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Potential conflict of interest: Nothing to report.
miR-17-5p is overexpressed in hepatocellular carcinoma (HCC), but the specific regulatory mechanisms of miR-17-5p in HCC remain unknown. We investigated the molecular basis of miR-17-5p as an oncogene in human HCC cell lines. Our in vivo and in vitro data indicate that miR-17-5p up-regulates the migration and proliferation of HCC cells. Interestingly, proteomic and western blotting assays revealed that miR-17-5p significantly activates the p38 mitogen-activated protein kinase MAPK pathway and increases the phosphorylation of heat shock protein 27 (HSP27). Our results also suggest that E2F1-dependent down-regulation of Wip1 regulates miR-17-5p-p38-HSP27 signaling. Furthermore, suppression of HSP27 expression by small interfering RNA or the p38 MAPK pathway-specific inhibitor SB203580 decreases the migration of HCC cells overexpressing miR-17-5p but does not reduce their proliferation. Finally, we show that miR-17-5p expression correlates well with HSP27 status in primary human HCC tissues with metastasis. Conclusion: Our findings suggest that the p38 MAPK pathway plays a crucial role in miR-17-5p-induced phosphorylation of HSP27 and, as a consequence, phosphorylated HSP27 enhances the migration of HCC cells. Our data highlight an important role of miR-17-5p in the proliferation and migration of HCC cells and support the potential application of miR-17-5p in HCC therapy. (Hepatology 2010;)
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MicroRNAs (miRNAs) are 21- to 25-nucleo tide RNA molecules that regulate cellular differentiation, apoptosis, proliferation, and migration.1, 2 Many studies have shown that miRNAs are implicated in many cancers, and altered miRNA levels can result in the aberrant expression of gene products that may contribute to cancer biology.3, 4 Indeed, some miRNAs have been classified as tumor suppressors or oncogenes.5 Recent studies that have used hybridization-based microarrays to investigate miRNA expression profiles in human hepatocellular carcinoma (HCC) have identified nonoverlapping signatures of a small number of miRNAs that are up-regulated and down-regulated in human HCC compared with paired peritumoral tissues.6-8 Although the roles of miRNAs in HCC have recently been postulated, their pathophysiological contributions to HCC are still largely unknown.
The miR-17-92 cluster (composed of miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92-1) first attracted attention following a series of observations linking these miRNAs to cancer pathogenesis.9 Overexpression of the miR-17-92 locus has been identified in lung cancer,10 B cell lymphoma,9 HCC,11 and stomach solid tumors.12 Moreover, elevated expression of the miR-17-92 polycistron in hepadnavirus-associated HCC contributes to a malignant phenotype.13 The association of miR-17-92 with a broad range of cancers not only underlines the clinical significance of this locus but also suggests that miR-17-92 may regulate fundamental biological processes. miR-17-5p, an important member of the miR-17-92 cluster, was found to be overexpressed in HCC.11, 13, 14 Some targets of miR-17-5p have been confirmed, such as E2F1,15 NCOA3,16 and RBL2.17 Many of these targets are known cell cycle regulators, but their identification does not sufficiently explain the oncogenic potential of miR-17-5p.
The introduction of proteomics has enabled the simultaneous analysis of changes in multiple proteins. Recently, some new proteomic approaches were used to measure changes in the synthesis of several thousand proteins in response to miRNA transfection or endogenous miRNA knockdown.18, 19 These data suggest that miRNAs can directly or indirectly regulate protein synthesis of thousands of genes. These approaches are powerful means by which to identify miRNA targets. However, beyond identifying specific targets, knowledge of large cohorts of proteins indirectly regulated by miRNAs may yield additional information. Using a two-dimensional differential in-gel electrophoresis (DIGE) approach, different samples prelabeled with mass-matched and charge-matched fluorescent cyanine dyes (Cy2, Cy3, and Cy5) can be coseparated in the same two-dimensional gel. An internal standard is used in every gel to negate the problem of intergel variation.20, 21 In this study, a two-dimensional DIGE proteomic approach was employed to compare the proteome of Huh-7 cells overexpressing either miR-17-5p or mock controls to identify clusters of proteins (and pathways) directly or indirectly regulated by miR-17-5p. We identified a cohort of proteins indirectly regulated by miR-17-5p during the stress response, signal transduction, and metabolism. A representative molecule that was differentially phosphorylated, heat shock protein 27, was further confirmed by western blotting. The specific regulatory mechanisms and the effects of miR-17-5p-induced up-regulation of phosphorylated heat shock protein 27 in HCC cells were also analyzed.
DIGE, differential in-gel electrophoresis; HCC, hepatocellular carcinoma; HSP27, heat shock protein 27; MAPK, mitogen-activated protein kinase; miRNA, microRNA; MMP-2, matrix metalloproteinase 2; siRNA, small interfering RNA.
Patients and Methods
For a description of the patients and methods used in this study, see the Supporting Information.
In Vitro miR-17-5p Up-regulation Model in Huh-7 Cells.
The miR-17-5p up-regulation model was created using Huh-7 cells stably expressing pEZX-17-5p (clone 1 and 2). The miR-17-5p levels of clone 1 and 2 cells, pEZX-MR01-Huh-7 cells, and untreated Huh-7 cells were determined using real-time polymerase chain reaction. We observed significant up-regulation of miR-17-5p in clone 1 and 2 cells compared with mock control or untreated cells (Fig. 1A).
miR-17-5p Regulates Proliferation and Intrahepatic Metastasis of HCC.
To determine whether miR-17-5p is associated with tumorigenesis or metastasis, clone 1 and pEZX-17-5p-Huh-7 cells were implanted through a subcutaneous route into the flanks of nude mice or orthotopically into the livers of nude mice (Fig. 1D). Up-regulation of miR-17-5p significantly facilitated overall tumor growth as assessed by measurements of tumor volume (Fig. 1B,C). The orthotopic tumor model yielded a similar tumor growth trend as seen in the original injection site. Clone 1 cells showed extensive hepatic invasion with a larger tumor volume than controls (Fig. 1E,F).
Analysis of Differentially Expressed Proteins.
After DIGE, the Cy2, Cy3, and Cy5 channels of each gel were individually imaged, and the images were analyzed using Decyder 5.0 software. On the basis of the average intensity ratios of protein spots, a total 30 protein spots were found to be dynamically changed in clone 1 cells (Fig. 2A), including 18 significantly up-regulated protein spots (ratioclone 1/pEZX-MR01 ≥ 1.3; P ≤ 0.05) and 12 significantly down-regulated protein spots (ratioclone 1/pEZX-MR01 ≤ 0.7; P ≤ 0.05).
Identification of Differentially Expressed Proteins.
To identify the differentially expressed proteins in clone 1 cells, 28 protein spots with a threshold greater than 1.3-fold were excised from silver-stained two-dimensional electrophoresis gels and subjected to in-gel trypsin digestion and subsequent matrix-assisted laser desorption/ionization time-of-flight identification. As shown in Supporting Table S3 and Fig. 2A, 15 differentially expressed spots were successfully identified. According to annotations from Genecards (http://www.genecards.org/index.shtml) and the Gene Ontology Database, the identified cellular proteins were involved in the stress response, the cytoskeleton, and signal transduction and metabolism.
Activation of the p38 Mitogen-Activated Protein Kinase-Heat Shock Protein 27 Signaling Pathway in Clone 1 and 2 Cells.
To verify the DIGE results, clone 1 and 2 cells were further analyzed by way of western blotting. The up-regulation of heat shock protein 27 (HSP27) was verified by way of western blotting (Figs. 2C, 3A). Several reports have shown that HSP27 is a terminal substrate of the p38 mitogen-activated protein kinase (MAPK) cascade,22, 23 and activation of p38 results in the phosphorylation of HSP27. Therefore, we assessed the level of phosphorylation of HSP27 (phospho-Ser15 and phospho-Ser82) and p38 MAPK (phospho-Tyr182). As shown in Fig. 3A,B, increased phosphorylation of HSP27 (phospho-Ser82 and phospho-Ser15) and p38 MAPK (phospho-Tyr182) were observed in clone 1 and 2 cells. Previous studies have reported that activated p38 MAPK increases the metastatic potential of cancer cell by up-regulating the expression of matrix metalloproteinase 2 (MMP-2). We quantified the levels of secreted MMP-2 in clone 1 and 2 cell culture. As shown in Fig. 3C, miR-17-5p increased MMP-2 secretion in HCC cells. These results indicate that the p38 MAPK-HSP27 signaling pathway might be activated in pEZX-17-5p-Huh-7 cells (clone 1 and 2). These phenomena were also observed in HepG2 cells (Supporting Fig. S1A).
miR-17-5p Enhances HSP27 Stability in HCC Cells.
We did not detect any changes in HSP27 transcription levels after miR-17-5p up-regulation (data not shown). To detect whether miR-17-5p enhances HSP27 stability, we treated clone 1 and 2 cells with the protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 as described in the Supporting Information. As shown in Fig. 3D, MG-132 abolished the changes in HSP27 expression levels between miR-17-5p overexpressed and control cells, but cycloheximide did not. Our data suggest that miR-17-5p reduces the rate of HSP27 degradation and enhances its stability.
Treatment with SB203580 or Small Interfering RNA Against HSP27 Reduces HSP27 Phosphorylation.
To elucidate the crucial role of the p38 MAPK pathway and total HSP27 levels in the activation of HSP27, we performed western blotting in clone 1 cells treated with the p38 MAPK inhibitor SB203580 or transfected with small interfering RNA (siRNA) against HSP27. As shown in Fig. 4A,B, HSP27 phosphorylation (both phospho-Ser15 and phospho-Ser82) was reduced by treatment with SB203580 and siRNAs against HSP27. These results indicate that activation of p38 MAPK and total HSP27 levels are essential for HSP27 phosphorylation.
Transcription Factor E2F1 and Phosphatase Wip1 Are Involved in p38 MAPK-HSP27 Signaling Pathway Activation.
One proven target gene of miR-17-5p is E2F1,15 which modulates p38 MAPK phosphorylation through transcriptional regulation of Wip1.24 We detected protein expression of E2F1 and Wip1 in clone 1 and 2 cells. As shown in Fig. 4C, E2F1 and Wip1 proteins were clearly down-regulated in miR-17-5p-overexpressing cells. To investigate whether Wip1 is responsible for the activation of the p38 MAPK-HSP27 signaling pathway, we overexpressed Wip1 in clone 1 cells. Figure 4D shows that the phosphorylation of HSP27and p38 MAPK is reduced when Wip1 is up-regulated. Together, these results suggest that E2F1-dependent down-regulation of Wip1 is necessary for p38 MAPK-HSP27 pathway activation by miR-17-5p.
miR-17-5p Facilitates Cell Growth Independent of the p38 MAPK-HSP27 Pathway.
To verify whether the p38 MAPK-HSP27 pathway contributes to miR-17-5p-facilitated cell growth, we performed CCK-8 assays to detect cell proliferation in vitro. In accordance with the results of the xenograft models in nude mice, clone 1 cells proliferated much faster than control cells, and SB203580 was able to reduce this effect in clone 1 cells. However, siRNAs against HSP27 had no effect (Fig. 5A). These results indicate that miR-17-5p facilitates cell growth, but this process does not involve the p38 MAPK-HSP27 pathway.
miR-17-5p-p38 MAPK-HSP27 Signaling Is Involved in HCC Cell Motility.
To verify whether the p38 MAPK-HSP27 pathway contributes to the miR-17-5p-induced increase in HCC cell motility, we performed a series of assays to detect cell migration in vitro. Migration of Huh-7 cells, as assayed by a scrape assay, was significantly increased in clone 1 and 2 cells compared with pEZX-MR01-Huh-7 cells. Treatment with SB203580 or the siRNA against HSP27 abolished this effect (Fig. 5B). Transwell experiments were then performed with clone 1, clone 2, and pEZX-MR01-Huh-7 cells with or without SB203580 or siRNA against HSP27. As shown in Fig. 6A,B, miR-17-5p induced Huh-7 cell migration in the transwell experiments, and this migration was inhibited by SB203580 or siRNA against HSP27. Together, these results indicate that p38 MAPK and HSP27 play a crucial role in the control of Huh-7 cell migration. Similar phenomena were also observed in HepG2 cells (Supporting Fig. 1B).
We investigated the effect of miR-17-5p on the organization of the actin cytoskeleton in Huh-7 cells. The actin cytoskeleton was stained with phalloidin (Fig. 6C). miR-17-5p increased the cortical localization of actin, which was inhibited by the siRNA against HSP27 or treatment with SB203580, demonstrating the involvement of miR-17-5p-p38 MAPK-HSP27 in actin cytoskeleton remodeling.
Inhibition of miR-17-5p Attenuates its Oncogenic Effect.
To address in greater detail the function of miR-17-5p in HCC cells and to avoid potential overexpression artifacts, we transfected clone 1 cells with 2′-Ome-modified antisense oligoribonucleotides against miR-17-5p. As shown in Fig. 7A, when introduced into clone 1 cells, phosphorylated HSP27, total HSP27, and phosphorylated p38 MAPK levels were reduced by 50%-60% compared with negative controls, indicating efficient up-regulation by miR-17-5p. Transfection with 2′-Ome-modified antisense oligoribonucleotides, but not the scrambled oligoribonucleotides, significantly reduced the migration of clone 1 cells (Fig. 7B,C), indicating the enhancing effect of this miRNA.
Taken together, these results show that overexpression of miR-17-5p is able to enhance the migration of HCC cells by activating the p38-HSP27 pathway. When miR-17-5p was inactivated, the migration of HCC cells was significantly reduced. These data substantially support the idea that miR-17-5p has a vital function in the migration of HCC cells.
Up-regulated Expression of miR-17-5p and HSP27 Profiling in Metastatic HCC.
miR-17-5p expression was analyzed in 13 metastatic HCCs (group 1), 12 nonmetastatic HCCs (group 2), and five normal liver tissue samples (group 3) by way of quantitative real-time polymerase chain reaction. Notably, miR-17-5p was up-regulated in the majority of examined metastatic HCCs (Fig. 8A), with nine of 13 (69.2%) metastatic HCC tissue samples displaying more than 50% up-regulation. Next, we analyzed the profiles of HSP27 and p38 MAPK in primary human HCC tissue by way of immunoblot analysis (groups 1-3). Among the 30 human samples analyzed, total HSP27 and phosphorylated HSP27 levels increased in 10 out of 13 metastatic HCC (group 1), but this was observed in only two out of 12 nonmetastatic HCCs (Fig. 8B,C). Phosphorylated p38 was also up-regulated in group 1 HCC tissues (Supporting Fig. 2). Collectively, these data suggest that deregulation of miR-17-5p and the profile of HSP27 may contribute to the progression of HCC.
Previous studies on the influence of miR-17-5p on protein expression were limited to single protein analyses, primarily using western blotting and reporter assays.15, 16, 25 It is unknown how much translational control is exerted by miR-17-5p at a genome-wide scale. We used DIGE to measure changes in the synthesis of several thousand proteins in response to miR-17-5p overexpression. Those changes may include direct and indirect effects of miR-17-5p. Two recent studies18, 19 analyzed changes in the proteomes of cells in response to individual miRNAs using quantitative mass spectrometry. The authors stated that this approach was a powerful means by which to identify miRNA targets. However, biosignal transduction is a cascade reaction, so the downstream effects are remarkably easy to detect. Therefore, in addition to information regarding specific targets, identification of proteins indirectly regulated by miRNAs may yield more information. In this study, the identified cellular proteins were indirectly regulated by miR-17-5p and were involved in the stress response, signal transduction, and metabolism (Supporting Table 3).
HSP27, a member of the small HSP family, is induced by stress and protects against heat shock, hypertonic stress, oxidative stress, and other forms of cellular injury in numerous cell types.26, 27 Overexpression of HSP27 has been reported in many kinds of tumor tissues and is found to be associated with poor prognoses for astrocytic brain tumor,28 breast cancer,29 ovarian carcinoma,30 and HCC.31-33 A previous study demonstrated that HSP27 expression is a powerful prognostic indicator and is related to the histologic grade and survival of patients with HCC.32 In another study, the authors detected HSP27 in HCC serum and validated the result by western blotting.34 Our finding that HSP27 is up-regulated in miR-17-5p overexpression in HCC cells further confirms these results. The capacity of HSP27 to regulate actin reorganization and cell migration is modulated by the phosphorylation of serine residues.35 We measured the levels of phosphorylation and found that the levels of HSP27 phosphorylation at serines 15 and 82 in miR-17-5p-overexpressing HCC cells were increased compared with control cells (Fig. 3A). We therefore propose that miR-17-5p overexpression increases total HSP27 and also up-regulates HSP27 activity.
It has also been reported that the MAPK cascade—in particular, the p38 MAPK pathway—leads to the phosphorylation of HSP27 through MAPK-activated protein kinase-2, one of the substrates of p38 MAPK.36, 37 Therefore, we investigated whether MAPKs are involved in HSP27 phosphorylation in HCC cells. We showed that the level of phosphorylated p38 MAPK was increased and inhibition of p38 MAPK activation results in the suppression of HSP27 phosphorylation in miR-17-5p-overexpressing HCC cells (Figs. 3B, 4A). We also found that HSP27 knockdown reduces the phosphorylation level of HSP27 in miR-17-5p-overexpressing HCC cells (Fig. 4B). These findings indicate the likelihood that up-regulation of miR-17-5p regulates the phosphorylation of HSP27 through p38 MAPK in human HCC cells.
It is not clear how miR-17-5p activates the p38 MAPK pathway, however. We hypothesized that miR-17-5p might indirectly modulate the p38 MAPK pathway through one of its target genes. Wip1, a serine/threonine phosphatase, is an E2F1-regulated gene that inactivates p38.24, 38 In addition, E2F1 is a proven target gene of miR-17-5p.15 Interestingly, we demonstrated that Wip1 is down-regulated in miR-17-5p-overexpressing HCC cells (Fig. 4C) and ectopic expression of Wip1 in miR-17-5p-overexpressing HCC cells can inactivate the p38 pathway (Fig. 4D). This result indicates that Wip1, an E2F1-regulated gene, is at least partly responsible for miR-17-5p-dependent activation of the p38 pathway.
Many studies have shown increased phosphorylation levels of HSP27 in different metastatic cancer cells and have indicated HSP27 activation correlates with the metastatic potential of cancer cells.26, 37, 39 Our results indicate that HCC cell motility is increased by miR-17-5p (Figs. 5-7). In addition, the increased cell motility induced by miR-17-5p was accompanied by an increase in p38 MAPK and HSP27 activity, suggesting that miR-17-5p may activate p38 MAPK-dependent pathways. Interestingly, treatment with a siRNA against HSP27, an inhibitor of p38 MAPK or 2′-Ome -modified antisense oligoribonucleotides against miR-17-5p blocked the miR-17-5p-induced up-regulation of HCC cell metastasis (Figs. 5-7). Additionally, we found increased concentration of secreted MMP-2 in miR-17-5p-overexpressing HCCs. The expression of MMP-2 can also be regulated by the p38 MAPK-HSP27 pathway.37 This finding further supports our hypothesis that miR-17-5p-induced changes in HCC cell metastasis depend on the activation of p38 MAPK and HSP27.
Collectively, our study identified a signaling pathway regulated by miR-17-5p using DIGE and confirmed this result by other molecular techniques. To our knowledge, this study is the first to take a nonbiased, broad-based approach to identify the downstream effects of overexpression of a miR and it provides functional data linking the miR to metastatic characteristics both in vitro and in vivo. It also provides data for a mechanism from direct effect to end effect. However, many protein spots remain unidentified. Further elucidation of the total physiological changes and the mechanisms responsible will require improvements in methodology (holistic analysis of all molecules) and miRNA regulation theory.
We thank Hong Lei and Xiu-hua Zhang for help with DIGE data analysis.