18β-glycyrrhetinic acid suppresses gastric cancer by activation of miR-149-3p-Wnt-1 signaling

18β-glycyrrhetinic acid (GRA) exerts anti-tumor effects on various types of cancer. In the present study, we found that GRA attenuated the severity of gastritis and suppressed gastric tumorigenesis in transgenic mice. We also discovered that miR-149-3p was downregulated in gastric cancer tissues and cell lines as compared to normal gastric tissues and epithelial cells, but was upregulated by GRA. miR-149-3p expression also correlated negatively with lymphnode metastasis. Our functional assays showed that miR-149-3p overexpression inhibited cell proliferation and cell cycle progression while inducing apoptosis, while inhibition of miR-149-3p had the opposite effects. In addition, we identified Wnt-1 as a direct target of miR-149-3p. These data suggest that GRA inhibits the initiation and progression of gastric tumors by ameliorating the inflammatory microenvironment through downregulation of COX-2 expression and by inhibiting Wnt-1 expression through the upregulation of tumor suppressor miR-149-3p. GRA may thus have the potential to serve as a useful therapeutic agent for the prevention and treatment of gastric cancer.


INTRODUCTION
Gastric cancer (GC) is the fifth most common malignancy (915,600 cases) and the third cause of cancer-related deaths (723,100 cases) worldwide [1].More specifically, GC ranks 2nd in cancer incidence (424,000 cases) and 3rd (298,000 cases) in cancer mortality in China [2].Most GC patients are diagnosed at an advanced-stage of the disease and given a poor prognosis.The main treatment consists in gastrectomy combined with chemotherapy and radiotherapy; however, recurrence and metastasis are common.This has prompted the rapid development of alternative targeted approaches, such as immunotherapy and treatment with antibodies or phytochemicals [3][4][5].
While gastric cancer is a multifactorial disease, inflammation is one of the main drivers of tumorigenesis [6].The most common histologic variant of gastric adenocarcinoma is the intestinal-type, and its typical cascade model consists of chronic superficial (nonatrophic) gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and finally gastric adenocarcinoma [7].The COX-2/PGE2 pathway acts as a tumor promoter during the generation of the inflammatory microenvironment.The transgenic mouse model K19-C2mE, engineered by the Oshima group [8], overexpressed Cox-2 and mPGES-1 in gastric mucosa epithelial cells, resulting in the development of hyperplasia, metaplasia and tumorigenesis in the proximal end of the gastric mucosa.In our previous study [9], Canolol decreased the inflammatory response in gastric mucosa epithelia, and inhibited gastric tumorigenesis by blocking the COX-2/ PGE2 pathway.
The major bioactive component in licorice root, 18β-glycyrrhetinic acid (GRA), exerts anti-inflammatory, anti-oxidative and anti-cancer effects [18][19][20].Indeed, GRA is contained in the compound Hongdoushan capsule (CHC), used to treat ovarian and breast cancers [21].Furthermore, previous studies revealed that GRA can act as a tumor suppressor in breast [19], lung [22] and gastric [23] cancers.Additionally, GRA significantly reduced cisplatin-induced nephrotoxicity through the Nrf2/NF-kappaB signaling pathway in BALB/c mice [24].In our previous study [18], GRA attenuated H. pylori-infected gastritis in gerbils; however, further studies are needed to elucidate the mechanisms by which GRA inhibits tumorigenesis in vivo and in vitro.
miRNAs are a class of small noncoding RNAs (18-25 nucleotides in length) that regulate gene expression post-transcriptionally. miRNAs play either a tumorsuppressive or an oncogenic role in cancer by regulating their target genes.Expression profiling has shown that genes in the COX-2/PGE-2 and Wnt/β-catenin pathway are affected by dysregulation of miRNAs in gastric cancer [25].For example, miR-7 suppressed active chronic gastritis and gastric cancer by targeting Cox-2 [26] and miR-1225-5p constrained GC growth and metastatic potential via inhibition of β-catenin signaling [27].In the present study, we hypothesized that miRNAs might be involved in the inhibitory effects of GRA on gastric carcinogenesis.We demonstrated that GRA changed miRNA expression profiles, improved the inflammatory microenvironment, inhibited gastric tumorigenesis in Tg mice, and suppressed cell proliferation in vitro.We also showed that GRA treatment upregulated miR-149-3p expression, which inhibited cell cycle progression and induced cell apoptosis in gastric tumor cells by suppressing the expression of its direct target gene Wnt-1.Our study suggests that GRA could be exploited as a chemoprophylactic and therapeutic agent against gastric cancer.

GRA suppressed chronic active gastritis and downregulated the inflammation-related genes in K19-C2mE Tg mice
The transgenic mice were divided into control (n = 40) and 0.05% GRA-treated groups (n = 40) randomly after genotyping (Figure 1A).At the end of the animal experiments, the survival rates were 90% (36/40) for the control group and 97.5% (39/40) for the GRAtreated group.GRA administration was well tolerated in Tg mice, and there were no significant differences in body weight or growth curves (Figure 1B).
We first conducted histopathological experiments to test for the effects of GRA on gastritis intransgenic mice.A four-point scale (G0, normal; G1, mild; G2, moderate; and G3, marked gastritis) was used to grade chronic active gastritis.HE staining showed active inflammatory mucosal changes, with many neutrophils, lymphocytes, and macrophages heavily infiltrating the gastric submucosa in the control group (Figure 1C).On the contrary, infiltration was reduced in the GRA-treated group (P = 0.001, Figure 1D).Then we assayed the mRNA levels of inflammatory cytokines, Cox-2, mPGES-1, Il-1β, and Il-12b by qRT-PCR.The results showed that GRA administration downregulated the expression of mPGES-1 to 41.95% and Il-1β to 35.41% (Figure 1E).

GRA markedly reduced the tumor incidence and development in vivo
The effects of GRA on GC development and progression were examined in Tg mice.The results showed that, in the control group, the incidence of gastric tumor was 77.8% (28/36), the mean length of tumor tissue was 12.1 ± 2.5 mm, and the mean width was 6.3 ± 1.3 mm.In contrast, in the GRA-treated group, the tumor incidence was 33.4% (13/39), the mean length was 7.4 ± 2.8 mm, and the mean width was 4.8 ± 1.6 mm.The results suggested that GRA suppressed the incidence (P = 0.002), length (P < 0.001) and width (P = 0.002) of gastric tumors (Table 1, Figure 2A).

GRA suppressed tumor cell viability and induced cell apoptosis in vitro
Considering the intestinal and liver metabolism of Tg mice, the gradient concentrations of GRA (50-300 μM) were designed in the preliminary experiment in vitro.Then, serial concentrations of GRA, 50 μM, 100 μM, 150 μM, 200 μM were adopted to treat gastric tumor cells BGC-823 and MKN-1 for 4 h, 24 h and 48 h.The human immortal gastric epithelial cell line GES-1 was used as control.
MTT assays showed that tumor cell proliferation was slightly increased by 50 μM GRA in BGC-823 (119.30%) and GES-1 (127.00%) cells for 4 h.On the other hand, proliferation was not influenced by 100 μM GRA in BGC-823, MKN-1 and GES-1 cells after 4 h and 24 h.However, when the GRA concentration reached to 150 μM, cell proliferation was inhibited in BGC-823 for 24 h (40.70%) and 48 h (25.44%), while GES-1 proliferation was 93.95% for 24 h and 27.47% for 48 h.Furthermore, MTT results also showed that 200 μM www.impactjournals.com/oncotargetGRA had measurable effects on cell viability.BGC-823 and MKN-1 proliferation was decreased to 64.76% and 33.59% independently.GES-1 proliferation was also decreased to 48.73% for 4 h.These results indicated that GRA inhibited gastric tumor cell proliferation in a doseand time-dependent manner, and that 150 μM was the ideal concentration causing less side-effects in normal epithelial cells (Figure 2B).
Flow cytometry analyses of the cell cycle showed that the number of cells at G0/G1 phase was 58.05% for MKN-1, and 52.93% for BGC-823.After exposure to 150 μM GRA for 24 h, the accumulation of cells reached a peak value of 65.57% for MKN-1 (Figure 2C), and 58.84% for BGC-823 (Figure 2E).Meanwhile, the number of cells in S phase was 25.36% for MKN-1, and 38.15% for BGC-823, while they were decreased to 16.45% in MKN-1, and 28.94% in BGC-823.The results showed that GRA inhibited cell cycle progression independently from the differentiation status of the cells.
In addition, flow cytometry analyses also showed that the apoptosis rate was 1.62% in MKN-1, increasing to 7.62% after 150 μM GRA treatment for 24 h (Figure 2D), and it was 2.61% in BGC-823, increasing to 23.30% after GRA treatment for 24 h (Figure 2F).
To further investigate the expression locations of COX-2, Wnt-1 and β-catenin, we analyzed immunohistochemically stained gastric tumor tissue.Immunostaining of COX-2 was observed mainly in the membrane and cytoplasm of gastric mucosa cells in the control group, while it was predominantly located on the cell membrane in the GRA-treated group (Figure 3B).β-catenin accumulated mainly in the cell cytoplasm and nucleus in the control group; however, the accumulation was much lighter in cytoplasm and absent in the nucleus after GRA treatment (Figure 3C).Similarly, Wnt-1, was mainly stained in the cell membrane and cytoplasm in the control group, and it was predominantly stained in the cytoplasm after GRA administration (Figure 3D).Furthermore, the H score indicated that the expression of COX-2, Wnt-1 and β-catenin was downregulated significantly by GRA administration (P < 0.001).
All the results of IHC, qRT-PCR and western blot indicated that COX-2 and the canonical Wnt pathway were the possible targets of GRA.

miR-149-3p was upregulated by GRA administration in a dose-and time-dependent manner
To examine whether miRNA expression is regulated after GRA administration, the miRNA profiles in the control group (n = 5) and GRA-treated group (n = 5) were examined (Figure 4A).Overall, the transcriptional levels of 38 miRNAs were altered, 22 miRNAs were upregulated (fold change ≥ 2, P < 0.05), and 16 miRNAs were downregulated (fold change ≤ 0.5, P < 0.05) in the GRA-treated group (Supplementary Table S1).Among these miRNAs, miR-149-3p was upregulated the most by 3.84-fold (P < 0.001) (Figure 4B).In addition, more gastric tumor tissues were used to confirm the results of the microarray analysis, and qRT-PCR showed that -ΔCt in the control group (n = 16) was 5.96, and 4.54 in GRA-treated group (n = 11) (Figure 4C), the results pointed out that GRA indeed upregulated the expression of miR-149-3p.
The miR-149-3p sequence is highly conserved in Mus musculus and Homo sapiens.Thus, gastric tumor cells BGC-823 and MKN-1 were chosen to detect the effects of GRA on the expressions of miR-149-3p, and qRT-PCR results demonstrated that miR-149-3p levels were upregulated by GRA in a time-and dose-dependent manner (Figure 4D and 4E).
To address the clinical impact of miR-149-3p expression in gastric cancer, we used qRT-PCR to analyze 11 cases of GC tissues and paired control tissues collected in our hospital.Compared with control tissues, the median level of miR-149-3p was lower in the gastric tumor tissue (P = 0.02, Figure 4H), consistent with TCGA data.In addition, to explore the relationship between miR-149-3p expression and GC progression, 74 cases of gastric cancer with intact clinical pathological information were divided into two groups according the median level of miR-149-3p expression.Results from our analyses showed that low levels of miR-149-3p correlated positively with the presence of lymphonode metastasis (P = 0.01, Figure 4K).However, we detected no correlation with tumor differentiation (Figure 4I), depth of invasion (Figure 4J), distant metastasis (Figure 4L) and pTNM stage (Figure 4M).

miR-149-3p suppressed GC cell proliferation and cell cycle progression in vitro
Next, we used MKN-1 and BGC-823 cells to monitor the impact of ectopic overexpression of miR-149-3p on cell cycle, apoptosis and reproducibility.Results from our analyses showed that miR-149-3p overexpression induced apoptosis, increased the numbers of cells in G0/ G1 phase and G2/M phase while decreasing those in S phase, and led to smaller and fewer colonies compared to miR-CK cells (P < 0.01) both in MKN-1 (Figure 5A-5C) and BGC-823 (Figure 5D-5F).

DISCUSSION
In the present study, we found that GRA ameliorated the inflammatory microenvironment by inhibiting the expression of inflammation-related genes.Additionally, the miRNA miR-149-3p was significantly upregulated by GRA in a dose-and time-dependent manner.We also confirmed that miR-149-3p played a tumor suppressor role by downregulating its direct target gene Wnt-1.
Chronic infection correlates with cancer in 15-20% of cases [10] and systemic inflammation is associated with poor prognosis in patients with advanced cancers [28].Inflammation promotes GC development through the induction of the COX-2/PGE2 pathway in K19-C2mE transgenic (Tg) mice [8].Here, our experiments with Tg mice showed that GRA inhibited the inflammatory response (Figure 1) and improved the inflammatory microenvironment in the gastric epithelial mucosa, where gastric cancer initiates and develops.Thiyagarajan and Chandrasekaran et al. showed that glycyrrhizin, from which GRA isextracted, did not exhibit anti-inflammatory activity inmurine macrophages (J774A.1)or human  neutrophils (HL-60) [29,30].Therefore, we hypothesized that GRA acts primarily on epithelial cells.
GutGard is a novel, flavonoid-rich extract derived from Glycyrrhiza glabra.Although this extract showed a marked improvement of Nepean dyspepsia index [31], glycyrrhizin did not exhibit anti-inflammatory activity in the GutGard experiment.On the contrary, Il-1β, Il-12b and Cox-2 were downregulated by GRA administration in our study (Figure 1E).Furthermore, the GutGard experiment only studied murine macrophages and human neutrophils and did not address the interaction of tumor cells with inflammatory microenvironments [29,30].On the other hand, the Tg mice used in our study developed gastric tumors spontaneously, emulating human gastric tumors morphologically and histochemically [8].Therefore, the results of our experiments using Tg mice show GRA's anti-tumor effects in vivo.
Recently, miRNAs have attracted considerable attention, partly because multiple drugs have been shown to upregulate or activate them, thereby eliciting tumor-suppressive effects and yielding therapeutic benefits [9,[37][38][39].In our study, GRA inhibited gastric tumorigenesis through the upregulation of tumor suppressor miR-149-3p.With the rapid development of miRNA profiling, an increasing number of miRNAs are being identified as therapeutic targets for various diseases.In addition to miR-149-3p, we found 13 other miRNAs that were upregulated and 22 that were downregulated in GC after GRA administration (Supplementary Table S1).For example, miR-328, a miRNA upregulated by GRA, inhibited cancer cell growth and impaired resistance to chemotherapeutic drugs by decreasing CD44 expression [40,41].Furthermore, miR-494, another miRNA upregulated by GRA, was shown to act as a tumor suppressor [42,43].In contrast, miR-223, a miRNA downregulated by GRA, was shown to function as an oncogene in human GC [44].
In conclusion, we propose that GRA inhibited the initiation and progression of gastric cancer by ameliorating the inflammatory microenvironment through inhibition of COX-2 expression, and by decreasing Wnt-1 expression through the upregulation of tumor suppressor miR-149-3p.
Our results highlight GRA as a potential preventive and therapeutic agent against gastric cancer and warrant further studies to elucidate other molecular mechanisms involving GRA and additional miRNAs in GC and other types of cancer.

Ethics statement
The use of the samples from animals and patients was approved by the Institutional Research Ethics Committee of Jilin University.Informed consent was obtained from all patients.

Chemicals
18β-glycyrrhetinic acid (GRA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).Stock solutions of GRA were dissolved in DMSO (vehicle) at 0.05% for in vivo studies and at 100 mM for in vitro studies as previously reported [18].

Animal model and experimental protocol
K19-C2mE transgenic mice produced by the Oshima group [8] were maintained by breeding male Tg mice with female C57BL/6 N mice at the Animal Facility of Jilin University.Tg mice over-expressing Cox-2 and mPGES-1 in gastric epithelial cells were housed in plastic cages with free access to food and water.DNA was extracted from the tails of 4-week old mice and subjected to PCR as previously reported [9].
The transgenic male animals aged 6 weeks were divided into two groups randomly after genotyping (Figure 1A).The GRA-treated group (n = 40) was given to drink distilled water containing 0.05% GRA during the entire experimental period (52 weeks).The GRA solution was freshly prepared three times per week.Control mice (n = 40) were fed with the same diet and distilled water but without GRA.

Tissue collection and histopathological assessment
The stomachs of Tg mice were resected, and pairs of tumor tissue and adjacent normal epithelial mucosa were preserved.The incidence, length, and width of tumors were calculated.All the samples were separated into two parts: one part was paraffin-embedded for hematoxylineosin (H&E) and immunohistochemical (IHC) staining, and the another part was flash-frozen in liquid nitrogen and preserved at -80°C until RNA extraction.For the grading of chronic active gastritis, a 4 point scale (G0, normal; G1, mild; G2, moderate; and G3, marked gastritis) was used according to criteria modified from the updated Sydney System [45].Immunohistochemical detection of COX-2 (1:200, Santa Cruz, USA), β-catenin (1:200, Abcam, Cambridge, USA), Wnt-1 (1:200, Abcam), and H score calculation were performed as previously described [9].

Patient samples and the cancer gnome atlas (TCGA) data analysis
For miRNA examination, 74 cases of gastric cancer and their paired control tissue (located > 3 cm away from the tumor) were obtained between September 2008 and January 2012 at First Hospital of Jilin University.The patient cohort consisted of 55 males and 19 females with a median age of 65.5 years (range, 39-90 years).The characteristics of the study patients are described in Table 2.For further verification, the miRNA data of 390 stomach adenocarcinoma and 42 paired normal gastric mucosa from the TCGA database (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp)were downloaded and analyzed.(These data had been obtained using Illumina HiSeq 2000 miRNA Sequencing System, Level 3).

Cell viability assay
The anti-proliferative effects of GRA were determined using MTT assay as previously reported [46].The gastric tumor cell lines BGC-823 and MKN-1 were seeded in 96-well plates and treated with serial dilutions of GRA (0, 50, 100, 150, 200 μM) (n = 10) for 4 h, 24 h and 48 h.The immortal gastric epithelia cell line GES-1 was used as a control.Cell viability was assessed using an MTT Kit (Promega, Madison, USA) and absorbance was measured at 490 nm using a micro plate reader (Thermo, MA, USA).

Colony formation assay
The ability of gastric tumor cells to divide unlimitedly after transfection with miR-149-3p or miR-149-3p inhibitor was tested using colony formation assay as previously described [47].The gastric tumor cell lines MKN-1 and BGC-823 were planted in 24-well plates with 4 × 10 5 cells per well, and transfected with 8 μg miR-149-3p/miR-CK or 200 nM miR-149-3p inhibitor/miR-NC.After 24 h, the cells were trypsinized and reseeded in 6-cm plates with 1 × 10 3 cells per well, and cultured for 10 days.The colonies were stained with 1% crystal violet for 15 min after fixation with 4% neutral formaldehyde for 15 min.All experiments were performed in triplicate.

Total RNA rich of miRNA isolation
Total RNA rich of miRNA isolation in Tg mouse gastric tumors, MKN-1 and BGC-823 cells with and without GRA treatment, as well as in tumors from gastric cancer patients, were extracted using the miRNeasy Kits (Qiagen, Hilden, Germany), and reversed transcribed using miScript II RT Kit (Qiagen) for miRNA quantification, or reversed transcribed using cDNA synthesis kits (Roche, Basal, Switzerland) for mRNA quantification according to the manufacturer's protocol.

MicroRNA microarray
Total RNA rich of miRNA isolation in the GRAtreated (n = 5) and control (n = 5) groups was extracted and analyzed by Affymetrix Mouse miRNA array (v.3.0).Each microarray chip was hybridized with a single sample labeled with either Cy3 or Cy5.The raw data were deposited at Shanghai Biotechnology Corporation (Shanghai, China).Background subtraction and normalization were performed using Expression Console software.The miRNAs that exhibited a significant difference in levels (≥ 2-fold, P < 0.05) were selected.

Figure 1 :
Figure 1: GRA treatment prevents gastritis and inflammatory cytokine production in transgenic mice.(A) Experimental design in this study.(B) Body weight of mice during the GRA treatment period.No significant difference was noted.(C) Histologic grades in Tg mice in the two groups.Arrows showed the infiltration of neutrophils, lymphocytes, and macrophages into the submucosa.(D) The percentage and quantification indicates the histopathology changes in Tg mice in the two groups.(E) Various inflammatory cytokines were decreased after GRA treatment.*P < 0.05.

Figure 2 :
Figure 2: GRA treatment inhibited the development and progression of gastric tumor in vivo, and suppressed cell proliferation and cell cycle progression in vitro.(A) Macroscopic appearance of the stomach of Tg mice in control and GRA-treated groups, compared with wild type mice (left).Representative quantification (right) of the tumor length and width in the GRA-treated and control groups (mean ± SD). (B) GRA decreased the viability of the tumor cell lines BGC-823 and MKN-1 in a time-and dose-dependent manner as measured by the MTT assay, and the normal mucosa cell line GES-1 was used as control.GRA induced G0/G1 phase arrest and enhanced apoptosis in (C-E).MKN-1 and (D-F) BGC-823 cells after exposure for 24 h.

Figure 4 :
Figure 4: miR-149-3p was upregulated after GRA administration, and frequently downregulated in GC tissues and cells.(A) MiRNA expression profile revealed upregulated and downregulated miRNAs in the GRA-treated group compared to the control group.(B) miR-149-3p was upregulated the most by GRA.(C) miR-149-3p upregulation was verified in enlarged samples of Tg mice.GRA upregulated miR-149-3p expression in (D) BGC-823 and (E) MKN-1 in a time-and dose-dependent manner.(F) miR-149-3p expression in STAD compared with normal tissue deposited in the TCGA database.(G) miR-149-3p expression in gastric tumor cells compared with normal mucosa cells.(H) miR-149-3p expression in gastric tumor tissues compared with paired normal epithelia mucosa.The correlation analysis of miR-149-3p expression with (I) differentiated grade of tumor, (J) T stage, (K) N stage, (L) M stage, and (M) pTNM stages.