MiR-1202 promotes cell migration and tumor metastasis in gastric cancer cells


 Introduction : Tumor metastasis significantly impacts the survival rate of gastric cancer (GC) patients. Increasing evidence has suggested that numerous microRNAs (miRNAs) are associated with tumor metastasis and could be potential candidate cancer biomarkers or therapeutic targets. Materials and Methods : Early GC cases were collected and divided into two groups according to the lymphatic metastasis (LM) situation. The microarray analysis was carried out to screen out differentially expressed miRNAs in the two groups, which were further evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The roles of miR-1202 on GC cells were determined through cell growth assay, cell migration assay in vitro and pulmonary metastasis assay in the mouse model. Bioinformatics analysis was used to explore the potential mechanisms of miR-1202-mediated biological effects. Results : MiR-1202 was first identified to be the most differentially expressed miRNA in GC patients with metastasis and those without metastasis. The overexpression of miR-1202 promoted GC cell migration in vivo whereas the knockdown of miR-1202 suppressed this process. However, miR-1202 did not affect GC cell growth or cell cycle distribution. The overexpression of miR-1202 promoted pulmonary metastasis and colonization after the tail-vein injection of GC cells. Pathways related to cell adhesion, collagen fibril organization, and positive regulation of mitogen-activated protein kinase (MAPK) cascade might be involved in miR-1202-mediated biological effects. Conclusion : Our results demonstrated a positive role of miR-1202 in regulating GC cell metastasis. MiR-1202 might be developed as a novel biomarker and a potential therapeutic target for GC metastasis.


Abstract
Introduction : Tumor metastasis signi cantly impacts the survival rate of gastric cancer (GC) patients.
Increasing evidence has suggested that numerous microRNAs (miRNAs) are associated with tumor metastasis and could be potential candidate cancer biomarkers or therapeutic targets. Materials and Methods : Early GC cases were collected and divided into two groups according to the lymphatic metastasis (LM) situation. The microarray analysis was carried out to screen out differentially expressed miRNAs in the two groups, which were further evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The roles of miR-1202 on GC cells were determined through cell growth assay, cell migration assay in vitro and pulmonary metastasis assay in the mouse model. Bioinformatics analysis was used to explore the potential mechanisms of miR-1202-mediated biological effects. Results : MiR-1202 was rst identi ed to be the most differentially expressed miRNA in GC patients with metastasis and those without metastasis. The overexpression of miR-1202 promoted GC cell migration in vivo whereas the knockdown of miR-1202 suppressed this process. However, miR-1202 did not affect GC cell growth or cell cycle distribution. The overexpression of miR-1202 promoted pulmonary metastasis and colonization after the tail-vein injection of GC cells. Pathways related to cell adhesion, collagen bril organization, and positive regulation of mitogen-activated protein kinase (MAPK) cascade might be involved in miR-1202-mediated biological effects. Conclusion : Our results demonstrated a positive role of miR-1202 in regulating GC cell metastasis. MiR-1202 might be developed as a novel biomarker and a potential therapeutic target for GC metastasis. Background GC is a common malignant tumor of the digestive system worldwide. The incidence of GC ranks fourth in cancers, and its mortality rate ranks second worldwide [1]. In the recent years, the incidence of GC has steadily declined mainly bene ting from improvements in sanitation, nutritional standards, and Helicobacter pylori treatment rate. However, due to its pathological complexity and diversity, the early diagnosis and survival rate of GC are still not ideal. In China, the incidence and mortality of GC are the second highest in all the cancers [2]. Because of the lack of speci c clinical pathological features in earlystage GC, patients diagnosed with GC are mostly already in the advanced stage and accompanied by metastasis. If it could be predicted whether GC will metastasize in early stage, speci c treatments would be taken so as to improve the survival rate of GC patients [1,[3][4][5]. Therefore, the identi cation of speci c biomarkers related to GC metastasis has a promising prospect in clinical therapy.
In the large amounts of RNAs produced by the genome, only the open reading frame of mRNAs is "coding sequence", while the rest mRNAs and other untranslated transcripts are called non-coding RNAs (ncRNAs). NcRNAs include "housekeeping" RNAs, like ribosomal RNA (rRNA), transfer RNA (tRNA), small cytoplasmic RNA (scRNA), among others. On the basis of the lengths, ncRNAs can be divided into long ncRNAs and short ncRNAs. MiRNAs are small RNAs with about 22 nucleotides in length, encoded by endogenous genes. MiRNAs are important to gene expression regulation in plants, animals, and fungi, participating in post-transcriptional regulation of protein-coding genes via translational repression, mRNA cleavage, and other mechanisms. A single miRNA can downregulate several target genes by guiding argonaute proteins to its target sites, which are generally located in the 3'UTR of mRNAs [6]. Emerging evidence demonstrates that miRNAs play an important regulatory role in cancer. Generally, miRNAs associated with cancer are divided into two major types, namely tumor suppressive miRNAs and oncogenic miRNAs. Usually, the tumor suppressive miRNAs prevent tumorigenesis and tumor development by targeting and downregulating the expression of oncogenes, normally exhibit downregulated expression pro les in tumor. In contrast, the oncogenic miRNAs' expressions are often increased in tumor and can inhibit tumor suppressor genes, directly or indirectly.
Recent studies have found that a number of miRNAs are involved in GC development. These miRNAs play diverse roles in cell apoptosis, proliferation, and metastasis of GC. For instance, miR-21, which is overexpressed in almost all the solid tumors, promotes tumor proliferation, migration, and invasion by targeting PTEN in GC [7,8]. Exosomal miR-21 induced mesothelial-to-mesenchymal transition and promoted cancer peritoneal metastasis by targeting SMAD7 [9]. MiR-375 was found to inhibit cell proliferation by targeting JAK2 and was frequently downregulated in GC [10].
MiR-1202 was rst reported in major depressive disorder (MDD). The expression of miR-1202 is low in the blood of MDD patients [11][12][13]. However, little is known about the biological function of miR-1202 during GC development. Here, we reported that miR-1202 was signi cantly upregulated in early-stage GC patients with metastasis compared with those without metastasis. To investigate the effect of miR-1202 on GC cells, lentivirus was utilized to establish stable cell lines. Nevertheless, miR-1202 did not affect cell cycle distribution or cell growth rate of GC cells. Cell migration assay in vitro and tumor metastasis assay in vivo demonstrated a positive regulatory role of miR-1202 in GC metastasis. Based on the data acquired from Oncomine, Gene Expression Omnibus (GEO), and 9 online miRNA targets prediction tools, we performed bioinformatics analysis to further explore the underlying mechanism of miR-1202's role in GC metastasis.

Clinical samples
The para n-embedded tumor specimens from 199 early GC cases were collected from Zhongshan Hospital a liated to Fudan University (Shanghai, China). These specimens were obtained by open or laparoscopic surgery. According to the neoplasm staging and LM situation by the pathology results, we divided specimens into two groups, 30 cases in the with LM group, and 169 cases in the non-lymphatic metastasis (NLM) group. This study was approved by the Ethics Committee of Zhongshan Hospital a liated to Fudan University.

Lentivirus infection
Recombinant plasmids carrying either pre-miR-1202 sequence or short hairpin (shRNA) targeting mature miR-1202 were subcloned at Shanghai Genechem Co., Ltd. (China). The lentiviral particles and the control lentivirus were further packaged in parallel. Cells were seeded into a 6-well plate (1 × 10 5 cells/well) and lentivirus infection was performed when the cells reached ~80% con uence. The multiplicity of infection (MOI) of SGC-7901 and AGS cells was 10.

RNA isolation
Total RNA was extracted from tissues or cells with Trizol reagent according to the manufacturer's instructions (Invitrogen, USA).

Microarray analysis
The total RNA from GC tissues was used for miRNA chip analysis at Genechem. The threshold we used to screen differentially expressed miRNAs with statistical signi cance was fold change ≥ 1.5 and P-value ≤ 0.05.

Quantitative reverse transcription PCR (qRT-PCR)
A total of 1 μg RNA was used for reverse transcription in the reaction mixture containing oligo (dT) primer, reverse transcriptase (Takara, Japan), U6 and miR-1202 stem-loop RT primers (Ribobio, China). The SYBR Green Supermix kit (Takara, Japan) was used in the qRT-PCR assay according to the manufacturer's instructions. PCR reaction was carried out in triplicates for each gene, while blank controls were performed in parallel to rule out contamination. The miR-1202 gene expression was normalized to U6.
The relative expression level of miR-1202 and others was calculated by the formula 2 -ΔΔCt [14]. Cycle parameters were 95℃ for a 3 min hot start, and 50 cycles of ampli cation: 95℃ for 10 s, 58℃ for 15 s and 72℃ for 20 s. Melting curve analysis was used to con rm the speci city of the PCR product.
Tumor xenograft and metastasis assays in vivo For the xenograft growth of subcutaneous animal assay, the tumor size was measured with a caliper every three days. The tumor volume was calculated using the following formula: length × width 2 × 0.5.
When the average tumor volume reached approximately 1000 mm 3 , all efforts were made to minimize suffering and mice were euthanized by carbon dioxide in a closed cage. The tumors were then excised and weighed. For in vivo metastasis assay, after 2.5 months' injection through the tail vein, the mice were euthanized, and the lung tissues were dissected and sectioned. Metastatic tumors were detected by hematoxylin and eosin (H&E) staining.
Differently expressed genes related to GC were also identi ed by using Oncomine (https://www.oncomine.org/resource/main.html) and GEO database (www.ncbi.nlm.nih.gov/geo/). Based on the interaction of miRNA-related and GC-related genes mentioned above, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were conducted by using the online tool (https://david.ncifcrf.gov/).

Statistical analysis
A two-tailed Student's t-test was applied to evaluate group-level differences. We considered two groups with a P-value < 0.05 (*) to be different and with a P-value < 0.01 (**) to be statistically signi cant.
To further con rm this nding, 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 signi cant (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 e ciency was con rmed by uorescence microscopy. More than 80% of cells had green uorescence, indicating that the infection e ciency was high enough for the following experiments ( Fig. 2A).
Flow cytometry analysis was rst 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 nonmetastatic 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 con rm 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 e ciency of AGS cells infected with LV-anti-miR-1202 was also con rmed by uorescence microscopy (Fig. 2D). Consistently, the knockdown of miR-1202 did not signi cantly 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 ve 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 con rmed 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.

Identi cation 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

Discussion
The occurrence and development of GC are a multi-stage process in uenced by numerous environmental and genetic factors. Environmental factors include unhealthy eating habits, excessive smoking and drinking, obesity, occupational exposure, Helicobacter pylori infection, and Epstein-Barr virus infection [1]. The in uence of genetic factors on tumorigenesis includes the accumulation of gene mutations, epigenetic alteration such as chromatin remodeling, histone modi cation, and ncRNAs action. Recently, the role of ncRNAs in GC development has gained more and more attention.
In our study, we rst performed a miRNA expression pro ling of metastatic and non-metastatic early GC samples. And it was found that miR-1202 was the most signi cantly differentially expressed miRNA associated with GC metastatic capability. MiR-1202 is a primate-speci c miRNA [11]. Although a certain level of miR-1202 can be detected in various human tissues, its expression is signi cantly enriched in the human brain. Previous studies concentrating on miR-1202 and MDD suggested that miR-1202 could be used as a potential biomarker of MDD with high sensitivity and speci city [15].
Several previous studies also reported the expression pattern of miR-1202 in different cancers but the role of miR-1202 during cancer development is only beginning to be explored. Some studies have suggested that miR-1202 is highly expressed in cancer tissues and may act as an oncogenic miRNA. For instance, Chen H. et al. investigated the expression level of miR-1202 in endometrial tissue from endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) patients. A high level of miR-1202 was found in EC compared with that of AEH [16]. In contrast, miR-1202 was downregulated in clear cell papillary renal cell carcinoma (CCPRCC) and cervical cancer [17,18]. Some other studies have suggested that miR-1202 might be associated with cancer metastasis. For instance, compared with that of papillary thyroid carcinoma (PTC) patients without lymph node metastasis (LNM), a higher expression level of miR-1202 was found in PTC patients with LNM [19]. Tokuhisa M. et al. found that miR-1202 was expressed in exosomes of malignant ascites samples, and it might serve as a biomarker of peritoneal dissemination [20]. Du B. et al. explored the role of miR-1202 in hepatocellular carcinoma (HCC), they found that elevated expression of miR-1202 weakened HCC cells migration and invasion abilities [21]. In glioma cells, it was found that miR-1202 inhibited cell proliferation and induced endoplasmic reticulum stress and apoptosis [22]. In summary, previous studies of miR-1202 in cancers found that miR-1202 played different roles in different cancer types and different clinical stages, which might be partially attributable to cancer heterogeneity.
After we revealed a correlation between miR-1202 expression and GC metastasis, cell experiments in vitro and animal experiments in vivo were used to further analyze the role of miR-1202 in GC. Our in vitro cell culture results showed that the overexpression of miR-1202 promoted GC cell migration but knockdown of miR-1202 suppressed cell migration. Our in vivo animal model results con rmed the function of miR-1202 in GC metastasis. The overexpression of miR-1202 in GC cells had no impact on primary tumor growth but promoted distal pulmonary metastasis.
To explore the molecular mechanism underlying miR-1202 functions, we performed a comprehensive bioinformatics analysis. We chose 139 genes on the basis of 9 miRNA-related databases, Oncomine, and GEO database for further analysis. According to the results of KEGG pathway and GO enrichment, several pathways and GO terms are potentially essential to GC metastasis. Certain hub target genes including NCAM1 are probably involved in the metastasis of GC. NCAM1 encodes a cell adhesion protein that is a member of the immunoglobulin superfamily. In 2015, Shi Y. et al. found a decreased level of NCAM1 in GC tissues, and NCAM1 protein is negatively associated with GC invasion [23]. In another research, match pairs of peritoneal tissues and peritoneal metastasis in GC patients were collected for whole-exome sequencing to characterize the mutation spectrum of GC with peritoneal metastasis and thus provide a basis for nding new biomarkers and treatment targets. The result showed that NCAM1 was the only mutated gene in metastatic cells and was expected to become a treatment target [24]. Our future study will try to explore the regulatory relationship between NCAM1 and miR-1202, as well as the mechanism of miR-1202-mediated GC metastasis. Declaration of Helsinki and approved by the institutional review boards of the centers participating in this study. All patients gave their signed informed consent for this study.

Consent for publication
Not applicable Availability of data and material The datasets used during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that no con ict of interest exists.

Supplementary Files
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