MicroRNA-223 functions as an oncogene in human gastric cancer by targeting FBXW7/hCdc4

Abstract

Aims: The aim of this study was (a) to determine the role of micro-223 (miR-223) in gastric cancer and (b) to elucidate its regulatory mechanism on the FBXW7/hCdc4 gene.

Materials and methods: Artificial miR-223 and control oligonucleotide was transfected into gastric cancer cell line SGC7901 by using Lipofectamine2000. Apoptosis of miR-223 group and control group cells was analyzed by flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and colony formation assays were performed to detect the cell viability, to survey migration of miR-223 group and control group cells; scratch wound-healing motility assays, Transwell Assay, and Western blot test were performed to measure the variance of hFBXW7. Luciferase Reporter Assay, which was done by pLUC-hFBXW7 WT-3'-UTR co-transfected with pLMP-hsa-miR-223 or pLMP plasmid (as control) into HEK293T cells, used to detect whether hFBXW7 is a direct target gene of miR-223. Gastric cancer cell line SGC7901 transfected with miR-223 or control oligonucleotide was resuspended in ECM gel and then was injected into the flank of nude mice, 4 weeks later, the nude mice were euthanized. The tumors were excised then were measured and weighted. SYBR-Green I-based real-time RT-PCR study was used to detect the level of miR-223 in 22 gastric cancer tissue and corresponding gastric mucosa tissues. Immunohistochemical method was applied to detect the protein of hFBXW7.

Results: Gastric cancer cell line SGC7901, transfected with miR-223, showed significant reduction in cellular apoptosis and increased proliferation and invasion in vitro. Similar results were found in tumorigenesis assays performed in nude mice. Moreover, 19 of 22 cancer tissue samples highly expressed miR-223, when compared with patient-matched normal gastric mucosa. Specifically, patients with lymph node metastasis or metastatic disease (M1) at an advanced pathological stage showed significantly higher expression of miR-223. FBXW7/hCdc4 protein (FBW7) levels in gastric cancer cases were inversely correlated with miR-223 expression. Overexpression of miR-223 in gastric cancer cell lines decreased FBW7 expression at the translational level and decreased FBXW7/hCdc4-driven luciferase-reporter activity.

Conclusion: In summary, the data indicated that miR-223 targets FBXW7/hCdc4 expression at the post-transcriptional level and appears to regulate cellular apoptosis, proliferation, and invasion in gastric cancer. MiR-223 may serve as a novel therapeutic target in gastric cancer.

Fig. 1

Differences in miR-223 expression levels between gastric cancer and matched non-tumor tissues (normal gastric mucosa) which were used as control (box-plot diagrams with median, 1st quartile, 3rd quartile and non-outlier range)

Fig. 2

MiR-223 expression in gastric cancer and its clinical significance. a Relationship between miR-223 expression and lymph node metastasis; when the number of lymph node metastasis increased, miR-223 expression level was upregulated. (N1 vs. N2, P = 0.428; N1 vs. N3, P = 0.012; N2 vs. N3, P = 0.078). b The miR-223 expression was correlated with metastatic disease (M1) (P = 0.002). c The miR-223 expression was similar among patients at stage IIIA, stage IIIB, and stage IIIC. There was a difference between patients at stage IIIA, stage IIIB and patients at stage IV (P = 0.01, P = 0.018)

Fig. 3

Overexpression of miR-223 promotes in vitro SGC7901 cell growth. SGC7901 cells were transfected with miR-223 or control oligonucleotide, respectively. a At 24, 48, 72, or 96 h transfection, MTT assay was performed to determine SGC7901 proliferation. Data represent the mean ± SD from three independent experiments. b Representative results of colony form SGC7901 cell transfected with miR-223 or control oligonucleotide. The results were reproducible in three independent experiments. *P < 0.05 and **P < 0.01

Fig. 4

Overexpression of miR-223 inhibits gastric cancer cell apoptosis. At 48 h after transfection, SGC7901 cells were collected for apoptosis analysis. The apoptotic rates of cells were detected by flow cytometry. All experiments were repeated in triplicate with similar results. *P < 0.05 and **P < 0.01

Fig. 5

MiR-223 regulates cell invasion ability in SGC7901 cells. a Quantification of SGC7901 cells that invaded through ECM gel-coated membrane after transfection with miR-223 or control oligonucleotide (P < 0.01). b miR-223 influences SGC7901 cell migration ability from 0 to 96 h after transfection. The distance between two edges of the scratch became increasingly narrow. There are notable difference between mimic group and control group (P < 0.05) at 96 h

Fig. 6

Overexpression of miR-223 promotes tumor growth in vivo. a Xenografts in the left flank of mice using cells transfected with control oligonucleotide. On the right flank, xenografts of mice using cells transfected with miR-223. b Tumor weight values are the average of tumor weight ±SD. c Tumor growth curves. The tumor volume was measured and calculated every 2–4 days. Each point is the average (n = 5); bars represent SD

Fig. 7

MiR-223 directly targets FBXW7/hCdc4 gene. a Putative miR-223-binding sequence in the 3′-UTR of FBXW7/hCdc4 mRNA. Mutation was generated on the FBXW7/hCdc44 3′-UTR sequence in the complementary site for the miR-223 seed region, as shown in (a). FBXW7/hCdc4 3′-UTR fragment containing wild type or mutant miR-223-binding sequence was cloned downstream of the luciferase reporter gene. b The wild type (FBXW7/hCdc4 3′-UTR-WT) or mutant (hFBXW7/hCdc44 3′-UTR-Mut) reporter plasmids were co-transfected into HEK263T cells with pLMP-hsa-miR-223, or negative control (pLMP-rno-miR-344). The normalized luciferase activity in the control group was set as relative luciferase activity. c The expression of FBXW7/hCdc4 mRNA (249 bp) was analyzed by RT-PCR assay. GAPDH was used as an internal control (180 bp). d The expression of hFBW7 protein was analyzed by western blot assay. β-actin was used as an internal control. All experiments were repeated in triplicate with similar results. e Immunostaining of FBW7 in primary gastric cancer tissue samples. Immunostaining of FBW7 was strongly positive in corresponding non-tumor mucosa tissues (a, b) but was very weakly positive gastric cancer tissues (c, d). Original magnification, 400×. f The immunoreactivity of FBW7 in gastric cancer tissues showed a statistically significant inverse correlation with the relative level of miR-223 expression. *P < 0.05, **P < 0.01