Endogenous and Extracellular Roles of a Tumor Suppressor miR-379-5p in Gastric Cancer Open Access

Gastric cancer remains to be the third leading cause of cancer-associated death worldwide [1]. More than two-thirds of the worldwide burden of gastric cancer is concentrated in Eastern Asia [2, 3]. Nowadays, gastrectomy with adjuvant chemotherapy is applied for most of the patients with gastric cancer [4, 5]. However, a large number of the patients will develop hematogenous metastasis subsequently even if they undergo a potential curative resection [6, 7]. Currently, most of these metastatic patients are diagnosed until they develop clinical signatures and/or diagnostic images. Therapies for these patients are limited, making the prognosis of patients with metastatic gastric cancer very poor [8]. Hence, metastasis is a fatal character of gastric cancer. Promising functional biomarkers for monitoring metastatic status of gastric cancer is in pressing need. Development of such biomarkers will be beneficial to predict development of metastasis of gastric cancer in order to adjust therapy, reduce mortality rate, and improve prognosis.

Numerous studies have revealed that dysregulation of miRNAs is actively involved in gastric cancer development [9]. MiRNAs belong to a group of small noncoding RNAs around 18–25 nucleotides in length. They bind to complementary sequences in the 3′-untranslated regions of target mRNAs to induce degradation or translational repression [10, 11]. MiRNAs are tissue specific, and even cell specific within those tissues. They are potentially useful for diagnosis, predicting clinical outcome, or acting as therapeutic targets in patients with cancer [12‒14]. The unique pattern of microRNAs in gastric cancer provides the possibility of applying miRNAs as biomarkers and therapeutic targets for monitoring gastric cancer [15, 16].

In addition, previous studies indicated that metastasis is an early event in cancer progression. Primary tumors create a favorable microenvironment in secondary organs and/or tissue sites for subsequent metastases. This is called the “seed” (refer to pre-metastatic niche) and “soil” (refer to secondary sites) theory. To transfer the “seed” to its appropriate “soil,” primary tumors secrete extracellular vesicles [17, 18]. Exosomes are an important type of extracellular vesicles. Intriguingly, exosomes released from primary cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function [19, 20].

In our previous study, we found that expression of exosomal miR-379-5p was significantly higher in serum of the patients with gastric cancer who developed hematogenous metastasis subsequent of resection. However, expression of endogenous miR-379-5p was significantly downregulated in gastric cancer tissue samples. Expression of exosomal miR-379-5p in cell culture medium was much higher than its endogenous expression in gastric cancer cells. This suggested that miR-379-5p might be translocated from gastric cancer cells to cell culture medium by exosomes [21].

In this study, we investigated the functional roles of miR-379-5p in primary gastric cancer. We also evaluated the expression of miR-379-5p in gastric cancer cell lines and cell culture medium. The culture medium of cell lines established from stomach (original site) was applied for culturing other cell lines (distant sites), to investigate whether exosomal miR-379-5p in the culture medium could be translocated in distant cells. This study will contribute to elucidate the mechanism of transportation of miRNAs in cancer development, as well as to provide a potential therapeutic target for gastric cancer.

Fifty-three pairs of human gastric cancer and non-tumor tissue samples were collected at Queen Mary Hospital, Hong Kong. All of the tumor tissue samples were validated to be malignant by experienced pathologist. None of the patients received preoperative treatment. All samples were immediately frozen in liquid nitrogen and stored at −70°C.

Cell lines AGS, SNU1, HEK293T (ATCC, Rockville, MD, USA), MKN45 (RIKEN, Japan), and BCG23 (from Beijing Cancer Institute) were used in this study. AGS, SNU1, BCG23 were established from gastric cancer tissues of original site (stomach). MKN45 was established from gastric cancer metastasized to liver. HEK293T was isolated from human embryo kidney tissue. It was immortalized but not malignant. Cells were cultivated in RPMI 1640 Medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% exosomes-depleted fetal bovine serum (SBI, System Biosciences, USA). All cells were incubated at 37°C in a humidified incubator which contains 5% CO2.

The specific miR-379-5p mimic (miRIDIAN^™ microRNA Mimics, C-300687-05-0020) and scrambled control were purchased from Dharmacon^™ (USA). Here, 10⁵ cells were seeded into a 6-well plate a day in advance of transfection and transfected with 20 nm mimic or scrambled control using HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. Transient transfected cells were applied for evaluation of expressions and functional assays.

Around 20 mg of each tissue sample or 10⁶ cells of each cell line were applied for miRNA extraction via miRNeasy Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. The concentrations of miRNA samples were quantified by NanoDrop 1000 (NanoDrop, Wilmington, DE, USA). Overall, 500 ng of total RNA from each sample was utilized for reversed transcript (RT).

Exosomal RNAs were extracted using the SeraMir Exosome RNA Kit (SBI, Mountain View, CA, USA) according to the manufacturer’s instructions. The quality and quantity of the RNAs were measured by NanoDrop 1000. The same amount of culture medium was applied for exosomal miRNA extraction. And the same amount of miRNAs according to NanoDrop concentration was applied for RT. So, the measurements among different groups were comparable.

U6 was applied as an internal control for miR-379-5p in tissue samples and cell lines. MiR-16-5p and MiR-93-5p were applied as internal controls for exosomal miR-379-5p.

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was applied for cell proliferation assay. A total of 5,000 cells/well were seeded in 96-well plates with transfection of miR-379-5p mimic or scrambled control. After 24 h and 48 h, the culture medium was discarded and restained with MTT (5 mg/mL) for 3 h. The absorbance was measured at 570 nm on a Multiskan^™ FC Microplate Photometer (Thermo Fisher Scientific).

Cells with transfection of miR-379-5p mimic or scrambled control for 24 h were harvested and suspended in RPMI 1640 Medium. A trans-well culture insert with 8 μm pore size membrane for 24-well plate (Cat no. 353097, Falcon, NY, USA) was used to analyze the migration activity. In total, 30,000 suspensions of cells in 300 μL of serum-free RPMI 1640 Medium were seeded into the upper inserted chamber, 700 μL of RPMI containing 10% fetal bovine serum was added to the well, and the plate was incubated at 37°C for 48 h. After incubation, the inner wall of the chamber was wiped with swabs to remove un-migrated cells. The outer wall of the chamber was gently rinsed with PBS and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Finally, the membrane was rinsed and allowed to air-dry. The stained membrane was photographed, and the number of migrated cells was counted. Matrigel-coated trans-well chambers with 8 μm pore size membrane for 24-well plate (Corning, NY, USA) were applied in invasion assay, and the following steps were performed as the same of migration assay.

Cells with transfection of miR-379-5p mimic or scrambled control for 48 h were harvested and applied for cell adhesion assay (CBA-070, Cell Biolabs, CA, USA). A 48-well plate with wells coated with collagen I, collagen IV, laminin, fibronectin, fibrinogen, or BSA was used. A total of 50,000 cells in 150 μL of serum-free RPMI 1640 Medium were seeded into the wells coated with extracellular matrix (ECM). The same number of cells was also seeded into wells coated with BSA as negative control. The plate was incubated at 37°C for 1 h, followed by aspiration of the medium from each well and washed by PBS to remove the un-adhesive cells. The wells were stained with the Cell Stain Solution for 10 min. The adhesive stained cells were photo-captured. The stained cells were extracted by the Extraction Solution. Finally, each extracted sample was transferred to a 96-well plate and measured the O.D. 570 nm on a Multiskan^™ FC Microplate Photometer (Thermo Fisher Scientific).

Potential targets of miR-379-5p were screened by TargetScan and miRDB. There were 124 predicted targets from TargetScan and 264 from miRDB. The overlap numbers of potential targets were 55 in total. Clusters of functions and signal pathways of the potential targets were analyzed by PantherDB.

Western blot was performed for validation of IGF-1R as a target of miR-379-5p. Briefly, protein was extracted and lysed by RIPA Buffer (Sigma Chemical Co., St Louis, MD, USA). Samples containing equal amounts of protein were separated by SDS-PAGE and electro-blotted onto Immobilon-P Transfer Membrane (Applied Biosystems). The membrane was blocked with 5% no-fat milk, followed by incubation with antibody specific for anti-IGF-1R (1:500, Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (1:10,000, Cell Signaling Technology, Beverly, MA, USA), respectively. Blots were then incubated with anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia, Cleveland, OH, USA) accordingly. The signals were captured by Fiji film development system.

Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) 24.0 for Windows (SPSS Inc., Chicago, IL, USA). Student’s t test was used to analyze the results of cell proliferation, cell migration, and invasion assays, expressed as mean ± SD. Wilcoxon sign rank test was used to analyze miR-379-5p in paired gastric cancer tissue samples. All p values were two-sided, and a value of p ≤ 0.05 is considered statistically significant.

Expression of miR-379-5p was evaluated in 53 paired gastric cancer/adjacent non-tumor tissue samples by RT-qPCR. The result showed miR-379-5p was significantly downregulated in gastric cancer tissues comparing with non-tumor samples (N = 53 pairs, downregulated in 37 pairs out of 53 pairs, 69.8%, *p = 0.0288, Fig. 1a). Moreover, miR-379-5p was significantly downregulated in gastric cancer cell lines AGS, BCG23, SNU1, and MKN45, comparing with its average expression in non-tumor tissues (Fig. 1b). The results indicated expression of miR-379-5p was significantly decreased in gastric cancer.

MiR-379-5p mimic was transfected into gastric cancer cell lines established from original site (stomach), including AGS and BCG23. Expression of miR-379-5p in the transfected cell lines was evaluated by RT-qPCR. The result indicated that expression of miR-379-5p in AGS and BCG23 was dramatically increased and at least lasted for 72 h (**p < 0.01, Fig. 2a). The increase of miR-379-5p was specific as there was almost no increase of other miRNAs in the same transfection (**p < 0.01, online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000546620). The transfected cell lines were then subjected to functional assays. Cells with miR-379-5p mimic or scrambled control were subjected to MTT assay to evaluate cell proliferation. The result showed enforced expression of miR-379-5p significantly inhibited gastric cancer cell proliferation by around 20% for BCG23 in 24 h and for AGS in 48 h (*p < 0.05, **p < 0.01, Fig. 2b).

Cells were pretreated with mitomycin to inhibit cell proliferation before subjecting to migration/invasion assay. The migrated cells were stained and counted under a microscopy. The representative images of stained cells with scrambled control or mimic of miR-379-5p are shown in Figure 2c. Migrated cell numbers are also indicated in Figure 2c. The migration assay indicated that migrated gastric cancer cells were significantly decreased by 50%–60% with enforced expression of miR-379-5p (*p < 0.05, **p < 0.01, Fig. 2c). For invasion assay, the trans-well chambers were coated with Matrigel to mimic the situation of basement membrane in tumor microenvironment. The invasion assay showed that the invasive gastric cancer cells were significantly decreased with enforced expression of miR-379-5p in AGS and BCG23 cells by around 40% (*p < 0.05, Fig. 2d). The results revealed that enforced expression of miR-379-5p inhibited gastric cancer cell proliferation, migration, and invasion.

On the other hand, the transfected cells were subjected to adhesion assay of ECM, including collagen I, collagen IV, laminin, fibronectin, and fibrinogen, with BSA as a blank control. Cells with miR-379-5p mimic or scrambled control were allowed to attach to ECM for an hour. Then, the cells were stained and washed. The stained cells were photographed under a microscopy and a camera (Fig. 3a). After that, the stained cells were extracted by solution to evaluate the colorimetric O.D. value. The result showed that miR-379-5p mimic enhanced AGS and BCG23 cell attachment to collagen I and collagen IV (Fig. 3b).

The above results showed that enforced expression of miR-379-5p inhibited gastric cancer cell proliferation, migration, and invasion. In contrast, overexpression of miR-379-5p enhanced cell attachment to collagen I and collagen IV. It indicated that miR-379-5p functioned as a tumor suppressor in gastric cancer.

Targets of miR-379-5p were predicted by TargetScan and miRDB. There were 124 predicted targets from TargetScan and 264 from miRDB. The overlap numbers of potential targets were 55 in total (online suppl. Table 1). Clusters of functions and signal pathways of the potential targets were analyzed by PantherDB. The analysis revealed that functions of the targets were associated with cancer initiation and progression, such as cell growth and cell death, cellular localization, and movement of cell. There were also functions associated with cell secretion, such as vesicle targeting and vesicle-mediated transport (Fig. 4a).

Among the targets, insulin-like growth factor 1 receptor (IGF1R) was associated with gastric cancer cell proliferation and mobility. Protein expression of IGF1R in gastric cancer cells was evaluated by Western blot. The result indicated that protein expression of IGF1R could be significantly suppressed by miR-379-5p in AGS and BCG23 cells, comparing with scrambled control (Fig. 4b, left panel). Protein expression of IGF1R also indicated that it was highly expressed in gastric cancer tissue samples comparing with non-tumor tissue samples (Fig. 4b, right panel). This suggested that IGF1R was a downstream target of miR-379-5p in primary gastric cancer. Downregulation of miR-379-5p increased the expression of IGF1R and further led to enhancement of gastric cancer cell proliferation and mobility.

The endogenous expression of miR-379-5p in AGS, BCG23, and SNU1, as well as expression of exosomal miR-379-5p in culture medium of these cells, was evaluated by qPCR. Both miR-16-5p and miR-93-5p were used as internal controls. The expression of these two miRNAs was abundant and relatively consistent in cell lines and exosomes of cell culture medium. This indicated that these two miRNAs were highly expressed in cells and were secreted by exosomes from cells to cell culture medium. The result showed that the expression of exosomal miR-379-5p in cell culture medium was significantly higher than its endogenous expression in gastric cancer cells (**p < 0.01, Fig. 5a). It suggested that miR-379-5p was secreted by exosomes from gastric cancer cells into cell culture medium. This could be the reason of downregulation of miR-379-5p in gastric cancer cells.

MiR-379-5p mimic or scrambled control was transfected into AGS or BCG23 cells for 7 days. The expression of exosomal miR-379-5p in culture medium of AGS or BCG23 was also evaluated by qPCR. The result showed that expression of exosomal miR-379-5p was significantly higher in the cell culture medium with overexpression of miR-379-5p comparing to that with scrambled control (**p < 0.01, Fig. 5b). It suggested that overexpression of miR-379-5p in gastric cancer cells might secrete miR-379-5p via exosomes to cell culture medium. The result was consistent with that expression of exosomal miR-379-5p was higher in cell culture medium than endogenous expression of miR-379-5p in gastric cancer cells.

As cell culture medium of AGS or BCG23 contained highly expressing exosomal miR-379-5p, such cell culture medium was used to culture MKN45 and a human embryonic kidney cell line HEK293T. The endogenous expression of miR-379-5p was evaluated in these cell lines cultured with AGS or BCG23 medium. The result showed that miR-379-5p was highly expressed (with lower Ct) in HEK293T with cell culture medium of AGS in week 1 and BCG23 since week 1 (*p < 0.01, **p < 0.05, Fig. 6a). The Ct values of miR-16-5p and miR-93-5p were also shown to indicate the alterations of miRNAs other than miR-379-5p (Fig. 6a). It suggested that miR-379-5p in exosomes from AGS or BCG23 culture medium could enter HEK293T cells to make higher endogenous expression of miR-379-5p. However, change of Ct value was not obvious in gastric cancer MKN45 cells (Fig. 6b). This suggested that attraction of exosomes into cells was different and depending on cell types.

In this study, we found that miR-379-5p was significantly downregulated in gastric cancer cell lines and tissue samples. On one hand, enforced expression of miR-379-5p inhibited gastric cancer cell proliferation, migration, and invasion. On the other hand, miR-379-5p mimic increased cell adhesion to certain ECM, such as collagen I and collagen IV. This evidence suggested that miR-379-5p was a tumor suppressor in primary gastric cancer. In our previous study, we found that exosomal miR-379-5p was highly expressed in patients with gastric cancer who developed hematogenous metastasis after operation, comparing with the patients with no distant metastasis [21]. In the current study, we further indicated that miR-379-5p was highly expressed in exosomes of gastric cancer cell culture medium than its endogenous expression in gastric cancer cells. This suggested that miR-379-5p could be secreted from gastric cancer cells into cell culture medium via exosomes. Endogenous expression of miR-379-5p, shown as Ct values, was not altered obviously in MKN45 cells (established from gastric cancer metastasized to liver), but in HEK293 cells (isolated from human embryo kidney tissue, immortalized but not malignant), when they were cultured with exosomes from cell culture medium of AGS or BCG23 cells. This suggested that exosomes containing miR-379-5p could enter certain recipient cells, depending on cell types.

In this study, IGF1R was a downstream target of miR-379-5p predicted by algorithm tools including TargetScan and miRDB. Previous studies reported that IGF1R plays an oncogenic role in various cancers, including breast cancer, colorectal cancer, glioma, and lung cancer [22‒26]. The expression of IGF1R has been shown to be overexpressed in gastric cancer [27, 28]. It has also been revealed that IGF1R functioned as an oncogene in gastric cancer. Upregulation of IGF1R contributed to cell proliferation, metastasis, and EMT in gastric cancer [29, 30]. In the current study, we found that enforced expression of miR-379-5p would inhibit protein expression of IGF1R in AGS and BCG23 cells by Western blot. As IGF1R contributed to cell proliferation, invasion, and migration, downregulation of miR-379-5p would lead to gastric cancer development through IGF1R and its associated signaling pathways. It might also increase gastric cancer cell movement in circulation to distant sites. This would further contribute to gastric cancer progression.

This study showed that miR-379-5p functions as a tumor suppressor in gastric cancer initiation and progression. It has also been revealed that miR-379-5p functions as a tumor suppressor in various cancers, including breast cancer [31, 32], colorectal cancer [33], osteosarcoma [34, 35], liver cancer [36, 37], and lung cancer [38‒40]. Another study even indicated that exosomal miR-379-5p was highly expressed in serum of patients with lung cancer [41]. This suggested that the tumor suppressor miR-379-5p could be translocated from intracellular to extracellular. It could also be the reason for downregulation of miR-379-5p in gastric cancer cells.

As tumor suppressive miRNAs are downregulated in various cancers, delivery of these miRNAs into the downregulation site can be a therapy for cancers [42‒44]. But the tumor suppressor miRNAs as therapeutic targets should be delivered into specific sites with low expression of these miRNAs. As in this study, we found that miR-379-5p could be translocated into HEK293T, but not into MKN45. Further study would also be needed to elucidate the repression of miR-379-5p in gastric cancer in order to restore the expression of miR-379-5p in stomach. Inhibition of secretion of tumor suppressive miRNAs by exosomes might be a better way for target therapy for gastric cancer.

Although miR-379-5p plays tumor suppressive roles in gastric cancer and IGF1R was a promising target of it, it needs further studies to validate IGF1R as a direct target and the roles of miR-379-5p in an in vivo model. Investigation of the mechanism of secretion of exosomal miR-379-5p is also in need. Further studies of other promising targets and associated signaling pathways of miR-379-5p will also elaborate the mechanism.

MiR-379-5p was significantly downregulated in gastric cancer. It functioned as a tumor suppressor in gastric cancer. MiR-379-5p was highly expressed in exosomes in cell culture medium than in gastric cancer cells. MiR-379-5p was translocated from intracellular to extracellular via exosomes. Exosomes may play significant roles in translocating miRNAs from primary site to distant sites through circulation. Exosomal miRNAs open a new avenue for elucidating the mechanism of downregulation of miRNAs and metastasis, also for providing therapeutic targets for gastric cancer.

This study protocol was reviewed and approved by Institutional Review Board of the University of Hong Kong, Approval No. UW 05-359 T/1022. All of the samples were obtained with the participants’ written informed consent.

The authors declare no conflict of interests for this article.

This work was supported by Seed Fund – Basic Research, University Research Committee (URC), The University of Hong Kong (Seed Fund No. 2202100962).

Prof. K.-M. Chu and Dr. Michelle X. Liu designed this project, performed data analysis, and wrote and revised the manuscript. Prof. K.-M. Chu collected the clinical samples. Dr. Michelle X. Liu performed experiments and collected data.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.