miR-589 promotes gastric cancer aggressiveness by a LIFR-PI3K/AKT-c-Jun regulatory feedback loop

A series of GC cell lines (GSE-1, BGC823, MKN45, MGC803, AGS, MKN28) were obtained from Foleibao Biotechnology Development (Shanghai, China). The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (NBCS) (PAA Laboratories, Inc., Pasching, Austria). All of these cell lines were incubated in a humidified chamber with 5% CO₂ at 37 °C. For inhibitor treatment, 10 mmol/L PI3K inhibitor LY294002 (Cell Signal Technology, Danvers, MA) was added in the cultured cells every two days.

LIFR plasmids, miR-589 mimic, anti-miR-589 oligos and all siRNA oligos including c-Jun specific siRNAs (si-1973, si-1554, si-2358 and si-1113) were purchased from GenePharma (Shanghai, China). GC cells at exponential growth phase were plated into 6-well plates for 24 h at a density of 0.5 × 10⁵ cells/mL, and transfected with 1 mg of siRNA or 4 μg cDNA using Lipofectamine 2000 reagent (Invitrogen; Carlsbad, Calif, USA) in reduced serum medium (OPTI-MEM-I; Invitrogen) according to the manufacturer’s protocol.

Fresh primary GC specimens with paired normal gastric tissues were obtained from the Tumor Tissue Bank of Nanfang Hospital. In each case, pathological diagnosis was made after elective surgery for GC in Nanfang Hospital during 2009 and 2014. All experiments performed are endorsed by the Ethics Committee of Southern Medical University and complied with the Declaration of Helsinki. No informed consent was required because data were going to be analyzed anonymously.

All animal experiments were carried out with the approval of the Southern Medical University Animal Care and Use Committee in accordance with the guidelines for the ethical treatment of animals. Nude nu/nu mice were maintained in a barrier facility in racks filtered with high-efficiency particulate air filter. The animals were fed with an autoclaved laboratory rodent diet. The mice in this study were purchased from the Experimental Animal Centre of Southern Medical University, which is certified by the Guangdong Provincial Bureau of Science. All animal experiments involved ethical and humane treatment under a license from the Guangdong Provincial Bureau of Science.

Protein expression was assessed by immunoblot analysis of cell lysates (20–40 μg) in RIPA buffer in the presence of rabbit antibodies to GAPDH (1:1000; Santa Cruz, California, USA); mouse antibody to LIFR (1:1000; Proteintech); rabbit antibodies to PI3K, p-PI3K, p-AKT(Ser473)(#4060), AKT(#4691) (1:1000; CST, Danvers, MA). Relative protein abundance of phosphorate proteins was determined by normalisation with levels of corresponding total protein. Relative protein abundance of total protein was determined by normalisation with levels of corresponding endogenous control protein.

Data were analyzed using SPSS version 19.0 software (SPSS; Chicago, USA). Statistical significance of difference between groups was determined by a two-tailed paired Student’s t test. Kaplan-Meier plots were performed to investigate the prognostic relevance of PIK3AP1 in univariate analysis. Statistical significance was established at P < 0.05.

The microarray result showed that miR-589 was upregulated an average of 1.25 folds (P < 0.001) in GC tumor tissues compared with adjacent normal tissues (Fig. 1a). We subsequently detected miR-589 mRNA expression in GC cell lines and normal gastric epithelium cell line GES-1. Real-time PCR assay showed higher level of miR-589 expression in five GC cell lines compared with GES-1 cell line (Fig. 1b). Consistent with this result, we found miR-589 is markedly up-regulated in GC tissues compared with matched normal tissues (Fig. 1c). As compared to tumor tissues without metastasis, metastatic tumors tissues showed relatively high miR-589 expression (Fig. 1d, P < 0.001). These data suggested miR-589 as an oncogene in GC tumorigenesis and metastasis. To evaluate the clinical relevance of miR-589 expression, we analyzed its relationship with pathological features. As shown in Additional file 1: Table S1, miR-589 expression in GC tissues was positively correlated with depth of tumor invasion (P = 0.016) and distant metastasis (P = 0.001), but had no correlation with the age and gender. Subsequently, we used TCGA data for survival analysis, the Kaplan-Meier survival curves displayed a significant trend towards poorer survival for patients whose tumors showed high miR-589 expression, compared with those tumors showed low miR-589 expression (Fig. 1e; P = 0.05).

In order to investigate the biological effect of miR-589 on GC cells, we chose MGC803 and BGC 823 cell lines to perform the gain- or loss-of function. We transfected miR-589 mimic oligonucleotides and anti-miR-589 into MGC803 and BGC823 cell lines. Real-time PCR assay confirmed the transfection efficiency (Fig. 1f). Wound healing and trans-well assays showed that miR-589-knockdown cells exhibited reduced migratory, metastatic and invasive capability, whereas an opposite result was observed in miR-589-overexpressing cells (Fig. 2a-c). Together, miR-589 played a positive role in GC cell migration, metastasis and invasion.

To evaluate the in vivo effect of miR-589 on GC cell metastasis, we established miR-589 stably expressing MGC803 cells by lentivirus injection. MGC803 cells stably overexpressing miR-589 were injected into nude mice via the lateral tail vein along with control cells. The bioluminescence images showed that miR-589 can significantly promote lung metastasis (Fig. 2d). The mice were sacrificed after 8 weeks and their lungs were dissected. Haematoxylin and eosin staining was conducted to evaluate the tissue morphology. We evaluated the number of lung metastasis nodules and found they were markedly increased in the MGC803-miR-589 group compared with the control group (Fig. 2e). These findings suggest that miR-589 increased GC metastasis and thus may act as a potential therapeutic target against metastatic GC.

Western blot analysis showed that miR-589 overexpression was positively correlated with PI3K and AKT phosphorylation. In contrast, knockdown of miR-589 significantly blocked AKT/PI3K pathway (Fig. 3a). These results suggested miR-589 as an upstream regulator of PI3K/AKT pathway. LY294002 was an inhibitor which was used to block AKT signaling pathway, western blot showed miR-589 significantly compensated the effect of LY294002 (Fig. 3b), indicating that miR-589 positively regulated AKT pathway. Meanwhile, miR-589 was sufficient to compensate the effects of LY294002 on GC cell migratory and invasive abilities (Fig. 3c & d). Taken together, miR-589 plays an important role in the activation of PI3K/AKT pathway, which may explain part of the mechanism underlying miR-589 function.

Bioinformatics approach based on the database TargetScan predicted that LIFR was the putative target of miR-589. The analysis of the 3’-UTR of LIFR mRNA revealed the potential binding sites for miR-589, which implied the existence of a regulative relationship between miR-589 and LIFR. Dual-luciferase reporter assays were performed to prove miR-589 regulation of LIFR. miR-589 markedly decreased the luciferase activity of wide-type LIFR 3’-UTR in both MGC803 and BGC823 cells, whereas the suppression effect was abrogated after the 3’-UTR binding site of LIFR was mutated (Fig. 4a). Next, Real-time PCR analysis showed an increased LIFR expression level in miR-589-knockdown cells, and a decreased LIFR level in miR-589-overexpressing cells. Correspondingly, LIFR protein levels coincided with the change of mRNA levels in miR-589-knockdown and miR-589-overexpressing cells (Fig. 4b). Therefore, we drew a conclusion that LIFR is the direct target of miR-589.

Given that miR-589 targeted LIFR and suppressed its expression, we investigated the biological function of LIFR in GC cells. Data from Kaplan-Meier plotter database (http://​kmplot.​com/​analysis/​) were utilized to visualize the association between LIFR expression and overall survival in GC patients. The results showed patients with high LIFR expression in tumors had a trend towards better survival when compared with patients showed low LIFR expression (Fig. 4c; HR = 0.7, P = 0.016). To evaluate the effects of LIFR on cellular process, we performed wound healing and transwell assays in MGC803 and BGC823 cells. LIFR-overexpressing cells displayed opposite phenotype compared with miR-589-overexpressing cells and showed markedly inhibited cell migratory and invasive abilities (Fig. 4d-f).

Western blot exhibited that LIFR efficiently revered miR-589-induced increase of p-PI3K and p-AKT (Fig. 5a), suggesting miR-589 regulates PI3K/AKT pathway via targeting LIFR. Subsequent rescue experiments showed that transiently transfecting LIFR into miR-589-overexpressing GC cells significantly weakened miR-589-midiated promotion of cell migration, metastasis and invasion (Fig. 5b-d). Collectively, these data indicate that LIFR attenuates miR-589-induced cell behavior and PI3K/AKT signaling activation.

Transcriptional factor c-Jun is known to be an downstream target of AKT signaling [24], intriguingly, we used JASPAR bioinformatics database to analyze the promoter region of miR-589 and found two putative c-Jun-binding sites (from − 2005 to − 1996 and from − 1075 to − 1060) within the region (Fig. 6d). Therefore, we predicted that c-Jun inversely regulated miR-589 and formed a miR-589-LIFR-PI3K/AKT-c-Jun feedback loop. Subsequently, we used 4 specific c-Jun siRNAs to interfere c-Jun expression in MGC803 and BGC823 cell lines. Indeed, Real-time PCR analysis showed a relatively low miR-589 expression in cells treated with c-Jun siRNAs (Fig. 6c). Moreover, LY294002 and LIFR overexpression significantly decreased miR-589 expression (Fig. 6a & b), suggesting the existence of a miR-589-LIFR-PI3K/AKT-c-Jun regulatory feedback loop.

Chromatin immunoprecipitation (ChIP) assays was performed to confirm the relationship between c-Jun and miR-589. DNA from the immunoprecipitated chromatin displayed a significant enrichment of the predicted region compared with negative control (IgG) pulldown (Fig. 6e). Furthermore, an increase of the wild-type miR-589 promoter luciferase activity was observed on upregulation of c-Jun in 293 T, BGC823 and MGC803 cell lines (Fig. 6d; P < 0.001). Thus, c-Jun increased miR-589 expression via binding to its promoter region. Collectively, these results offer a conclusion that miR-589 induced cell migration, metastasis and invasion via a miR-589-LIFR-PI3K/p-AKT-c-Jun feedback loop.