MiR-27a-3p functions as an oncogene in gastric cancer by targeting BTG2

Abstract

microRNA-27a (miR-27a) is frequently dysregulated in human carcinoma, including gastric cancer. The B-cell translocation gene 2 (BTG2) has been implicated in gastric carcinogenesis. However, till now, the link between miR-27a and BTG2 in gastric cancer has not been reported. Here, we found that two isoforms of mature miR-27a, miR-27a-5p and miR-27-3p, were both frequently overexpressed in gastric cancer tissues and cell lines, whereas the expression level of miR-27-3p in gastric cancer was significantly higher than that of miR-27a-5p. And overexpression of miR-27a-3p, but not miR-27a-5p, markedly promoted gastric cancer cell proliferation in vitro as well as tumor growth in vivo. Further experiments revealed that BTG2 was a direct and functional target of miR-27a-3p in gastric cancer and miR-27a-3p inhibition obviously up-regulated the expression of BTG2. In turn, overexpression of BTG2 triggered G1/S cell cycle arrest, induced subsequent apoptosis, and inhibited C-myc activation following Ras/MEK/ERK signaling pathway, which involved in the biological effects of miR-27a-3p/BTG2 axis on gastric carcinogenesis and cancer progression. Overall, these results suggested that the miR-27a-3p/BTG2 axis might represent a promising diagnostic biomarker for gastric cancer patients and could be a potential therapeutic target in the management of gastric cancer.

Keywords: BTG2; apoptosis; cell proliferation; gastric cancer; miR-27a-3p.

Figure 1. miR-27a-3p and miR-27a-5p are overexpressed in GC tissues and cell lines

A. MiR-27a-5p expression in 20 pairs of gastric cancer tissue (Tumor) and their corresponding nontumorous tissue (NT). MiR-27a-5p expression levels were calculated by the MiR-27a-5p/U6 expression ratio (i.e., 2-ΔCt). (p< 0.0001, Mann–Whitney test). B. Comparison of MiR-27a-5p expression level between gastric cancer tissues and their corresponding non-tumorous tissues. C. MiR-27a-3p expression in20 pairs of gastric cancer tissue (Tumor) and their corresponding nontumorous tissue (NT). MiR-27a-3p expression levels were calculated by the MiR-27a-3p/U6 expression ratio (i.e., 2^−ΔCt). (p< 0.0001, Mann–Whitney test). D. Comparison of MiR-27a-3p expression level between gastric cancer tissues and their corresponding non-tumorous tissues. E. MiR-27a-5p expression in the NT, Tumor, normal gastric cancer cell line GES-1 and gastric cancer cell lines AGS, NCI-N87, BGC-823, HGC-27, SGC-7901 and MGC-803. F. MiR-27a-3p expression in the NT, Tumor, normal gastric cancer cell line GES-1 and gastric cancer cell lines AGS, NCI-N87, BGC-823, HGC-27, SGC-7901 and MGC-803. Case numbers were listed below the chart of (A) and (C).

Figure 2. miR-27a-3p promotes GC cell growth in vitro and in vivo

A. The verification of plasmid activation in MGC803 and NCI-N87 cells. B. Growth curve of GES-1 cells treat with miR-27a (+)/pEGFP-C1 or pEGFP-C1. C. Growth curve of MGC-803 cells treat with miR-27a (−)/pEGFP-C1 or pEGFP-C1. D. Sphere formation assay of stable cell lines in Soft agar. E. Sphere formation assay of gastric or gastric cancer cells. Equal numbers cell lines were seeded into soft agar in 6-well. After 2 weeks of culture, the number of microspheres was counted. F. GES-1/Vector and GES-1/miR-27a(+) subcutaneous xenograft in nude mice. G. MGC-803/Vector and MGC-803/miR-27a(−) subcutaneous xenograft in nude mice. H. Growth curve of GES-1, BGC-823, and MGC-803 cells treat with miR-27a-3p mimics or inhibitor and miR-27a-5p mimics or inhibitor.

Figure 3. BTG2 is a direct functional target of miR-27a-3p in GC cells

A. Schematic representation of the binding between miR-27-3p and BTG2 with mutated sites labeled with gray shading. B. Expression patterns of BTG2 with miR-27a-3p in gastric cancer tissues (p< 0.0001, spearman correlation) All data were presented as mean±SD and as representative of an average of three independent experiments. C. Verify the activation of BTG2-pcDNA by Real-time PCR and western blot. D. The effect of miR-27a-3p on BTG2 luciferase activity by dual-luciferase assay. Data were calculated by Student t test (^**p< 0.01, ^***p< 0.001). E. Western Blotting was performed to determine the protein expression level of BTG2 in miR-27a-3p-upregulated or miR-27a-3p-inhibited cells, β-actin was used as the loading control. All data were presented as means ± SD and as representative of an average of three measurements.

Figure 4. miR-27a-3p/BTG2 axis regulates cell cycle progression in GC cells

A. Flow cytometry was performed to determine the cycle arresting of GES-1. B. Flow cytometry was performed to determine the sub-G1 of GES-1. C. Western Blotting was performed to determine the protein expression level of cyclin D and cyclin E. β-actin was used as the loading control.

Figure 5. miR-27a-3p/BTG2 axis regulates apoptosis in GC cells

A. TUNEL staining image of intervertebral disc histotomy from the first-batch SD rats. Original magnification is all 100×. B. Western Blotting was performed to determine the protein expression level of caspase3 and PARP1. β-actin was used as the loading control.

Figure 6. BTG2 inhibits C-myc expression through Ras/MEK/ERK pathway in GC cells

A. Expression patterns of BTG2 with C-myc in gastric cancer tissues (p< 0.0001, spearman correlation) All data were presented as means±SD and as representative of an average of three independent experiments. B. Western Blotting was performed to determine the protein expression level of BTG2, Ras, MEK, P-MEK, ERK, P-ERK, c-Myc. β-actin was used as the loading control.