Yonsei Med J.  2018 Dec;59(10):1166-1173. 10.3349/ymj.2018.59.10.1166.

RPS15a Silencing Suppresses Cell Proliferation and Migration of Gastric Cancer

• Shi D
• Liu J

• KMID: 2426330
• DOI: http://doi.org/10.3349/ymj.2018.59.10.1166

Abstract

PURPOSE
Information on the possible role of the ribosomal protein S15a (RPS15a) in gastric cancer is scarce. The aim of this study was to evaluate the impact of RPS15a gene expression on the growth and cell cycle of gastric cancer cells in vitro and in vivo.
MATERIALS AND METHODS
RPS15a mRNA expression was examined in cancer tissues and their corresponding adjacent normal tissues of 40 gastric adenocarcinoma patients. Next, RPS15a was knocked down using a lentivirus-mediated RNA interference (short hairpin RNA) system in the gastric cancer cell line BGC823. The effect of RPS15a knockdown was examined using CCK-8 assay, cell scratch test, colony formation assay, and flow cytometry. Finally, in nude mice, a tumorigenicity test was performed, and the tumor volume and weight were measured.
RESULTS
RPS15a expression in tumor tissue was significantly greater than that in the adjacent normal tissue of gastric cancer patients. After RPS15a silencing, the BGC823 cell proliferation rate decreased significantly; most cells were arrested in the G0/G1 phase, cell growth was inhibited, and the migration rate was decreased. Colony formation assay showed that the number and size of clones in the RPS15a-silenced cells were fewer and smaller, compared to control cells. The nude mouse tumorigenicity test showed that RPS15a silencing had an inhibitory effect on tumor volume and mice weight.
CONCLUSION
The present study found RPS15a expression to be higher in gastric tumors and its silencing in gastric cancer cells to inhibit the proliferation, growth, and migration thereof. Accordingly, RPS15a may be considered as a potential therapeutic target in gastric cancer.

Keyword

Gastric cancer; RPS15a; BGC823 cells; gene silencing

MeSH Terms

Adenocarcinoma
Animals
Carcinogenicity Tests
Cell Cycle
Cell Line
Cell Proliferation*
Clone Cells
Flow Cytometry
Gene Expression
Gene Silencing
Humans
In Vitro Techniques
Mice
Mice, Nude
Ribosomal Proteins
RNA Interference
RNA, Messenger
Sincalide
Stomach Neoplasms*
Tumor Burden
RNA, Messenger
Ribosomal Proteins
Sincalide

Figure

• 

  Fig. 1 Ribosomal protein S15a (RPS15a) expression in gastric cancer and normal tissue as detected by quantitative PCR. The relative expression levels of RPS15a were significantly greater in gastric cancer tissues compared with normal tissues (n=40, *p<0.05). M, mark; C, gastric cancer tissues, N: adjacent tissues. β-actin was used as a reference.

• 

  Fig. 2 Ribosomal protein S15a (RPS15a) expression in siRNA-transfected gastric cancer and control cells as detected by quantitative PCR. PCR analysis of RPS15a mRNA expression in BGC823 cells transfected with different siRNAs and control cells showed that the RPS15a fragments were virtually not amplified in cells treated with siRPS15a-2, which was proven to be better than siRPS15a-1 in the silencing experiments (*p<0.01). ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a-1 and siRPS15a-2, BGC823 cells transfected with siRPS15a-1- and siRPS15a-2-containing siRNA, respectively.

• 

  Fig. 3 Ribosomal protein S15a (RPS15a) expression after RPS15a silencing in each group as detected by western blot analysis. Western blot analysis of RPS15a protein expression in BGC823 cells transfected with different siRNAs and control cells demonstrated that the highest protein expression was present in the wild-type BGC cells and the lowest in the siRPS15a-2-transfected cells. ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a-1 and siRPS15a-2, BGC823 cells transfected with siRPS15a-1- and siRPS15a-2-containing siRNA, respectively.

• 

  Fig. 4 Colony formation experiments. After ribosomal protein S15a (RPS15a) interference, BGC823 cells were grown for 2 weeks, and cell colonies were compared. ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a-1 and siRPS15a-2, BGC823 cells transfected with siRPS15a-1- and siRPS15a-2-containing siRNA, respectively (*p<0.01).

• 

  Fig. 5 Cell proliferation assay. The proliferation rate of BGC823 cells was decreased significantly in siRPS15a-1- and siRPS15a-2-transfected cells (*p<0.05, †p<0.01). RPS15a, ribosomal protein S15a; ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a- 1 and siRPS15a-2, BGC823 cells transfected with siRPS15a-1- and siRPS15a-2-containing siRNA, respectively.

• 

  Fig. 6 Cell cycle analysis. The cell cycle distribution after ribosomal protein S15a (RPS15a) interference for 48 h was determined (*p<0.05). ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a-1 and siRPS15a-2, BGC823 cells transfected with siRPS15a- 1- and siRPS15a-2-containing siRNA, respectively.

• 

  Fig. 7 Cell migration assay. The migration rate of BGC823 cells was decreased significantly in siRPS15a-1- and siRPS15a-2-transfected cells (*p<0.01). RPS15a, ribosomal protein S15a; ck, normal BGC823 cells; siNC, BGC823 cells transfected with the negative control sequence; siRPS15a-1 and siRPS15a-2, BGC823 cells transfected with siRPS15a-1- and siRPS15a-2-containing siRNA, respectively.

• 

  Fig. 8 Cell transfection efficiency and the effect of knocking down ribosomal protein S15a (RPS15a) on tumor growth in nude mice. (A) The fluorescence analysis shows that tumor cells were successfully transfected with the shRNA (magnification: ×100). (B) Quantitative PCR analysis of RPS15a mRNA expression in BGC823 cells transfected with shRNA and control cells (*p<0.01). (C) Western blot analysis of RPS15a protein expression in BGC823 cells transfected with shRNA and control cells demonstrated that the highest protein expression was present in the wild-type BGC cells. (D) Tumor growth curve (*p<0.01). (E) Tumor weight (*p<0.01). vector, BGC823 cells transfected with the empty plasmid; ck, normal BGC823 cells; shRPS15a, BGC823 cells transfected with the shRPS15a plasmid; pCDH, noncoding sequence-transfected BCG823 cells; rps15a-shRNA, RPS15a knock-down group.

Reference

1. Zhang Y, Zhang G, Li X, Li B, Zhang X. The effect of ribosomal protein S15a in lung adenocarcinoma. PeerJ. 2016; 4:e1792. PMID: 26989627.

2. Li G, Zhang L, Liu J, Xiao T, Liu G, Wang J, et al. shRNA-mediated RPS15A silencing inhibits U937 acute myeloid leukemia cell proliferation and enhances apoptosis. Mol Med Rep. 2016; 13:4400–4406. PMID: 27035327.

3. Chen J, Wei Y, Feng Q, Ren L, He G, Chang W, et al. Ribosomal protein S15A promotes malignant transformation and predicts poor outcome in colorectal cancer through misregulation of p53 signaling pathway. Int J Oncol. 2016; 48:1628–1638. PMID: 26847263.

4. Xu M, Wang Y, Chen L, Pan B, Chen F, Fang Y, et al. Down-regulation of ribosomal protein S15A mRNA with a short hairpin RNA inhibits human hepatic cancer cell growth in vitro. Gene. 2014; 536:84–89. PMID: 24334120.

5. Zhang C, Zhang T, Song E, Himaya SW, Chen X, Zheng L. Ribosomal protein S15A augments human osteosarcoma cell proliferation in vitro. Cancer Biother Radiopharm. 2014; 29:451–456. PMID: 25409460.

6. Lian Z, Liu J, Li L, Li X, Tufan NL, Wu MC, et al. Human S15a expression is upregulated by hepatitis B virus X protein. Mol Carcinog. 2004; 40:34–46. PMID: 15108328.

7. Zhao X, Shen L, Feng Y, Yu H, Wu X, Chang J, et al. Decreased expression of RPS15A suppresses proliferation of lung cancer cells. Tumour Biol. 2015; 36:6733–6740. PMID: 25833696.

8. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136:E359–E386. PMID: 25220842.

9. Akhondi-Meybodi M, Ghane M, Akhondi-Meybodi S, Dashti G. Five-year survival rate for gastric cancer in Yazd Province, Central Iran, from 2001 to 2008. Middle East J Dig Dis. 2017; 9:39–48. PMID: 28316765.

10. Qiao YF, Chen CG, Yue J, Ma MQ, Ma Z, Yu ZT. Prognostic significance of preoperative and postoperative CK19 and CEA mRNA levels in peripheral blood of patients with gastric cardia cancer. World J Gastroenterol. 2017; 23:1424–1433. PMID: 28293089.

11. Baniak N, Senger JL, Ahmed S, Kanthan SC, Kanthan R. Gastric biomarkers: a global review. World J Surg Oncol. 2016; 14:212. PMID: 27514667.

12. Lazăr DC, Tăban S, Cornianu M, Faur A, Goldis¸ A. New advances in targeted gastric cancer treatment. World J Gastroenterol. 2016; 22:6776–6799. PMID: 27570417.

13. Jomrich G, Schoppmann SF. Targeted therapy in gastric cancer. Eur Surg. 2016; 48:278–284. PMID: 27795701.

14. Zhang C, Fu J, Xue F, Ryu B, Zhang T, Zhang S, et al. Knockdown of ribosomal protein S15A induces human glioblastoma cell apoptosis. World J Surg Oncol. 2016; 14:129. PMID: 27130037.

15. Yao Y, Liu Y, Lv X, Dong B, Wang F, Li J, et al. Down-regulation of ribosomal protein S15A inhibits proliferation of human glioblastoma cells in vivo and in vitro via AKT pathway. Tumour Biol. 2016; 37:4979–4990. PMID: 26537582.