MiR-1202 was up-regulated in tissues of GC patients with metastasis
We first carried out miRNA microarray analysis on three GC tissues (clinical phase I) with metastasis and three tissues without metastasis. As shown in Fig. 1A, 19 miRNAs exhibited significantly different expression levels between metastatic group and non-metastatic group, including miR-23b, miR-24, miR-1225-5p, miR-1207-5p, miR-4281, miR-1202, miR-150, miR-30c, miR-130a, miR-4270, miR-142-3p, miR-132, miR-486-5p, miR-4291, miR-766, miR-3651, miR-140-5p, miR-139-5p, and miR-582-5p.
To further confirm this finding, other 20 GC tissues with metastasis and 20 GC tissues without metastasis were utilized in the qRT-PCR experiment. It was shown that 10 out of the 19 miRNAs were differentially expressed in the two groups with miR-1202 being the most significant (Fig. 1B). Therefore, we focused our attention on the functional study of miR-1202 in GC cells in the following work.
MiR-1202 played a regulatory role in GC cell migration
To assess the biological function of miR-1202 on regulating cell growth and migration, SGC-7901 cells were infected with lentivirus expressing pre-miR-1202 (LV-miR-1202) and control (LV-miR-Ctl) lentivirus at a MOI of 10. The infection efficiency was confirmed by fluorescence microscopy. More than 80% of cells had green fluorescence, indicating that the infection efficiency was high enough for the following experiments (Fig. 2A).
Flow cytometry analysis was first performed and it was found that there was no statistical difference in cell cycle distribution (supplementary Figure 1A). A similar result was gained from a cell growth curve in cell proliferation assay (supplementary Figure 1B).
Based on the differential expression pattern of miR-1202 between metastatic GC tissues and non-metastatic GC tissues, we further assessed the potential function of miR-1202 on cell migration using the transwell assay. Fig. 2B-C shows that, compared with those of the control group, SGC-7901 cells infected with LV-miR-1202 showed an increased migration ability. Furthermore, we performed miR-1202 inhibition experiments to confirm the potential role of miR-1202 in GC cells migration. The target sequence of the lentiviral vector of miR-1202-inhibitor (LV-anti-miR-1202) complemented mature miR-1202 and thus inhibited its function. The efficiency of AGS cells infected with LV-anti-miR-1202 was also confirmed by fluorescence microscopy (Fig. 2D). Consistently, the knockdown of miR-1202 did not significantly affect cell cycle and cell growth rate (Supplementary Figure 2A and 2B), but suppressed the cell migration ability of GC cells (Fig. 2E-F). These results suggested that miR-1202 was able to regulate cell migration ability in vitro in GC cells.
Overexpression of miR-1202 promoted distal pulmonary metastasis in nude mice
To further investigate the effect of miR-1202 in vivo, we selected SGC-7901 cells to perform the tumor xenograft and lung metastasis on BALB/c nude mice. The body weight and overall health status of the nude mice were assessed before mice were randomly divided into two groups. Equal numbers of cells overexpressing miR-1202 and control cells were injected subcutaneously into the mice and the tumor size was measured every three days. As displayed in Fig. 3A, the volumes of the tumors formed by SGC-7901 cells overexpressing miR-1202 and the control cell did not show statistical difference during the experimental period. Consistent with the tumor volumes, weights of tumors in the two groups were not statistically different, either (Fig. 3B-C).
The pulmonary metastasis assay was carried out to investigate the role of miR-1202 in tumor metastasis. Two groups of SGC-7901 cells were injected into nude mice through the lateral tail vein. After 2.5 months, the lungs of mice were isolated. While no metastatic tumor was observed in the lungs of all the control mice, four of the five mice injected with cells overexpressing miR-1202 had multiple visible lung metastases. The representative photos were shown in Fig. 3D. The results of H&E staining further confirmed lung metastases in the miR-1202 group (Fig. 3E). Collectively, the results suggested that miR-1202 had no effect on primary GC cell growth but promoted distal pulmonary metastasis in vivo.
Identification of miR-1202 target genes and bioinformatics analysis
The information about the regulatory target of miR-1202 is limited, we, therefore, utilized 9 miRNA target prediction databases, such as Oncomine database and GEO database, in order to get a better understanding of miR-1202 mechanism. As shown in Fig. 4A-B, 139 potential target genes of miR-1202 were selected. According to the KEGG pathway analysis and GO enrichment in DAVID, 4 KEGG pathways, 39 GO terms of biological processes (BP), 13 GO terms of cellular components (CC), and 14 GO terms of molecular function (MF) were identified. Four KEGG pathways and the top 10 GO annotations of each section were listed in Fig. 4C. According to the KEGG pathway results, 17 target genes were likely involved in the cytokine-cytokine receptor interaction, CAMs (cell adhesion molecules), valine, leucine and isoleucine degradation, and glutathione metabolism. In the GO enrichment, the target genes accumulated in the BP of cell adhesion, collagen fibril organization, and positive regulation of mitogen-activated protein kinase (MAPK) cascade, in the CC of the external side of the plasma membrane, melanosome, apical plasma membrane, and in the MF of peroxidase activity, signal transducer activity, and fatty-acyl-CoA binding.