Myeloid ecotropic viral integration site 1 inhibits cell proliferation, invasion or migration in human gastric cancer

Abstract

Myeloid ecotropic viral integration site 1 (MEIS1) has been identified to be a potential tumor suppressor in some cancers. However, the mechanisms underlying MEIS1-induced cancer development and progression were not clear. Here, we investigated the expression and role of MEIS1 in gastric cancer. In vivo, we analyzed tumor growth using nude mice model. In the present study, MEIS1 expression was obviously decreased in GC cell lines compared with that in normal gastric cell lines (all p<0.001). MEIS1 overexpression inhibited cell proliferation and G1/S transition accompanied by decreased Cyclin D1 and Cyclin A expression. Furthermore, MEIS1 overexpression decreased the expression of Survivin, and induced cell apoptosis (p<0.001). Transwell migration assay revealed that MEIS1 affects cell invasion and migration, and inhibited epithelial-mesenchymal transition (EMT). Finally, MEIS1 inhibits MKN28 cell growth in nude mice model. In conclusion, our study suggested that MEIS1 plays an important role in regulating cell survival, proliferation, anchorage-independent growth, cell cycle, apoptosis and metastasis. Thus, MEIS1 might be recommended as an effective target for GC patients.

Keywords: MEIS1; gastric cancer; invasion; migration; proliferation.

Figure 1. MEIS1 expresses in GC cell line SGC7901, MKN28, and normal gastric cell line RGM-1, and GES-1

(A) Total protein extracted from the indicated cell lines were analyzed by western blot. GAPDH was chosen as a loading control. (B) The relative protein level was shown as mean ± SD from three independent experiments with similar results. (C) The mRNA level of Meis1 in 10 cases of gastric cancer tissues and matched normal tissues was determined by real-time RT-PCR. The GC cell line, MKN28 (D) or SGC7901 (E) were infected with Ad-control or Ad-MEIS1. The expression of MEIS1 in GC cells was shown as photographs or relative expression level. ^*p < 0.05 versus cells infected Ad-control.

Figure 2. MEIS1 attenuates GC cell proliferation or survival

MKN28 (A) or SGC7901 (B) cells infected with Ad-control, Ad-MEIS1 or the parental cells were grown in regular medium, and harvested at indicated time points. Cell number was determined by MTT. Then, colony formation for MKN28 (C) and SGC7901 (D) cells infected with Ad-control or Ad-MEIS1 were measured. All values were shown as photograph (C, D) or mean ± SD (E, F) of triplicate measurements with similar results. ^*p < 0.05 versus parental cells or cells infected Ad-control.

Figure 3. MEIS1 siRNA promotes GES-1 cell proliferation or survival

GES-1 cells (A) infected with Ad-control or Ad-MEIS1 were grown in regular medium, and harvested at the indicated time points. Cell numbers were determined by MTT assay at 490 nm. The expression of MEIS1 was identified by western blot (B, C) Next, colony formation for GES-1 cells (D) was measured. All values were shown as photograph (D, E) or mean ± SD (F) of triplicate measurements with similar results. ^* p < 0.05 versus with Ad-control.

Figure 4. MEIS1 inhibits anchorage-independent growth of GC cells

MKN28 (A) or SGC7901 (B) cells, infected with Ad-control or Ad-MEIS1 were analyzed by soft agar. Results were shown as photograph or mean ± SD of triplicate measurements with similar results. ^* p < 0.05 versus with Ad-control.

Figure 5. MEIS1 induces non-apoptotic cell death of GC cells

(A, B) Representative flow-cytometer of cells stained with Annexin V and PI in (A) MKN28 or (B) SGC7901 cells infected with Ad-control or Ad-MEIS1. Data were also shown as mean ± SD of three experiments with similar experiments. ^* p < 0.05 versus with Ad-control.

Figure 6. MEIS1 reduces the protein level of pro-survival and enhances the expression of pro-apoptosis regulator

MKN28 (A) or SGC7901 (B) cells were infected with Ad-control or Ad-MEIS1. Then, cells were harvested for western blot. Protein level of pro-survival regulators cIAP-1, cIAP-2 or Survivin and pro-apoptosis regulator BAX was detected by antibodies. GAPDH was chosen as loading controls. Relative protein level is shown as mean ± SD of three experiments with similar results. ^* p < 0.05 versus cells with Ad-control.

Figure 7. MEIS1 induces the cell cycle arrest at G1/S in GC cells

(A, B) Cell cycle of MKN28 (A) or SGC7901 (B) cells infected with Ad-control or Ad-MEIS1 were detected by flow-cytometry. The proportion of cells in each cell cycle phase was shown (A, B). The G0/G1 cells in MKN28 (C) or SGC7901 cells (D) was also shown as mean ± SD of three experiments with similar results. ^* p < 0.05 versus Ad-control.

Figure 8. MEIS1 reduces the positive G1/S transition regulators, Cyclin D1 and Cyclin A

Representative WB for Cyclin D1 or Cyclin A in MEIS1 overexpressing MKN28 (A) or SGC7901 (B) cells. Data were also shown as mean ± SD of three experiments with similar results. ^* p < 0.05 versus Ad-control.

Figure 9. MEIS1 inhibits metastasis of high aggressive GC cells MKN28

MKN28 cells, infected with Ad-control or Ad-MEIS1, were analyzed by transwell formation assays. Invasion (A) or migration (B) was shown as photograph or mean ± SD of triplicate measurements with similar results. ^* p < 0.05 versus Ad-control.

Figure 10. MEIS1 inhibits EMT process

MKN28 cells were infected with Ad-control or Ad-MEIS1. The expression of epithelial marker E-cadherin and two mesenchymal markers, N-cadherin and Vimentin was detected by antibodies (A) Relative protein level is shown as the mean ± SD of three experiments with similar results (B) ^* p < 0.05 versus Ad-control.

Figure 11. MEIS1 inhibits in vivo growth of MKN28 cells

MKN28 cells were infected with Ad-control or Ad-MEIS1. Next, cells were injected in to BALB/c nude mice. The effect of MEIS1 on MKN28 cells in vivo growth was showed as tumor volumes (A) or tumor weight (B) ^* p < 0.05 versus Ad-control.