RGS1 serves as an antitumor target to inhibit proliferation of NICN87-DR cells and tumor growth in the gastric cancer mouse model

Abstract

Gastric cancer is becoming the 4th leading cause of cancer-associated death worldwide. The purpose of this study was to investigate the role of RGS1 in gastric cancer in vitro and in vivo. Proliferation, migration, invasion, and colony formation of NCIN87 cells and drug-resistant NCIN87 cells (NCIN87-DR) were determined. Cell apoptosis and cell cycle were examined using a flow cytometry assay. RGS1 gene knock-down vector (pLVshshRGS1) and Xenograft tumor mouse model was generated. RGS1 and epithelial-mesenchymal transition (EMT) associated markers, including E-cadherin (E-cad), N-cadherin (N-cad), Slug, and Vimentin were detected using a western blotting assay. Tumor size of Xenograft tumor mouse was measured and Ki67 expression was detected using the immunohistochemical assay. NCIN87-DR cells demonstrated significantly lower proliferation, migration, and invasion compared to those of NCIN87 cells (p < 0.05). NCIN87-DR cells showed obvious early apoptosis and displayed obvious alterations for the cell cycle. NCIN87-DR cells exhibited predominantly higher RGS1 expression than that in NCIN87 cells (p < 0.01). E-cad expression was markedly decreased (p < 0.01) and N-cad (p < 0.05), Slug (p < 0.01), Vimentin (p < 0.05) expressions were significantly increased in NCIN87-DR cells than those in NCIN87 cells. RGS1 gene silence remarkably reduced NCIN87-DR proliferation compared to that in NCIN87-DR cells without treatment (p < 0.01). RGS1 gene-silenced NCIN87-DR cell immunization predominantly inhibited tumor growth in Xenograft tumor mouse than that without RGS1 silence (p < 0.05). RGS1 gene-silenced NCIN87-DR cell immunization significantly downregulated Ki67 expression in tumor tissues compared with that without RGS1 silence. In conclusion, RGS1 gene silence reduced the proliferation of NCIN87-DR cells in vitro and inhibited tumor growth in vivo. Therefore, RGS1 served as an antitumor target for the gastric cancer treatment.

Keywords: RGS1; drug-resistance; gastric cancer; proliferation; treatment.

Figure 1

Proliferation characteristics of NCIN87 cells and drug-resistant NCIN87 cells (NCIN87-DR) (n = 3). A. Cell proliferation of NCIN87 and NCIN87-DR cells determined using CCK-8 assay. B. Migration of NCIN87 and NCIN87-DR cells determined using transwell assay. C. Invasion of NCIN87 and NCIN87-DR cells determined using transwell assay. D. Colony formation determined in NCIN87 and NCIN87-DR cells using colony formation analysis. ^*p < 0.05 versus NCIN87 cells.

Figure 2

Cell cycle and cell apoptosis were analyzed using flow cytometry (n = 3). A. Cell cycle analysis and statistical analysis for different phage cells. B. Cell apoptosis analysis and statistical analysis for both NCIN87 and NCIN87-DR cells. ^*p < 0.05 versus NCIN87 cells.

Figure 3

Determination for the RGS1 and EMT-associated molecules expression in both NCIN87 and NCIN87-DR cells (n = 3). A. Western blotting images for RGS1, E-cadherin, N-cadherin, Slug, and Vimentin expression. B. Statistical analysis and comparison for above molecules. ^*p < 0.05 and ^**p < 0.01 versus NCIN87 cells.

Figure 4

Effects of pLVshRGS1 and/or T-mab treatment on proliferation of NCIN87-DR cells (n = 3). A. RGS1 gene transcription verified with real time PCR assay. ^**p < 0.01 versus NCIN87-DR group, ^##p < 0.001 versus pLV group. B. Proliferation evaluation of NCIN87-DR cells using CCK-8 assay and statistical analysis. ^***p < 0.001 versus NCIN87 group, ^###p < 0.001 and ^#p < 0.05 versus NCIN87-DR group, ^$$$p < 0.001 versus NCIN87-DR+T-mab group, ^&&&p < 0.001 versus NCIN87-DR+pLVshRGS1+T-mab group.

Figure 5

Tumor growth in Xenograft tumor mouse model (n = 3). A. Images of the in vivo tumors in body and isolated tumors. B. Statistical analysis for tumor size. ^***p < 0.001, ^**p < 0.01 and ^*p < 0.05 versus NCIN87 group. ^###p < 0.001 ^##p < 0.01 and ^#p < 0.05 versus NCIN87-DR group, ^$$p < 0.01 and ^$p < 0.05 versus NCIN87-DR group, ^&&&p < 0.001, ^&&p < 0.01 and ^&p < 0.05 versus NCIN87-DR+T-mab group.

Figure 6

Effects of pLVshRGS1 and/or T-mab treatment on Ki67 expression in tumor tissues of Xenograft tumor mouse model (n = 3). A. Western blotting assay and statistical analysis for RGS1 expression. B. Ki67 expression in tumor tissues using immunohistochemical analysis. ^***p < 0.001 versus NCIN87 group. ^##p < 0.01 ^###p < 0.001 versus NCIN87-DR group. ^$$$p < 0.001 versus NCIN87-DR+T-mab group.