MicroRNA-216a inhibits the metastasis of gastric cancer cells by targeting JAK2/STAT3-mediated EMT process

Abstract

MicroRNAs (miRNAs), a group of small, non-protein coding, endogenous RNAs, play critical roles in the tumorigenesis and progression of human cancer. miR-216a has recently been reported to play an oncogenic role in human cancer. While, the expression of miR-216a, its biological function and underlying molecular mechanisms in gastric cancer (GC) are largely unknown. In this study, we revealed that miR-216a was underexpressed in GC tissues compared to matched noncancerous tissues. Decreased levels of miR-216a were confirmed in GC cell lines compared with a normal gastric epithelium cell line. miR-216a underexpression was associated with malignant prognostic features including lymph node metastasis, venous infiltration, invasive depth and advanced TNM stage. GC patients with low miR-216a level showed an obvious shorter overall survival. miR-216a overexpression restrained migration and invasion of MGC-803 cells, while its knockdown exerted opposite effects on metastatic behaviors of SGC-7901 cells. In vivo experiments found that miR-216a restoration reduced metastatic nodes of GC cells in nude mice liver. miR-216a notably suppressed epithelial-mesenchymal transition (EMT) of GC cells. Janus kinase 2 (JAK2) was recognized as a direct target and downstream mediator of miR-216a in GC cells. Interestingly, JAK2/signal transducer and activator of transcription 3 (STAT3) pathway was prominently inactivated by miR-216a and probably mediated the role of miR-216a in the regulation of migration, invasion and EMT process of GC cells. In conclusion, these data suggest that miR-216a functions as a tumor suppressive miRNA in the development of GC possibly by targeting JAK2/STAT3-mediated EMT.

Keywords: JAK2/STAT3; epithelial-mesenchymal transition; gastric cancer; miR-216a; tumor metastasis.

Figure 1. The expression and prognostic value of miR-216a in GC

(A) The relative expressions of miR-216a in 90 pairs of GC tissues and matched noncancerous tissues. Data were presented as log2 of fold change of GC relative to matched noncancerous tissues and analyzed by student’s t-test. (B) qRT-PCR analysis of miR-216a expression in GC cell lines (SGC-7901, MGC-803, MKN-28, and BGC-823) and the normal human gastric epithelium cell line (GES-1). n = three repeats with similar results, ^*P<0.05 by ANOVA. (C) GC patients were divided into miR-216a high expression group and miR-216a low expression group based on the median level of miR-216a expression. GC patients with low miR-216a level (n = 45) had an obvious shorter overall survival compared to those with high miR-216a level (n = 45). P<0.05 by Log-rank test.

Figure 2. miR-216a inhibits migration and invasion of GC cells

(A) MGC-803 cells that were transfected with precursor miR-216a (pre-miR-216a) or scrambled controls (miR-control) were subjected to qRT-PCR for miR-216a expression. n = three repeats with similar results, ^*P<0.05 by t test. (B) Wound healing assays indicated that miR-216a overexpression inhibited migration of MGC-803 cells. n = three repeats with similar results, ^*P<0.05 by t test. (C) The number of invaded cells was reduced after miR-216a restoration in MGC-803 cells. n = three repeats with similar results, ^*P<0.05 by t test. (D) SGC-7901 cells that were transfected with miR-216a inhibitors (anti-miR-216a) or scrambled controls (anti-control) were subjected to qRT-PCR for miR-216a expression. n = three repeats with similar results, ^*P<0.05 by t test. (E) Wound healing assay indicated that miR-216a knockdown promoted migration of SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test. (F) The number of invaded cells was increased after miR-216a loss in SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test.

Figure 3. miR-216a suppresses liver metastasis of GC cells in mice

MGC-803 cells that were transfected with precursor miR-216a (pre-miR-216a) or scrambled controls (miR-control) were intravenously injected into tail vein in nude mice. HE staining revealed that miR-216a overexpression significantly reduced the number of metastatic nodes of MGC-803 cells in nude mice livers. Scale bar: 1mm. n = 6, ^*P<0.05 by t test.

Figure 4. miR-216a retrains EMT process of GC cells

(A) MGC-803 cells that were transfected with precursor miR-216a (pre-miR-216a) or scrambled controls (miR-control) were subjected to immunoblotting for E-cadherin, N-cadherin and Vimentin expression. n = three repeats with similar results, ^*P<0.05 by t test. (B) IF data revealed that miR-216a overexpression enhanced E-cadherin staining while reduced Vimentin staining in MGC-803 cells. (C) SGC-7901 cells that were transfected with miR-216a inhibitors (anti-miR-216a) or scrambled controls (anti-control) were subjected to immunoblotting for E-cadherin, N-cadherin and Vimentin expression. n = three repeats with similar results, ^*P<0.05 by t test. (D) IF data revealed that miR-216a knockdown reduced E-cadherin staining while enhanced Vimentin staining in SGC-7901 cells.

Figure 5. JAK2 is a direct target of miR-216a in GC cells

(A) The potential miR-216a binding site in wild type (wt) 3’-UTR sequence of JAK2. The underlined part is the mutant site designed for mutant (mt) 3’-UTR sequence of JAK2. (B) and (C) MGC-803 and BGC-823 cells that were transfected with precursor miR-216a (pre-miR-216a) or scrambled controls (miR-control) were confirmed by qRT-PCR and immunoblotting for JAK2 expression. n = three repeats with similar results, ^*P<0.05 by t test. (D) miR-216a overexpression decreased the luciferase activity of wt 3’-UTR of JAK2. In contrast, no changes in relative luciferase activity were observed when the miR-216a binding site was mutated. n = three repeats with similar results, ^*P<0.05 by t test. (E) An inverse correlation between the levels of JAK2 mRNA and miR-216a expression was observed in GC tissues. n = 90, P<0.05 by Spearman’s rank correlation test. (F) Representative IHC staining showed that miR-216a low expressing GC tissue (n = 45) showed strong staining of JAK2, while weak staining of JAK2 was observed in miR-216a high expressing case (n = 45). The JAK2 levels in high-miR-216a PADCs were significantly lower than that of those low-miR-216a GCs. Scale bar: 50μm. ^*P<0.05 by t test.

Figure 6. miR-216a inhibits activation of JAK2/STAT3 pathway

MGC-803 cells that were transfected with precursor miR-216a (pre-miR-216a) or scrambled controls (miR-control) were detected by immunoblotting. miR-216a overexpression led to reduced levels of JAK2, phophorylated STAT3, Slug, Snail and Twist in MGC-803 cells. On the other hand, SGC-7901 cells that were transfected with miR-216a inhibitors (anti-miR-216a) or scrambled controls (anti-control) were subjected to immunoblotting. miR-216a knockdown promoted activation of JAK2/STAT3 pathway in SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test.

Figure 7. JAK2/STAT3 functions in miR-216a inhibited EMT process and metastasis of GC cells

(A) MGC-803 cells that were transfected with corresponding vectors were subjected to immunoblotting for JAK2, p-STAT3, STAT3, E-cadherin, N-cadherin and Vimentin. n = three repeats with similar results, ^*P<0.05 by ANOVA. (B) JAK2 re-expression abolished the inhibitory effect of miR-216a on migration of MGC-803 cells. n = three repeats with similar results, ^*P<0.05 by ANOVA. (C) JAK2 restoration facilitated invasion of miR-216a overexpressing MGC-803 cells. n = three repeats with similar results, ^*P<0.05 by ANOVA.

Figure 8. The JAK2 inhibitor SAR317461 reverses the effects of miR-216a knockdown in GC cells

(A) SGC-7901 cells that were transfected with miR-216a inhibitors (anti-miR-216a) were treated with the JAK2 inhibitor SAR317461 and DMSO, respectively. Western blotting data showed that SAR317461 treatment reduced the level of phosphorylated STAT3 and inhibited EMT process of SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test. (B) SAR317461 treatment abrogated the promoting effect of miR-216a knockdown on migration of SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test. (C) SAR317461 treatment restrained invasion of miR-216a silenced SGC-7901 cells. n = three repeats with similar results, ^*P<0.05 by t test.