Screening And Fuctional Study Of Metastasis-related MicroRNAs In Gastric Cancer

【Background】The five-year survival rate for early gastric cancer (GC) has been improved significantly with the advances in diagnosis and therapy approaches. However, the prognosis of advanced GC with extensive invasion and metastasis remains poor. Owing to the mechanisms of GC metastasis are not fully understood, there is a lack of early diagnosis and effective treatment of GC metastasis. Further understanding the molecular mechanisms of metastasis and improving the prognosis of patients with GC are the most important and urgent issues in the field of oncology research.Recently, microRNAs (miRNAs), a set of naturally occurring short non-coding RNA molecules, have been identified to play important roles in tumor metastasis by negatively regulating target genes expression. Compared with protein-coding genes, miRNAs is more effective in inhibition of tumor metastasis because each miRNA can regulate more than one target genes involved in tumor growth, invasion and metastasis. Thus, we wondered whether there was a specific miRNA signature for GC metastasis, what kind of roles the miRNAs play in the process of GC metastasis, what is their mechanism of action and whether therapeutic intervention on miRNA expression could effectively reverse the metastatic phenotype of GC cells. It is clear that all these issues still need to be clarified.【Aims】To screen for the metastasis-related miRNAs in GC and to clarify their roles and the underlying mechanisms in metastasis process, with the aim of better elucidating the mechanisms of GC metastasis and providing a new theoretical basis for early diagnosis and treatment of GC metastasis.【Methods】1. We generated cell sublines with high-invasive (MKN28-M and SGC7901-M) or low-invasive potential (MKN28-NM and SGC7901-NM) from established human GC cell lines MKN28 and SGC7901 by using the repeated Transwell approach in vitro. Then MTT assay and tumour formation in nude mice were used for evaluating cell-growth and survival; Flow Cytometry were used for analysis of Cell Cycle; Transwell migration and invasion assays, would-healing assay and tail vein injection metastasis assay were employed for investigating the metastatic properties of each cell subline.2. The differential expression of miRNA between the high-invasive (MKN28-M and SGC7901-M) and low-invasive potential (MKN28-NM and SGC7901-NM) cell sublines were detected by miRNA microarray. According to miRNA bioinformatical analysis, We focused on identifying miR-218 because it might be involved in both tumorigenesis and metastasis. QRT-PCR was carried out to verify the differential expression of candidate miRNAs.3. The expression levels of miR-218 in 40 GC tissues and corresponding non-tumor mucosa were detected by qRT–PCR. Correlations between the miR-218 expression level and clinicopathologic characteristics of GC were analyzed.4. miR-218 was overexpressed in MKN28-M with a miR-218 expression vector (pGenesil-1-miR-218) or silenced in MKN28-NM cells with antisense oligonucleotide respectively. The effect of miR-218 expression on cell growth in vitro and tumorigenicity in vivo were determined by MTT assay and nude mice.Transwell migration and invasion assays and tail vein injection metastasis assay were performed to observe the effect of miR-218 expression on metastasis of GC.5. To ascertain how the low expression of miR-218 contributes to the migration and metastasis of GC, we searched for its potential regulatory targets by computative predicting tools, including miRanda, Pictar, and TargetScan. Although hundreds of different targets were predicted, those involved in migration or invasion may be critical for the pathological functions of miR-218. We then performed a functional classification of the predicted targets using the DIVID program (http://david.abcc.ncifcrf.gov/ ). Of these genes, Robo1 is regarded as a proto-oncogene and harbors migration-promoting activity, suggesting that Robo1 could be a possible target for miR-218.6. To determine whether Robo1 is one of miR-218 target genes, we analyzed the expression of miR-218 and Robo1 mRNA in GES, non-invasive (MKN28-NM and SGC7901-NM), and invasive (MKN28-M and SGC7901-M) GC cells by qRT–PCR. Then we observed the Robo1 mRNA and protein levels when miR-218 was upregulated by transfecting pGenesil-1-miR-218 in MKN28-M cells or was knocked down by transfecting antisense oligonucleotide in MKN28-NM cells. The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry in 40 cases of GC and matched adjacent normal tissues which also were used in clinicopathological studies, as well as 29 matched metastases.7. We investigated the binding site of miR-218 in the Robo1 mRNA 3′-UTR by bioinformatics, and constructed a luciferase reporter (Luc-Robo1) in which the fragment containing the nucleotide base sequences 971-978 of the Robo1 3′-UTR complementary to miR-218 were inserted into the pMIR-REPORT miRNA expression reporter vector. Correspondingly, we also generated both the mutant reporter (Luc-Robo1-mu), in which the first 6 nucleotides in the miR-218 seed-region complementary sites were deleted, and the control reporter, which contained a non-related fragment of cDNA (Luc-Ctrl). miR-218 plasmids were co-transfected with Luc-Robo1 or Luc-Robo1-mu or Luc-Ctrl into MKN28-M cells for assessing the impact of miR-218 on the expression of Robo1. To test whether Robo1 is functionally regulated by miR-218, we generated a Robo1 expression construct containing only a fragment of the predicted miR-218 binding site and Robo1 mutant expression vector entirely lacking the 3′-UTR. We also made the Robo1 siRNA. MKN28-M-miR-218 cells, which stably expressed miR-218 ectopically, were transiently transfected with the Robo1 construct or the mutant construct (with no miR-218 binding site), and MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA.8. qRT-PCR was employed to examine the expression of the miR-218-1 precursor, the miR-218-2 precursor, mature miR-218, Slit2 mRNA, and Slit3 mRNA in the GC tissues used in the survival analysis. Statistical analysis of the correlation coefficient of the qRT-PCR results uncovered the reason for the downregulation of miR-218 in GC.【Results】1. The metastatic properties of each cell subline were characterized in vitro and in vivo. The results shown that the migration and invasion abilities of MKN28-M was approximately 4-fold greater than that of MKN28-NM cells. In the in vivo studies, tumor cell metastasis was observed in nude mice. Almost no metastatic GC cells were detected in the lungs or livers of nude mice at 10 weeks after injection of MKN28-NM cells, whereas most of the mice injected with MKN28-M cells displayed obvious lung or liver metastases. Similar results were observed for SGC7901-M and SGC7901-NM cells. No significant differences in cell proliferation or cell-cycle distribution were observed among these cell sublines.2. The microarray results revealed that the expression of 124 miRNAs significantly differed between the highly invasive variant MKN28-M and the non-invasive cell subline MKN28-NM. Of these, 83 were upregulated and 41 were downregulated. Compared with SGC7901-NM, 62 miRNAs were differentially expressed in the SGC7901-M cell subline, including 47 downregulated and 15 upregulated miRNAs. In total, 11 miRNAs were found to be upregulated and 34 miRNAs were downregulated in both MKN28-M and SGC7901-M cells. Of the 45 differentially regulated miRNAs, miR-218 was one of those that displayed significantly differential expression. miR-218 has been reported to be downregulated in cervical cancer, GC, lung cancer and prostate cancer, indicating possible involvement in both oncogenic transformation and tumor metastasis. However, miRNA-218 was only one of the many potential miRNAs of interest in cancers. In this study, miR-218 has been investigated in much greater detail. To validate the microarray results, we assessed miR-218 expression in the GC cell sublines previously mentioned and in the immortal gastric epithelial cell line GES by qRT–PCR. It was found that miR-218 expression was significantly decreased in MKN28-M and SGC7901-M cells and was lower in all four GC cell sublines compared to immortalized human gastric epithelial GES cells. Furthermore, we compared miR-218 expression between the primary GC tumor and the metastatic lymph nodes in 10 patients with stage III/ⅣGC using qRT–PCR. The results showed that mature miR-218 levels were significantly decreased in 7 out of 10 metastatic lymph nodes.3. The data of miR-218 expression in GC tissues verified that the miR-218 expression level in GC (-13.81±0.15, mean±SE) tissues was significantly lower than that in non-neoplastic mucosa (-11.62±0.15, mean±SE) (P < 0.0001, t = 10.62, paired t-test). Analysis of correlation between the miR-218 expression level and clinicopathologic characteristics of GC showed that there were statistically significant associations between the miR-218 expression level and clinical stage and between the miR-218 expression level and GC metastasis. The median expression of miR-218 was -14.25±0.17 in the 22 cases with advanced stage (stage III andⅣ) disease, whereas the median expression was -13.27±0.20 (P = 0.0010, Mann-Whitney test) in the 18 cases with early-stage (stages I and II) disease. In the 29 cases of GC with lymph node metastasis, the median expression of miR-218 was -14.09±0.16, which was significantly lower than the median expression (-13.07±0.24 ) in the 11 non-metastatic GC cases (P = 0.0036). The expression of miR-218 in GC patients did not correlate with age, gender, tumor size, or cell differentiation. Moreover, we examined whether the level of miR-218 expression was associated with survival in patients with GC. Patients were subsequently divided into low expression (n = 20) and high expression groups (n = 20) based on miR-218 levels greater or less than the mean (-13.81). Kaplan–Meier survival analyses revealed that patients whose primary tumors displayed low expression of miR-218 had a shorter median survival time. The three-year survival rate of patients with low miR-218 expression was 30%, which was significantly lower than the survival rate in patients with high miR-218 expression (65%; P = 0.0012, log-rank test).4. Functional studies of miR-218 revealed that ectopic expression of miR-218 resulted in an approximately three-fold reduction in migration and invasiveness. Consistent with the above data, there was a three- to four-fold increase in cell migration and invasiveness when we silenced miR-218 with an antisense oligonucleotide inhibitor in the MKN28-NM cells. To test if inhibition of tumor invasion by miR-218 is caused by impairing the invasive ability of tumor cells, we excluded the effect of miR-218 on the proliferation and cell cycle distribution of GC cells. Over-expression of miR-218 did not affect the proliferation and the cell cycle distribution of MKN28-M cells in vitro. To further investigate the inhibition of in vivo tumor metastasis by miR-218, we implanted MKN28-M-miR-218 cells that were stably expressing miR-218 or control cells into nude mice through the lateral tail vein. Lung and liver metastasis of GC was apparent in mice injected with MKN28-M-miR-control cells. In contrast, few metastatic tumors were detected in mice injected with MKN28-M-miR-218 cells. Furthermore, we simultaneously observed the growth of the primary tumors and the incidence of distant metastasis in the nude mice injected subcutaneously with MKN28-M-miR-218 cells or control cells. The results showed lung or liver metastasis was apparent in 3 out of 10 mice injected with MKN28-M-miR-control cells; in stark contrast, no metastasis were found in mice injected with MKN28-M-miR-218 cells.5. Bioinformatics predicted that Robo1 may be a target for miR-218. To further test the hypothesis, we analyzed the expression of miR-218 and Robo1 in GC cell lines. The results showed a negative correlation between the levels of miR-218 and Robo1 mRNA in these cells. Furthermore, we observed that Robo1 mRNA and protein levels were decreased when miR-218 was expressed by pGenesil-1-miR-218 in MKN28-M cells. The reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry in 40 cases of GC, in matched adjacent normal tissues that were also used in clinicopathological studies, and in 29 matched metastases. The results show that Robo1 was upregulated in GC, especially in metastatic GC, in which miR-218 has a relatively low expression.6. Luciferase reporter assay showed showed that the luciferase activity in the Luc-Robo1-transfected cells was significantly decreased compared to the luciferase activity in the mutant and negative control cells (P < 0.05), suggesting that miR-218 reduced the luciferase activity of Luc-Robo1 but had no effect on Luc-Robo1-mu. Furthermore, MKN28-M-miR-218 cells transfected with the Robo1 mutant construct showed a 3.8-fold increase in invasion ability compared to cells transfected with the Robo1 construct. These results indicate that introduction of mutant Robo1 cDNA that lacked the miR-218 binding site into the miR-218-overexpressing cells reversed the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion, which fell to levels similar to those observed after transfection with the miR-218-expressing vector.7. miR-218 is an intronic miRNA. Two genes code for mature miR-218, miR-218-1 and miR-218-2, which are located within intron 15 of Slit2 and intron 14 of Slit3, respectively. The intronic location of the two miR-218 genes prompted us to ask whether miR-218-1 and miR-218-2 are transcribed together with their host gene mRNAs. To test this hypothesis, we used qRT-PCR to examine the expression of the miR-218-1 precursor, the miR-218-2 precursor, mature miR-218, Slit2 mRNA, and Slit3 mRNA in the GC tissues used in the survival analysis. Statistical analysis of the correlation coefficient of the qRT-PCR results revealed a significant positive correlation between the levels of Slit2 mRNA and miR-218-1 and between the levels of Slit3 mRNA and miR-218-2. These results indicate that the miR-218 coding genes, miR-218-1 and miR-218-2, are transcribed together with their host genes, Slit2 and Slit3, respectively. A significant positive correlation between the levels of miR-218 and miR-218-2 was seen in GC; however, no such correlation was seen between the levels of miR-218 and miR-218-1. These results indicate that downregulation of miR-218 in GC is promoted by a decrease in miR-218-2, but not in miR-218-1. Consistent with this conclusion, Slit3 expression was significantly reduced in GC (-22.43±0.21, mean±SE) compared to normal gastric tissue (-20.79±0.23, mean±SE), (P < 0.0001, t = 7.67, paired t-test) (Figure 6F), whereas Slit2 expression was not significantly different (P = 0.0772, paired t-test).【Conclusions】1. We successfully established a GC metastatic model, which would provide a research platform for study of GC metastasis.2. Many miRNAs were differentially expressed between the highly invasive variants and the low-invasive cell sublines of GC, suggesting that miRNAs play important roles in GC metastasis。 3. miR-218 was downregulated in GC, especially in metastatic GC. Ectopic expression of miR-218 inhibited tumor cell invasion and metastasis by targeting the Robo1 receptor.4. miR-218 is part of a regulatory circuit involving the Slit-Robo1 pathway. miR-218 coding genes, miR-218-1 and miR-218-2, are transcribed together with their host genes, Slit2 and Slit3, respectively. In metastatic tumor cells, miR-218 was suppressed along with Slit3, one of its host genes. Meanwhile, Robo1, one of several Slit receptors, is upregulated in response to the decrease in miR-218, which in turn induced a reactive upregulation of the Slit-Robo1 pathway through an interaction with Slit2, thus facilitating tumor cell migration and invasion.