells. Pancreatology 2010; 10: 19-26.

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24614_sm_SuppInfo.doc1472KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.