Metastasis is a major clinical obstacle in the treatment of gastric cancer (GC) and it accounts for the majority of cancer-related mortality. MicroRNAs have recently emerged as regulators of metastasis by acting on multiple signaling pathways. In this study, we found that miR-7 is significantly downregulated in highly metastatic GC cell lines and metastatic tissues. Both gain-of-function and loss-of-function experiments showed that increased miR-7 expression significantly reduced GC cell migration and invasion, whereas decreased miR-7 expression dramatically enhanced cell migration and invasion. In vivo metastasis assays also demonstrated that overexpression of miR-7 markedly inhibited GC metastasis. Moreover, the insulin-like growth factor-1 receptor (IGF1R) oncogene, which is often mutated or amplified in human cancers and functions as an important regulator of cell growth and tumor invasion, was identified as a direct target of miR-7. Silencing of IGF1R using small interefering RNA (siRNA) recapitulated the anti-metastatic function of miR-7, whereas restoring the IGF1R expression attenuated the function of miR-7 in GC cells. Furthermore, we found that suppression of Snail by miR-7, through targeting IGF1R, increased E-cadherin expression and partially reversed the epithelial–mesenchymal transition (EMT). Finally, analyses of miR-7 and IGF1R levels in human primary GC with matched lymph node metastasis tissue arrays revealed that miR-7 is inversely correlated with IGF1R expression. The present study provides insight into the specific biological behavior of miR-7 in EMT and tumor metastasis. Targeting this novel miR-7/IGF1R/Snail axis would be helpful as a therapeutic approach to block GC metastasis.
Advances in diagnostic techniques, chemotherapeutic agents and operative management have increased the early detection and decreased the mortality rate of gastric cancer (GC); however, it is still the second leading cause of cancer-related death worldwide, and patients with advanced disease frequently develop recurrence.1 The major cause of death and relapse from GC is metastasis, which greatly hampers the success of treatment modalities. Metastasis is a complex process comprising multiple sequential steps. In the metastatic cascade, local invasion can be considered an initial, essential step in the malignancy of carcinomas leading to the generation of a distant metastasis.2 The changes that occur during tumor invasion are reminiscent of an important developmental process termed the epithelial–mesenchymal transition (EMT). EMT is thought to be a critical step in the metastatic process that confers certain fundamental abilities to cancer cells, such as migration, invasion and anoikis resistance, which initiate and increase metastasis.3, 4 However, the molecular mechanisms that promote EMT and metastasis in GC cells are still poorly understood.
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that suppress gene expression at the post-transcriptional level by blocking mRNA translation or degrading target mRNAs.5 Mounting evidence has shown that mutations or aberrant expression of miRNAs correlates with various human cancers, and that miRNAs can function as tumor suppressors and oncogenes.6 Recently, miRNAs have been shown to be implicated in both the promotion and suppression of metastasis.7 In particular, miR-21 stimulates cell invasion and metastasis in breast cancer, colon cancer and gliomas;8, 9, 10 miR-10b promotes in vitro and in vivo migration, invasion and metastasis in breast cancer cells;11 and let-7 family members inhibit cell adhesion, migration and invasion in lung cancer, GC and breast cancer.12, 13, 14 In addition, miRNAs have key roles in regulating the EMT process. miRNAs from the miR-200 family and miR-205 target both the ZEB1 and ZEB2 EMT-inducing transcription factors, which function as transcriptional repressors of E-cadherin expression, thereby reducing the aggressiveness of cancer cells.15
Interestingly, some miRNAs can be multitasking by interacting with different target genes in various cellular contexts. miR-7 is a member of these miRNAs, which has been characterized as a tumor suppressor in several human cancers by targeting critical cancer-related pathways. It is shown that miR-7 inhibits Schwannoma tumor growth by targeting the EGFR, Pak1 and Ack1 oncogenes,16 and it also inhibits the growth of human A549 non-small-cell lung cancer cells by targeting BCL-2.17 Recent studies also indicated that miR-7 is associated with tumor metastatic capability and can inhibit the motility and invasiveness of breast cancer and urothelial cancer;18, 19, 20 however, its roles and mechanism remain largely unknown. This study provides the first evidence for a role of miR-7 in GC metastasis and partially elucidates the molecular mechanism underlying this effect.
Insulin-like growth factor-1 receptor (IGF1R), which belongs to a family of tyrosine kinase receptors, mediates the biological actions of both IGF1 and IGF2, and has pivotal roles in cellular processes such as proliferation, survival, cell migration and differentiation.21 Furthermore, mounting evidence indicates that IGF1R is a prerequisite for oncogenic transformation and is involved in critical steps of the metastatic cascade, including cell adhesion, migration, invasion, angiogenesis and metastatic growth at distant organ sites.22, 23 These findings suggest that IGF1R may be a valuable therapeutic target, and IGF1R-antagonistic monoclonal antibodies have been developed to block its activities.24
In the present study, we comprehensively investigated the biological function and its underlying molecular mechanism of miR-7 in GC metastasis. We identified significant downregulation of miR-7 in metastatic GC cell lines and tissues. Furthermore, enhanced expression of miR-7 potently inhibited the ability of GC cells to metastasize in vitro and in vivo, whereas loss of miR-7 promoted GC cell migration and invasion. In addition, we identified IGF1R as a direct and functional target of miR-7. Intriguingly, ectopic expression of miR-7 induced the restoration of invasion suppressor E-cadherin. We further demonstrated that downregulation of the E-cadherin suppressor Snail by miR-7, through targeting IGF1R, inhibited EMT in GC cells, which might contribute to the anti-metastatic role of miR-7. Finally, we confirmed an inverse correlation between miR-7 and IGF1R expression in human GC tissues.
miR-7 is downregulated in GC cell lines, and its expression is inversely correlated with GC metastasis
To explore the expression and significance of miR-7 in GC carcinogenesis, we measured the expression of miR-7 in four human GC cell lines (AGS, SGC7901, MKN28 and MKN45) and GES, an immortalized gastric epithelial cell line. Quantitative reverse-transcriptase PCR (qRT–PCR) showed that miR-7 expression was significantly decreased in GC cell lines compared with GES (Figure 1a). To further identify the role of miR-7 in GC metastasis, we selected GC9811-P and MKN28M, which are highly metastatic GC cells, and GC9811 and MKN28NM, which have weak invasive potential, for further study. These two pairs of GC cell lines were established and characterized in our lab previously.25, 26 We found that miR-7 expression was markedly higher in GC9811 and MKN28NM compared with their counterparts (Figure 1b). Furthermore, tissues from lymph node metastases expressed lower levels of miR-7 compared with primary GC tissues and the adjacent normal tissue, indicating an inverse relationship between the expression of miR-7 and the metastatic status of GC tissues (Figure 1c). Taken together, these results suggest that downregulation of miR-7 is correlated with increased GC metastasis and that miR-7 might inhibit GC progression.
miR-7 suppresses GC cell invasion and metastasis in vitro and in vivo
To determine whether miR-7 regulates GC cell invasion and metastasis, we first performed in vitro gain-of-function analyses by overexpressing miR-7 with a lentiviral vector in GC9811-P cells. Transwell migration and invasion assays were performed on the miR-7-infected cells. We found that ectopic expression of miR-7 significantly suppressed the invasion of GC9811-P cells in Transwell assays with Matrigel and reduced the migration of GC9811-P in Transwell assays without Matrigel (Figure 2a). In contrast, the migration and invasion of GC9811 cells increased when endogenous miR-7 was silenced with antisense oligonucleotides (Figure 2b). These observations suggest that miR-7 can suppress GC cell migration and invasion in vitro. To further determine whether miR-7 could suppress metastatic behaviors in vivo, GC9811-P cells stably expressing miR-7 were delivered into nude mice by tail vein injection. We found that the number of metastatic nodules in the liver was dramatically decreased in miR-7 groups when compared with negative controls (Figure 2c). Taken together, these results indicate that miR-7 has a suppressor role in GC metastasis.
miR-7 downregulates IGF1R through interaction with its 3′-untranslated region
To understand the underlying molecular mechanism by which miR-7 suppress GC invasion and metastasis, we searched for miR-7 targets using different computational methods, such as TargetScan and miRanda. These methods identified 181 candidate genes that were commonly predicted to be possible targets of miR-7. To uncover the biological functions of these miR-7 target candidates, Gene Ontology Analysis was carried out. Genes classified as having molecular functions involved in ‘cell motion’ or ‘cell migration’ were identified. Several of these genes that have roles in cell migration or invasion, including RRAS2, IRS1, IRS2, EGFR and IGF1R, were selected for further analysis. We were particularly interested in IGF1R because of its positive roles in cancer cell proliferation and invasion.22 Analysis of the 3′-untranslated region (UTR) sequence of IGF1R identified three possible binding sites for miR-7, two of which are poorly conserved among different species, whereas the third is highly conserved. To determine whether IGF1R is a direct target of miR-7, we constructed its 3′-UTR fragments, in which wild-type and mutant binding sites were inserted into the region immediately downstream of the reporter gene (Figure 3a). Luciferase reporter assays showed that miR-7 transfection caused a remarkable decrease in relative luciferase activity in HEK293T, GES and GC9811 cells when the IGF1R plasmid containing wild-type 3′-UTR was present, but the luciferase activity did not drop as sharply in the 3′-UTRs that contained mutant binding sites as the wild-type one (Figure 3b). Furthermore, qRT–PCR showed that overexpression or inhibition of miR-7 had no effect on IGF1R mRNA level (Figure 3c). However, western blotting demonstrated that overexpression of miR-7 significantly suppressed IGF1R expression in GC9811-P cells and that silencing of miR-7 increased IGF1R expression in GC9811 cells (Figure 3d), which indicating that miR-7 regulates IGF1R at the post-transcriptional level. Taken together, these results suggest that miR-7 regulates IGF1R expression by directly targeting its 3′-UTR.
Downregulation of IGF1R inhibits GC cell invasion and metastasis
Next, we investigated whether downregulation of IGF1R represses cell migration and invasion in GC cells. To this end, GC9811-P cells were infected with lentiviral constructs containing siRNA against IGF1R or the negative control (Figure 4a). Remarkably, silencing of IGF1R inhibited GC9811-P cell migration and invasion (Figure 4c). In contrast, overexpression of IGF1R in GC9811 cells (Figure 4b) increased cell migration and invasion (Figure 4d). In vivo experimental metastasis assays also showed that downregulation of IGF1R suppressed GC cell invasion and metastasis (Figure 4e).
Restoration of miR-7 inhibits IGF1R-mediated GC cell invasion and metastasis
Because IGF1R can promote GC cell migration and invasion, and miR-7 can post-transcriptionally regulate the expression of IGF1R, we hypothesized that miR-7-mediated downregulation of IGF1R directly inhibits GC invasion and metastasis. To test this hypothesis, we first transfected GC9811 cells with IGF1R plasmids containing the wild-type 3′-UTR or lacking 3′-UTR (Figure 5a). When miR-7 was cotransfected into GC9811 cells, IGF1R expression was markedly reduced in the cells transfected with the wild-type 3′-UTR, but not in those transfected with the construct lacking 3′-UTR (Figure 5b). More important, both Transwell assays and in vivo metastasis assays indicated that the restoration of miR-7 expression significantly reduced the IGF1R-induced GC cell migration, invasion and metastasis (Figures 5c). Taken together, these results suggest that IGF1R is a functional target of miR-7.
miR-7 inhibits EMT in GC cell
In GC9811-P cells, we observed that increased miR-7 resulted in morphological changes from an extended, fibroblast-like morphology to highly organized cell–cell contacts (Figure 6a). These changes represent the reverse processes of EMT, which has a central role in the process of cancer cell dispersion. Because extensive evidence suggests that growth factors can initiate the EMT process,27 we speculated that suppression of IGF1R by miR-7 might impact EMT. To investigate this hypothesis, we examined the expression of the epithelial makers E-cadherin and β-catenin, as well as the mesenchymal maker vimentin. We found that E-cadherin and β-catenin expression dramatically increased in GC9811-P cells infected with miR-7, whereas silencing miR-7 suppressed E-cadherin and β-catenin expression, and induced vimentin in GES (Figure 6b). Immunofluorescent staining also showed that miR-7 infection led to the upregulation of E-cadherin and the downregulation of vimentin (Figure 6c). In addition, β-catenin was primarily located in the nucleus in GC9811-P cells; however, following miR-7 infection, β-catenin was absent from the nucleus, and instead, was localized at the plasma membrane, associated with E-cadherin (Figure 6d). These results suggest that expression of miR-7 can reverse EMT in the metastatic GC cell.
Among the previously described makers, E-cadherin is a central component that involved in the conversion between mesenchymal and epithelial phenotypes. Thus, we examined the mRNA expression of Snail, Slug, ZEB1 and ZEB2, which are transcriptional repressors of E-cadherin and potent EMT inducers, in GC9811-P cells expressing miR-7. We found that Snail showed the largest degree of reduction by miR-7 (Figure 6d). IGF1R can increase the expression of Snail in mammary epithelial cells.28 Therefore, we next sought to determine whether Snail could be induced in GC9811 cells by transfection with an IGF1R plasmid with or without the 3′-UTR. More important, we found that miR-7 suppressed Snail, which was induced by wild-type IGF1R, and subsequently increased the expression level of E-cadherin in GC9811. In contrast, this suppression was abrogated when the wild-type IGF1R was substituted by the one without 3′-UTR (Figure 6e). These results suggest that suppression of Snail by miR-7 is mediated through targeting IGF1R and that this enhances E-cadherin expression and eventually inhibits the EMT process in GC cell.
miR-7 and IGF1R are inversely expressed in GC specimens
Finally, we determined whether miR-7 expression was associated with IGF1R expression in GC specimens from GC patients to evaluate its clinical relevance. To detect the expression patterns of miR-7 and IGF1R in the same type of commercialized tissue microarrays, we employed in situ hybridization and immunohistochemistry. The tissue microarrays contained 40 pairs of primary GC specimens and their matched lymph node metastatic tissues. The in situ hybridization analysis showed an overt reduction of miR-7 in the lymph node metastatic tissues compared with their corresponding primary GC tissues (Figure 7a, Table 1). By contrast, immunohistochemistry staining revealed that IGF1R was higher in metastatic tissues than in primary GC tissues (Figure 7b, Table 2). Furthermore, statistical analysis revealed that IGF1R expression was inversely correlated with miR-7 expression (Table 3, R=−0.6786, P<0.01). Taken together, these observations suggest that IGF1R expression is elevated in GC metastatic tissues and that its enhancement is correlated with reduced miR-7.
miRNAs have emerged as important regulators of gene expression at the post-transcriptional level and regulate a wide range of physiological and developmental processes. Over the past several years, it has become clear that alterations in the expression of miRNAs contribute to the pathogenesis of most human cancers, where they act as either oncogenes or tumor suppressors.29 Recently, accumulating data indicate that miRNAs are involved in advanced stages of cancer progression and that they can act as activators or suppressors of metastasis. In the present study, we investigated the biological role of miR-7 in human GC metastasis.
Aberrant miR-7 expression is correlated with carcinogenesis. miR-7 is downregulated in malignant mesothelioma, Schwannoma, GC and non-small-cell lung cancer.30, 31, 32 Moreover, several lines of evidence indicate that miR-7 may perform novel functions in tumor invasion and metastasis. miR-7 has been associated with aggressiveness and metastasis in breast cancer and urothelial carcinoma.18, 19 Kefas et al.33 showed that transfection with miR-7 decreases the viability and invasiveness of primary glioblastoma cell lines. miR-7 also inhibits the motility, invasiveness, anchorage-independent growth and tumorigenic potential of highly invasive breast cancer cells.19 In this study, we identified the expression of miR-7 in several GC cell lines, in the immortal gastric epithelial cell line GES, and in two pairs of low and high metastatic GC cell lines using qRT–PCR. miR-7 expression was significantly decreased in all four GC cell lines and was lower in GC9811-P and MKN28M cells compared with GES. Furthermore, the results obtained from clinical GC tissue also confirm that miR-7 was downregulated in advanced stages of GC, indicating its possible involvement in both oncogenic transformation and tumor metastasis. We subsequently confirmed that miR-7 significantly suppressed GC cell invasion and metastasis both in vitro and in vivo.
Similar to classical transcription factors, miRNAs exert their effects via regulating specific target genes. Generally, one gene can be repressed by multiple miRNAs, and one miRNA may repress multiple target genes,34 which suggests that one specific miRNA could carry out a variety of functions by targeting different genes in different cell contexts. miR-7 targets α-synuclein mRNA in neurons and protects cells against oxidative stress.35 Splicing factor SF2/ASF promotes miR-7 maturation, and mature miR-7 targets the 3′-UTR of SF2/ASF to repress its translation, forming a negative-feedback loop.36 Furthermore, several known oncogenes have been reported to be the targets of miR-7. miR-7 functions as a tumor suppressor in three major oncogenic pathways by targeting EGFR, Pak1 and Ack1, respectively.16, 19, 37 However, few studies have been performed to determine which genes miR-7 targets to modulate the behavior of GC metastasis. In this study, potential targets of miR-7 were analyzed using different prediction algorithms and Gene Ontology Analysis. Our search to unravel the biological role of miR-7 in GC metastasis identified IGF1R as a critical downstream target. We found that IGF1R was upregulated in GC cells, and exogenous miR-7 downregulated the expression of IGF1R protein. Furthermore, luciferase reporter assays revealed that miR-7 could directly target the 3′-UTR of IGF1R mRNA.
IGF1R belongs to a family of tyrosine kinase receptors whose distinctive structural feature is to function as covalently linked tetramers of two α- and two β-subunits.21 IGF1R is expressed in most transformed cells, where it displays potent anti-apoptotic, pro-survival and transforming activities.38, 39, 40 In recent years, mounting evidence indicates that IGF1R and its ligands may be involved in human cancer progression. Dunn et al.41 have shown that impairment of IGF1R function by expression of a dominant-negative mutant significantly suppresses the adhesion and invasion potentials of two estrogen receptor-negative breast cancer cell lines. In vivo, functional impairment of IGF1R does not suppress the growth of the primary tumor but significantly decreases tumor metastases to the lungs, liver, lymph nodes and lymph vessels, suggesting that IGF1R can regulate tumor metastasis independently of tumor growth.22 These observations indicate that IGF1R promotes cancer cell invasion and metastasis, which is opposite of the role of miR-7. In this study, we identified IGF1R as a direct and functional target of miR-7. We confirmed the positive effects of IGF1R protein on GC cell migration, invasion and metastasis in vitro and in vivo using RNA interference. Moreover, miR-7-mediated suppression of IGF1R depended on the 3′-UTR. Finally, IGF1R-induced cell migration, invasion and metastasis were reversed by miR-7. Taken together, these results establish a functional connection between miR-7 and IGF1R, and confirm that miR-7 functions as an anti-metastatic miRNA in GC cells by targeting IGF1R.
Metastasis arises through a multistep process that begins when cancer cells within primary tumors detach from neighboring cells and invade the basement membrane.42 This local invasion may frequently be triggered by contextual signals that cancer cells received from the nearby stroma, causing them to undergo an EMT process.3 The most important event of EMT is the loss of E-cadherin, which is a prerequisite for epithelial tumor cell invasion. The consequences of E-cadherin loss are impairment of the cell–cell adhesion and nuclear translocation of β-catenin which induces a gene expression pattern favoring tumor invasion.43 In this study, overexpression of miR-7 in GC9811-P cells induced morphological changes from an extended, fibroblast-like morphology to highly organized cell–cell contacts. Inhibition of miR-7 in GES greatly diminished E-cadherin expression but increased the expression of vimentin, whereas overexpression of miR-7 significantly induced E-cadherin and β-catenin expression in GC9811-P cells. Furthermore, miR-7-mediated E-cadherin induction led to the recruitment of β-catenin to the plasma membrane and inhibition of its nuclear translocation. Because β-catenin functions in a dual manner in epithelial cells, depending on its intracellular localization, the phenomena we observed further suggest that miR-7 has multiple roles in EMT and GC metastasis.
Loss of E-cadherin expression has been identified as a critical event in EMT, and Snail, ZEB and some basic helix-loop-helix factors can directly bind to the E-cadherin promoter and repress its transcription.44 Among these factors, Snail is a strong repressor of E-cadherin expression and a potent EMT effector.45 The interaction of extracellular signals, including components of the extracellular matrix and soluble growth factors, has a major role in the EMT process.46 Lee et al.28 have shown that IGF1R induces EMT through upregulation of Snail. In this study, IGF1R transfection in GES was sufficient to induce Snail expression; however, co-transfected miR-7 was able to suppress Snail and increase E-cadherin. As further evidence that the effect of miR-7 on Snail is IGF1R-dependent, the negative regulation relationships were abrogated upon expression of IGF1R lacking the 3′-UTR, that is, lacking miR-7-binding sites. Previous studies have partially described the mechanism of action of IGF1R in metastasis. Brodt et al.47 identified IGF1R as a positive regulator of vascular endothelial growth factor-C production and lymphatic metastasis. Several groups also identified IGF1R as a promoter of matrix metalloprotease-2 synthesis and tumor invasion.48 In addition, IGF1R regulates urokinase-type plasminogen activator expression in breast cancer cells and both urokinase-type plasminogen activator and its receptor in pancreatic cancer cells.49, 50 Our findings suggest that suppression of Snail by miR-7, through targeting IGF1R, inhibits the EMT process and blocks metastasis in the early stage. Furthermore, restoring E-cadherin expression by inactivating Snail is likely to contribute to the miR-7-mediated reduction of GC cell metastasis.
In summary, we have demonstrated that miR-7 is significantly downregulated in highly metastatic cells and tissues. miR-7 overexpression can inhibit GC cell migration, invasion and metastasis both in vitro and in vivo. Furthermore, IGF1R is a direct and functional target of miR-7, and IGF1R-mediated Snail expression and loss of E-cadherin can be reversed by miR-7 in GC cells. This novel miR-7/IGF1R/Snail axis provides new insight into the mechanisms underlying tumor metastasis, and restoration of miR-7 expression may be a potential therapeutic strategy for the treatment of GC in the future.
Materials and methods
Cell culture and tissue collection
GC9811, GC9811-P, MKN28NM, MKN28M, AGS, SGC7901, MKN28, MKN45 and GES cells were cultured in RPMI-1640 medium (Thermo Scientific HyClone, Beijing, China). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (HyClone). Paired samples of primary GC, adjacent normal tissues and lymph node metastatic tissues were obtained from patients who had undergone GC surgery at Xijing Hospital, Xi’an, China. All samples were clinically and pathologically shown to be correctly labeled. This study was approved by the Hospital’s Protection of Human Subjects Committee, and informed consent was obtained from every patient.
RNA extraction and real-time RT–PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RT and PCR primers for miR-7 and U6 were purchased from RiBoBio (Guangzhou, China). The PCR primers for IGF1R were 5′-GAGAAGGAGGAGGCTGAATACCG-3′ and 5′-GTGATGTTGTAGGTGTCTGCGGC-3′. The primers for Snail were 5′-CACTATGCCGCGCTCTTTC-3′ and 5′-GGTCGTAGGGCTGCTGGAA-3′. The primers for Slug were 5′-TGTTGCAGTGAGGGCAAGAA-3′ and 5′-GACCCTGGTTGCTTCAAGGA-3′. The primers for ZEB1 were 5′-GCCAATAAGCAAACGATTCTG-3′ and 5′-TTTGGCTGGATCACTTTCAAG-3′. The primers for ZEB2 were 5′-CAAGAGGCGCAAACAAGC-3′ and 5′-GGTTGGCAATACCGTCAT-3′. The first-strand cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa) and measured in a LightCycler 480 system (Roche, Basel, Switzerland). Expression of U6 or glyceraldehyde 3-phosphate dehydrogenase was used as internal control. All of the reactions were run in triplicate.
Lentivirus infection and oligonucleotide transfection
The miR-7 and IGF1R siRNA were purchased from GeneChem (Shanghai, China). The constructs containing the pre-miR-7 or IGF1R siRNA sequence, and 100 bases of upstream and downstream flanking these sequences were cloned into the pGCSIL-GFP vector. Target cells (1 × 105) were infected with 1 × 107 lentivirus transducing units in the presence of 10 μg/ml polybrene. Empty lentiviral vector was used as negative control. The miR-7 inhibitor and negative control were designed and synthesized by RiBoBio. Target cells were transfected with miR-7 inhibitor and negative control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were collected 48 h after transfection.
The plasmid pcDNA3-IGF1R-wt UTR was constructed by inserting the IGF1R cDNA into the pcDNA3 vector (Invitrogen) at Xhol and Xbal sites. pcDNA3-IGF1R-no UTR contained the full-length IGF1R coding region, but removed its 3′-UTR beginning at codon 4155.
To construct a luciferase reporter vector, the wild-type 3′-UTR of IGF1R, containing three putative binding sites for miR-7, was PCR-amplified using genomic DNA from 293T, GES and GC9811 cells as templates. The corresponding mutant constructs were created by mutating the seed regions of the miR-7-binding sites. Both wild-type and mutant 3′-UTRs were cloned downstream of the luciferase gene in the psiCHECK-2 Luciferase vector. The constructs were verified by sequencing.
Migration and invasion assays
For migration assays, infected or transfected cells were harvested and resuspended in serum-free RPMI-1640 medium, and 1 × 105 cells were placed into Boyden chambers (Corning, Cambridge, MA, USA) with an 8.0 μm pore membrane. For invasion assays, 1 × 105 cells were placed into chambers coated with 150 μg of Matrigel (BD Biosciences, Bedford, MD, USA). The chambers were then inserted into the wells of a 24-well plate and incubated for 24 h in RPMI-1640 medium with 10% fetal bovine serum before examination. The cells remaining on the upper surface of the membranes were removed, whereas the cells adhering to the lower surface were fixed, stained in a dye solution containing 0.05% crystal violet and counted under a microscope (Olympus Corp., Tokyo, Japan) to calculate their relative numbers. The results were averaged among three independent experiments.
In vivo metastasis assays
For in vivo metastasis assays, 2 × 106 GC9811-P cells infected with miR-7 or IGF1R siRNA lentivirus and negative control were suspended in 200 μl phosphate-buffered saline and injected into the tail vein of nude mice (six in each group, female nu/nu). After 4 weeks, the mice were killed, their livers were dissected, and tumor nodules were counted under a stereomicroscope (Olympus). The nude mice were provided by the Experimental Animal Center of the Fourth Military Medical University. All animal studies complied with the Fourth Military Medical University animal use guidelines and the protocols approved by the Fourth Military Medical University Animal Care Committee.
Luciferase reporter assays
For luciferase reporter assays, 293T, GES and GC9811 cells were seeded in 24-well plates and transiently transfected with appropriate reporter plasmid and miRNA using Lipofectamine 2000. After 48 h, the cells were harvested and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla-luciferase was used for normalization. For each plasmid construct, the transfection experiments were performed in triplicate.
Whole-cell lysates were prepared in RIPA buffer (Byotime, Haimen, China), and western blotting was performed as previously described.51 The primary antibodies used were IGF1R (Abcam, Cambridge, UK), E-cadherin (BD Biosciences), β-catenin (BD Biosciences), vimentin (Thermo Scientific), Snail (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (Sigma, St Louis, MO, USA).
Indirect immunofluorescence staining for E-cadherin, β-catenin and vimentin was performed in stable GC9811-P cells as previously described.52
GC tissue microarrays (No. ST810) were purchased from Alenabio (Xi’an, China). Each array included 40 cases of gastric malignant tissues and their matched lymph node metastatic tissues.
Immunohistochemistry and in situ hybridization
Immunohistochemical staining was performed as previously described,53 using anti-IGF1R antibody (Abcam). In situ hybridization was performed using a miR-7 probe from Exiqon (miRCURY LNA detection probe 5′ and 3′-DIG (digoxigenin)-labeled). The probe was detected using digoxigenin antibody (Abcam), LSAB2 System-HRP (Dako Denmark A/S, Glostrup, Denmark) and liquid DAB+ Substrate Chromogen System (Dako) according to the manufacturer’s instructions.
The results of immunostaining and hybridization were independently scored by two pathologists (Zengshan Li and Zhe Wang) in a blind manner. The scoring was based on the intensity and extent of staining and was evaluated according to the following histological scoring method. The mean proportion of staining cells per specimen was determined semi-quantitatively and scored as follows: 0 for staining <1%, 1 for 1–25%, 2 for 26–50%, 3 for 51–75%, and 4 for >75% of the examined cells. Staining intensity was graded as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. The histological score (H-score) for each specimen was computed by the formula: H-score=Proportion score × Intensity score. A total score of 0–12 was calculated and graded as negative (−, score: 0), weak (+, score: 1–4), moderate (++, score: 5–8) or strong (+++, score: 9–12). The correlation between IGF1R and miR-7 was analyzed using Spearman’s rank test.
The SPSS12.0 program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Experimental data are expressed as the means±s.e. The differences between groups were analyzed using Student’s t-test when comparing only two groups or assessed by one-way analysis of variance when more than two groups were compared. For comparison of paired tissues, a paired Student’s t-test was used to determine the statistical significance. Differences were considered statistically significant at P<0.05, *P<0.05 and **P<0.01.
We acknowledge Dr Tiziana Deangelis from Tomas Jefferson University for providing the pcDNA3-IGF1R plasmid. We thank Professor Zengshan Li and Professor Zhe Wang from Xijing Hospital for their help with pathological analyses. We thank Qing Ye from the Fourth Military Medical University for excellent statistical assistance. This work was supported by the National 973 Project of China (No. 2010CB529300, 02, 05, 06) and the National Natural Science Foundation of China (No. 81030044, 30970149, 30900675).
About this article
MicroRNA-7 as a potential therapeutic target for aberrant NF-κB-driven distant metastasis of gastric cancer
Journal of Experimental & Clinical Cancer Research (2019)