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Keywords:

  • matrix metalloproteinase-9 (MMP-9);
  • microRNA-15a;
  • migration;
  • neuroblastoma;
  • reversion-inducing cysteine-rich protein with Kazal motifs (RECK)

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we found that the expression of miR-15a was positively correlated with neuroblastoma (NB) clinical pathological stage and was negatively correlated with reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression. Using the enhanced green fluorescent protein (EGFP) reporter construct carrying the 3′-UTR of RECK, we identified RECK as a direct target of miR-15a. Suppression of miR-15a significantly decreased the migration ability of GI-LA-N and SK-N-SH cell lines, whereas overexpression of miR-15a increased the migration ability; these effects could be partly reversed by RECK inhibition or ectopic expression. Moreover, inhibition of miR-15a significantly increased secreted matrix metalloproteinase-9 expression in culture medium through regulating the expression of RECK. These findings provide new insights into the characteristics of the miR-15a–RECK–matrix metalloproteinase-9 axis in NB progression, especially in NB migration and invasion.


Abbreviations
ASO

antisense oligonucleotides

EGFP

enhanced green fluorescent protein

miRNA

microRNA

MMP

matrix metalloproteinase

NB

neuroblastoma

RECK

reversion-inducing cysteine-rich protein with Kazal motifs

SD

standard deviation

siRNA

small interfering RNA

WB

western blot

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Approximately 60% of children (aged > 1 year) with neuroblastoma (NB) present with metastatic disease at diagnosis with a poor outcome, despite intensive treatment protocols [1]. NB metastasis is a multistep process, among which the migration and invasion of tumor cells from the local area into the surrounding normal tissues is the most critical step [2]. Although clusters of genes associated with the ability of NB cells to metastasize and invade have been recorded [3], the precise mechanisms, especially the role of microRNAs (miRNAs), in NB metastasis are still not yet completely understood.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was isolated with an expression-cloning strategy designed to isolate genes inducing flat morphology in v-Ki-ras-transformed NIH3T3 cells [4]. It encodes a membrane-anchored glycoprotein of Mr ~ 110 000 with multiple epidermal growth factor-like repeats and serine protease inhibitor-like domains, associated with the membrane through a C-terminal glycosylphosphatidylinositol modification [5]. RECK is expressed ubiquitously in adult human tissues, and decreased expression or loss of expression of RECK has been shown to be significantly correlated with poor survival in hepatocellular carcinoma and colorectal cancer patients [6-8]. Previous studies showed that restored expression of RECK decreased tumor angiogenesis, invasion and metastasis in vitro and in vivo [9, 10]. Moreover, Qian Dong et al. in our group found that the rate of expression of the RECK protein in the NB sample was low, and was reduced with the increase in invasion depth and distant metastasis in NB [11].

MicroRNAs are typically excised from a hairpin RNA structure of 60–100 nucleotides termed precursor miRNA that is transcribed from a large primary transcript (primary miRNA) with a final size of ~ 22 nucleotides [12]. By imperfect base pairing with complementary sequences, which located mainly in the 3'-UTR of target mRNAs with two manner- translational repression and transcript degradation, miRNAs can regulate its downstream gene expression. miRNAs can regulate its downstream gene expression [13, 14]. The relationship between miRNAs and various cancer metastases has been widely explored. Most recently, it has been reported that miR-21 promotes glioma invasion by targeting the matrix metalloproteinase (MMP) regulators RECK and TIMP3 [15]. Hence, whether there are some other miRNAs acting as upregulators of RECK and that contribute to NB metastasis is still unknown.

Our previous study investigated expression profiles of miRNAs in metastatic NB as compared with primary NB, and explored the potential roles of these screened miRNAs in the NB metastatic process [16]. Five mature miRNAs – miR-15a, miR-107, miR-145, miR-92a, and miR-92b – were chosen as study targets because of their upregulation in metastatic NB tissues and because they were predicted to be potential upstream regulator of RECK by the targetscan and pictar algorithms (Fig. 1). As a result, miR-15a was found to show a positive correlation with increased NB clinical stage. Hence, in this study, we further investigated whether miR-15a was an important candidate miRNA for regulating NB migration capabilities in vitro through the potential target gene RECK and regulation of MMP-9 expression.

image

Figure 1. miR-15a is a candidate miRNA for regulation of RECK expression in NB metastasis. Both left frames in A and B: our previous study showed dysregulated miRNAs between metastatic and primary NB. (A) Upper right frame: the miRNAs predicted by pictar to target the RECK 3′-UTR. (B) Lower right frame: a similar analysis was performed by targetscan.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

MicroRNA-15a, a candidate regulator of RECK, is positively correlated with NB clinical stage

As shown in Fig. 1, miR-107, miR-92a/b, miR-145, and miR-15a, which are upregulated in metastatic NB, are predicted to be potential regulators of RECK by the targetscan and pictar algorithms. Moreover, miR-15a was the only one that was upregulated in metastatic NB and that potentially targets RECK as predicted by the pictar and targetscan algorithms simultaneously. To further investigate the relationship between RECK expression and metastatic capability in NB samples, we determined RECK expression levels in different stages of NB by using western blot (WB) analysis (Fig. 2A). The results showed that the expression level of RECK protein was reduced significantly from stage 1 to stage 4 in NB samples. Then, we evaluated the expression levels of miR-107, miR-92a/b, miR-145 and miR-15a in the different stages of NB samples according to the above prediction results. As shown in Fig. 2B, miR-15a showed a positive correlation with increasing stage in NB samples, whereas other miRNAs, e.g. miR-107, which promotes tumor progression by targeting let-7 [17], did not show an apparent correlation with the NB metastatic process. The fact that expression of miR-15a showed an inverse correlation with RECK expression but a positive correlation with increased clinical stage in NB led us to presume that miR-15a, as a potential regulator of RECK, may be involved in the NB metastatic process.

image

Figure 2. RECK is negatively correlated with miR-15a expression in different stages of NB. (A) WB was performed to measure the expression level of RECK in each stage of NB, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (B) Real-time PCR was used to measure the expression level of five miRNAs in each stage of NB, with the U6 RNA normalized as a loading control. Three independent assays were performed. Data are presented as mean ± SD.

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MicroRNA-15a directly targets the RECK 3′-UTR and negatively regulates its expression

With the help of the pictar and targetscan databases, we picked out the putative binding site and depicted its mutated version in the 3′-UTR of RECK mRNA (Fig. 3A). Next, using the enhanced green fluorescent protein (EGFP) reporter assay, we studied the direct interaction between miR-15a and the RECK mRNA 3′-UTR in the GI-LA-N and SK-N-SH cell lines. When the EGFP reporter vector with the wild-type RECK 3′-UTR was used, suppression of miR-15a in GI-LA-N and SK-N-SH cells led to an increase in EGFP intensity (Fig. 3D). The binding was specific, because the EGFP reporter vector with the mutated RECK 3′-UTR was not affected by the change to miR-15a (Fig. 3D). These data indicated that RECK is directly and negatively regulated by miR-15a.

image

Figure 3. MicroRNA-15a directly targets the RECK 3′-UTR. (A) Sequence alignment of miR-15a with the putative binding sites within the wild-type and mutant 3′-UTR regions of RECK mRNA. (B) The expression level of RECK mRNA in four NB cell lines was significantly increased following transfection with miR-15a ASO, as determined by quantitative RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. (C) The protein level of RECK was determined by immunoblotting in four NB cell lines and two non-NB cell lines after transfection with miR-15a ASO. (D) EGFP fluorescence reporter assay demonstrated that suppression of miR-15a could enhance the intensity of EGFP fluorescence in GI-LA-N and SK-N-SH cells transfected with the RECK 3′-UTR construct, while having no effect on the mutant RECK 3′-UTR construct. Three independent assays were performed, and data are presented as mean ± SD. *P < 0.05.

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In order to further determine whether miR-15a can regulate endogenous RECK expression, four NB cell lines – GI-LA-N, SK-N-SH, IMR-32, and CLB-P – and two other non-NB cell lines – HEK293 and HeLa – were transfected with miR-15a ASO or oligonucleotide control. As shown in Fig. 3C, inhibition of miR-15a significantly increased RECK protein expression in GI-LA-N (~ 2.5-fold), SK-N-SH (~ 4-fold), CLB-Pe (~ 1.5-fold) and HEK293 (~ 2.0-fold) cells, but resulted in only a slight increase in IMR-32 cells (~ 1.3-fold). To our surprise, inhibition of miR-15a slightly decreases RECK protein expression in HeLa cells. Moreover, real-time PCR analysis showed varying degrees of increase in RECK mRNA expression level in all miR-15a ASO-transfected cells (Fig. 3B). These results suggest that miR-15a could regulate endogenous RECK expression at least partly by degrading its mRNA in NB cells.

MiRNA-15a regulates NB cell migration by targeting RECK

In order to investigate the potential role of miR-15a in modulating NB cell migration, two in vitro methods –transwell and scratch wound healing assays – were used. As shown in Fig. 4A, in the migration assay, the migration ability of SK-N-SH and GI-LA-N cells was significantly inhibited when they were transfected with miR-15a ASO. Moreover, SK-N-SH and GI-LA-N cells transfected with miR-15a ASO migrated more slowly to the wound healing first created in a cell monolayer at regular intervals, especially at 24 h (Fig. 4B). These results demonstrate that endogenous miR-15a can regulate the migration ability of NB cells.

image

Figure 4. Suppression of miR-15a reduced NB cell migration ability. (A) The migration assay was performed in GI-LA-N and SK-N-SH cells transfected with either miR-15a ASO or the control oligonucleotide. Cells that migrated to the lower chamber were fixed, stained, and enumerated with a light microscope. Representative images and randomly selected fields are shown. A numerical representation of the data was obtained by counting average numbers of cells from three different fields for each treatment. Data are represented as mean ± SD. (B) Almost 80% confluent GI-LA-N and SK-N-SH cells were transfected with either miR-15a ASO or the control oligonucleotide. At 5 h post-transfection, the cell monolayer was scratched with a scratch spatula (time zero), and migration of the cells towards the ‘wound’ was visualized. Images were taken at 0, 12 and 24 h after the monolayer had been scratched. The software metavue was used to determine the migration distance. On the right, the distance of each group at 0 h is normalized as 100%, and the distance of each group measured at 12 h and 24 h is taken as relative wound breadth remaining. Data are presented as mean ± SD *P < 0.05, **P < 0.01.

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To further investigate whether miR-15a regulates NB cell migration by directly targeting RECK, two groups of rescue experiment were performed. In one, 100 nm siRNA against RECK or control small interfering RNA (siRNA) was cotransfected with miR-15a ASO or control oligonucleotide into SK-N-SH and GI-LA-N cells; in the other, pcMV6/RECK construct or control vector was cotransfected with miR-15a mimic or mimic control into the two NB cell lines, and RECK protein expression and cell migration ability were then determined. As shown in Fig. 5A, transfection with RECK siRNA significantly inhibited RECK expression in SK-N-SH and GI-LA-N cells. Furthermore, suppression of miR-15a led to an increase in RECK expression, which could be alleviated by transfection with RECK siRNA in SK-N-SH and GI-LA-N cells. As was expected, inhibition of RECK resulted in an increase in the numbers of penetrated SK-N-SH and GI-LA-N cells. Moreover, when SK-N-SH and GI-LA-N cells were cotransfected with RECK siRNA and miR-15a ASO, the decreased migration ability of the two NB cell lines caused by transfection of miR-15a ASO could be partly restored (Fig. 5B). On the other hand, overexpression of miR-15a significantly enhanced the migration ability of the NB cells by decreasing the RECK expression level, and ectopic expression of RECK with the pcMV6/RECK vector partly rescued this phenotype (Fig. 5C,D). These results demonstrate that miR-15a regulates NB cell migration at least partly by directly targeting RECK expression.

image

Figure 5. MicroRNA-15a regulates NB cell migration by targeting RECK. (A) GI-LA-N and SK-N-SH cells were cotransfected with the RECK siRNA plasmid with or without miR-15a ASO. At 48 h post-transfection, the RECK protein level was measured by WB. (B) Transwell assays without Matrigel were used to evaluate the potential for cell migration ability. (C) GI-LA-N and SK-N-SH cells were cotransfected with the pcMV6/RECK plasmid with or without miR-15a mimics. At 48 h post-transfection, the RECK protein level was measured by WB. (D) Transwell assays without Matrigel were used to evaluate the potential for cell migration ability. Three independent experiments were performed, and representative images are shown. Data are presented as mean ± SD. *P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Knockdown of RECK enhances the migration ability of SK-N-SH and GI-LA-N cells by influencing MMP-9 secretion

Previous studies have indicated that overexpression of RECK decreases the amounts of active MMP-2, MMP-9 and MMP-14 in conditioned medium, and inhibits metastatic activity in vitro and in vivo [5, 18]. Hence, in the following study, we investigated whether RECK also influenced the amounts of active MMP-2, MMP-9 and MMP-14 in medium secreted by SK-N-SH and GI-LA-N cells. Conditioned medium from SK-N-SH and GI-LA-N cells transfected with either RECK siRNA or siRNA control were collected, concentrated, and immunoblotted with antibody against MMP-2, MMP-9, or MMP-14. As shown in Fig. 6A, transfection with RECK siRNA caused a significant reduction in RECK protein levels, up to almost 70%, in both SK-N-SH and GI-LA-N cells. Inhibition of RECK expression increased the cell migration ability of NB cells as compared with control cells (Fig. 6B). Furthermore, siRNA-mediated inhibition of RECK enhanced the amount of secreted MMP-9 in culture medium for both SK-N-SH and GI-LA-N cells, but had no significant effect on MMP-2 and MMP-14 (Fig. 6C). Knockdown of MMP-9 significantly decreased the endogenous expression level of MMP-9, by up to almost 75%, in both SK-N-SH and GI-LA-N cells (Fig. 6D). Moreover, the in vitro migration assay indicated that siRNA-mediated inhibition of MMP-9 decreased the migration ability of both SK-N-SH and GI-LA-N cells (Fig. 6E). These results suggest that RECK regulates the migration ability of SK-N-SH and GI-LA-N cells by influencing MMP-9 expression and secretion into the medium.

image

Figure 6. RECK regulates the migration ability of SK-N-SH and GI-LA-N cells by regulating MMP-9 proteolysis activities. (A) RECK protein level was measured by WB 48 h after transfection of the siR-RECK plasmid into SK-N-SH and GI-LA-N cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading/transfer control and for normalization of values. (B) The histogram indicates the migration ability of SK-N-SH and GI-LA-N cells when transfected with the siR-RECK plasmid. (C) The levels of MMP-2, MMP-9 and MMP-14 collected and concentrated from conditioned medium were measured by WB 48 h after transfection of siR-RECK into SK-N-SH and GI-LA-N cells. GAPDH was used as the loading/transfer control and for normalization of values. (D) The level of MMP-9 was measured by WB 48 h after transfection of siR-MMP-9 into SK-N-SH and GI-LA-N cells. GAPDH was used as the loading/transfer control and for normalization of values. (E) The histogram indicates the migration ability of SK-N-SH and GI-LA-N cells after transfection with MMP-9 siRNA. Three independent experiments were performed, and the data are presented as mean ± SD. *P < 0.05. NS, not significant.

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MicroRNA-15a regulates MMP-9 proteolysis activities by targeting RECK

The above results showed that RECK decreases the amount of active MMP-9 but not of MMP-2 and MMP-14 in conditioned medium for SK-N-SH and GI-LA-N cells. We next examined whether miR-15a in these two NB cell lines inhibits the proteolytic activity of MMP-9 by directly targeting RECK. Conditioned media from SK-N-SH and GI-LA-N cells transfected with either miR-15a ASO or the control oligonucleotide were collected, concentrated, and immunoblotted with antibody against MMP-2, MMP-9, or MMP-14. As shown in Fig. 7A,B, suppression of miR-15a significantly inhibited the amount of MMP-9 secreted into the medium, but had no effect on the amounts of secreted MMP-2 and MMP-14. This result is in accordance with that shown in Fig. 6C, when cells were transfected with RECK siRNA. In order to further explore whether the effect of miR-15a on the secretion of MMP-9 occurs through the targeting of RECK, 100 nm siRNA against RECK or control siRNA was cotransfected with miR-15a ASO or control oligonucleotide into SK-N-SH and GI-LA-N cells. As shown in Fig. 7C, transfection with RECK siRNA significantly increased the amount of secreted MMP-9 for SK-N-SH and GI-LA-N cells. Moreover, suppression of miR-15a led to an increase in RECK expression level, and resulted in a decrease in the amount of secreted MMP-9; both of these effects could be partly alleviated by transfection with RECK siRNA. The fact that the change in the amount of secreted MMP-9 showed an opposite trend to RECK expression demonstrates that miR-15a regulates MMP-9 proteolytic activity by targeting RECK, which may result in the change in migration ability of NB cells.

image

Figure 7. MicroRNA-15a regulates MMP-9 expression by directly targeting RECK. (A) WB was used to detect the expression of MMP-2, MMP-9 and MMP-14 pooled and concentrated from conditioned medium in GI-LA-N and SK-N-SH cells 48 h after transfection with miR-15a ASO. (B) The histogram reflects the relative expression levels of MMP-2, MMP-9 and MMP-14 in GI-LA-N and SK-N-SH cells transfected with the miR-15a ASO as compared with the oligonucleotide control. (C) WB was used to detect the expression of MMP-9 and RECK in GI-LA-N and SK-N-SH cells 48 h after transfection with the RECK siRNA plasmid with or without miR-15a ASO. Three independent experiments were performed, and representative images are shown. Data are presented as mean ± SD. *P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Over the past few years, various miRNA profiles have indicated that alterations in the expression levels of particular miRNAs may play a critical role in cancer initiation and progression, including the metastatic process in NB [19]. The miRNA expression profiles in our previous study [16] revealed a list of dysregulated miRNAs in metastatic NB tissues as compared with the primary NB tissues. To obtain potentially useful information about the function of altered miRNAs in NB metastasis, an important suppressor protein, RECK, which has been demonstrated to play an important role in the NB metastatic process in another study [11], was chosen for further study. Two computational algorithms, targetscan and pictar, and the previously established miRNA profiles led us to focus on five miRNAs: miR-92a, miR-92b, miR-107, miR-104, and miR-15a (Fig. 1). The final quantitative real-time PCR results obtained with different stages of NB tissues encouraged us to pick out miR-15a as a candidate upstream regulator of RECK for further research (Fig. 2).

The prompting role of miR-15a in NB migration is different from its general function in other types of tumor

Extensive research on the functions of miRNA-15a in various cancers and other pathological processes has been widely reported. For example, miR-15a was initially reported as a signature associated with prognosis and progression in chronic lymphocytic leukemia [20]. The mechanism by which miR-15a regulates MCL1, BCL2, ETS1 and JUN gene expression may be involved in chronic lymphocytic leukemia development [21, 22]. Several other studies have also shown that miR-15a is deleted or downregulated in tumors such as non-small cell lung cancer, prostate cancer, pituitary adenoma, and pancreatic cancer, suggesting that it has a ‘hot spot’ roles in cancer transformation [23-26]. Up to now, most reports have indicated a suppressor role of miR-15a in various human tumor transformations. However, interestingly, miR-15a/miR-16 is reported to be frequently upregulated in cervical cancer, which normally expresses an inactive form of Rb [27]. Hence, a dual role of miR-15a as a tumor suppressor or oncogene, depending on the active or inactive form of Rb, is still controversial. Indeed, some miRNAs have dual roles in tumor biology in different types of tumor. The most representative one is miR-373, which is a tumor suppressor in estrogen receptor negative-breast cancer, but acts as an oncogene in hepatocellular carcinoma [28, 29]. Other miRNAs that may have dual roles in the tumorigenesis of different tumors include miR-9 [30-32] and miR-214 [33, 34]. Our previous study suggested that there is a 2.5875-fold change in the expression of miR-15a in metastatic NB as compared with the primary NB [16]. In this study, we found that the expression of miR-15a gradually increased, and that this increase was positively correlated with NB clinical pathological stage, categorized mainly according to lymph node metastasis capability. Further in vitro migration and wound healing assays demonstrated that miR-15a can enhance NB cell migration ability, which was consistent with the phenotype in NB tissues. It is generally believed that tumor onco-miRNAs can promote the migration and invasion of cancer, whereas tumor suppressor miRNAs inhibit metastatic progression. Hence, these results seem to support a role of onco-miRNAs in NB tumorigenesis, although Rb activation was not determined.

MiR-15a regulates NB migration ability by directly targeting RECK

Several lines of evidence have demonstrated that, as a novel tumor suppressor gene, RECK can inhibit tumor invasion, angiogenesis, and metastasis [35, 36]. Our previous study also indicated a coincidental role of RECK in NB metastasis [11]. Although it has been reported that RAS can induce RECK gene silencing through DNMT3b-mediated promoter methylation, and TIMP-2, AP-1 can upregulate RECK expression in endothelial cells [38-40], the precise mechanism of its regulation, especially post-transcriptional regulation mediated by miRNAs, is still not well understood. In the present study, the EGFP reporter assay demonstrated that miR-15a can directly target the RECK 3′-UTR and downregulate its expression (Fig. 3). These results support our previous suggestion that miR-15a may play an important role in NB metastasis procedure by targeting RECK expression. The subsequent in vitro migration and scratched wound healing assays further confirmed our hypothesis that miR-15a promotes NB migration by post-transcriptional regulation of RECK (Figs 4 and 5). It has also been reported that miR-21 can promote glioma cell migration and invasion by targeting RECK expression in glioblastoma [15]. However, as a newly discovered RECK upregulator, miR-15a targets the RECK 3′-UTR at position 811–817, which does not overlap with the binding sites within miR-21 (positions 1122–1144, 2355–2392, and 1017–1039). Whether miR-21 and miR-15a have a synergistic effect in regulating NB migration and invasion ability by targeting the same RECK is an interesting problem, and requires further investigation.

An miR-15a–RECK–MMP-9 axis plays an important role in regulating NB migration

RECK can suppress the invasive, metastatic and angiogenic activities of malignant cells by regulating the secretion of pro-MMP-9 [5]. In this study, we demonstrated that RECK has a negative regulatory role in NB cell migration ability by influencing the MMP-9 secretion pathway (Fig. 6). Furthermore, suppression of miR-15a could lead to an increase in RECK expression, and reduced secretion of MMP-9 but not of MMP-2 and MMP-14. The evidence provided by this study shows that miR-15a regulates NB migration ability by directly targeting RECK. Hence, a speculative representation of the hypothetical molecular mechanism by which miR-15a joins the axis of the miR-15a–RECK–MMP-9-mediated NB metastatic process in NB is shown in Fig. 8, and this may play an important role in NB progression, especially in NB migration and invasion.

image

Figure 8. Schematic representation of the hypothetical molecular mechanism by which miR-15a regulates NB migration through the miR-15a–RECK–MMP-9 axis.

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In conclusion, our results can be summarized as follows: (a) the expression of miR-15a is positively correlated with NB clinical pathological stages, and negatively correlated with RECK expression; (b) miR-15a directly targets the RECK 3′-UTR, and regulates its mRNA and protein expression levels in SK-N-SH and GI-LA-N cells; (c) suppression of miR-15a reduces NB cell migration ability by directly targeting RECK; (d) overexpression of miR-15a enhances NB cell migration ability by suppressing RECK expression; and (e) miR-15a regulates MMP-9 secretion by targeting RECK. Consequently, the identification of miR-15a as regulating NB metastatic ability may help us to understand potential molecular mechanisms of tumorigenesis, and may provide new prognostic markers for the metastasis of NB in the future.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

NB samples, cell culture, and plasmids

Fresh human NB tissues of four stages were obtained from the Affiliated Hospital of the Medical College of Qingdao University, and were snap-frozen in liquid nitrogen and stored at −80 °C until use. All of the human NB samples used were taken with the understanding and written consent of the subjects and their legal guardians. The study methodology conformed to the standards set by the Declaration of Helsinki, and was approved by the Qingdao medical ethics committee in Shandong province, China. Six human NB cell lines – CLB-Pe, IMR-32, GI-LA-N, SK-N-SH, HEK293, and HeLa – obtained from the ATCC were used in this study. All of these cell lines were cultured at 37 °C in 5% CO2 in DMEM (Multicell; Wisent, St Bruno, Quebec, Canada) with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA), 50 U·mL−1 penicillin, and 50 mg·mL−1 streptomycin (Gibco, Carlsbad, CA, USA). The RECK expression vector pcMV6/RECK (catalogue number: sc112572) was purchased from OriGene Technologies. The MMP-9 siRNA product (catalogue number: sc-29401) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Quantitative RT-PCR analysis of mRNA and miRNA expression

To detect the level of expression of mature miRNA, a stem–loop RT-PCR assay was performed [41]. Briefly, 2 μg of RNA was reverse transcribed with cDNA by use of Moloney murine leukemia virus reverse transcriptase (Promega, Beijing, China). The cDNA was used for the amplification of mature miR-15a and an endogenous control U6 snRNA for all PCR reactions. PCR cycles were as follows: 94 °C for 4 min, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 40 s. An SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) was used, according to the manufacturer's instructions, and real-time PCR was performed, and the results were analyzed, with a 7300 Real-Time PCR system (ABI, Beijing, China).

EGFP reporter assay

To confirm the direct interaction of miR-15a and the target RECK mRNA, NB cells were transfected with miR-15a ASO or control oligonucleotide in 48-well plates, along with the reporter vector pcDNA3/EGFP–RECK 3′-UTR or pcDNA3/EGFP–RECK 3′-UTR mut. A relative amount of red fluorescent protein expression vector pDsRed2-N1 was cotransfected into each group to be used for normalization. At 48 h post-transfection, cells were lysed with 0.25 mL of RIPA lysis buffer (50 mm Tris/HCl, pH 7.2, 150 mm NaCl, 1% Triton X-100, 0.1% SDS), and total protein was harvested. The intensities of EGFP and red fluorescent protein fluorescence were detected with an F-4500 Fluorescence Spectrophotometer (Hitachi).

In vitro migration assay

The protocol for the in vitro migration assay is described Doc. S1.

Bioinformatics

The target genes of miRNA were predicted by the following two computer-aided algorithms: targetscan Release 5.2 (http://www.targetscan.org) and pictar (http://pictar.mdc-berlin.de/cgi-bin/168 new_PicTar_vertebrate.cgi).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Student's t-test was used for analysis, and P-values < 0.05 were considered to be significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the National Natural Science Foundation of China (No. 30872702).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12074-sup-0001-DocS1.zipZip archive286KDoc S1. Primers used in this study and the in vitro migration assay protocol.

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