Oncogenesis. 5(11):e268(2016) 

SALL4 promotes gastric cancer progression through activating CD44 expression

X Zhang,W Xu


摘要:

The stem cell factor SALL4 (Sal-like protein 4) plays important roles in the development and progression of cancer. SALL4 is critically involved in tumour growth, metastasis and therapy resistance. However, the underlying mechanisms responsible for the oncogenic roles of SALL4 have not been well characterized. In this study, we demonstrated that SALL4 knockdown by short hairpin RNA greatly inhibited the proliferation, migration and invasion of gastric cancer cells. We further confirmed the inhibitory effects of SALL4 knockdown on gastric cancer cells by using a tetracycline-inducible system. Mechanistically, SALL4 knockdown downregulated the expression of CD44. The results of luciferase assay and chromatin immunoprecipitation study showed that SALL4 bound to CD44 promoter region and transcriptionally activated CD44. The results of rescue study revealed that CD44 overexpression antagonized SALL4 knockdown-mediated inhibition of gastric cancer cell proliferation, migration, and invasion in vitro and gastric cancer growth in vivo. Collectively, our findings indicate that SALL4 promotes gastric cancer progression through directly activating CD44 expression, which suggests a novel mechanism for the oncogenic roles of SALL4 in gastric cancer and represents a new target for gastric cancer therapy.To demonstrate the biological roles of SALL4 in gastric cancer, we suppressed SALL4 expression in gastric cancer cells by using two short hairpin RNAs (shRNAs) that specifically target SALL4. The knockdown efficacy of shRNAs in SALL4 gene and protein expression was verified by using quantitative RT-PCR and western blot (Figure 1a). The knockdown of SALL4 gene by shRNAs downregulated the expression of both SALL4A and SALL4B isoforms (Supplementary Figure 1). We observed significant changes in cell morphology in SALL4 knockdown cells after cell replating (Figure 1b). We then checked the effects of SALL4 knockdown on the malignant phenotypes of gastric cancer cells. We examined the role of SALL4 knockdown in gastric cancer cell growth by using cell counting assay. As shown in Figure 1c, SALL4 knockdown greatly retarded the growth of gastric cancer cells. We further confirmed the inhibitory effect of SALL4 knockdown on the growth of gastric cancer cells by using cell colony formation assay (Figure 1d). We found that SALL4 knockdown induced apoptosis and cell cycle arrest at G1 phase in gastric cancer cells (Figures 1e and f). We next detected the effects of SALL4 knockdown on the motility of gastric cancer cells. Compared to the control cells, SALL4 knockdown cells showed a greatly reduced motility ability (Figure 1g). Finally, we determined the effects of SALL4 knockdown on cell migration and invasion. As shown in Figures 1h and i, SALL4 knockdown greatly reduced the migration and invasion abilities of gastric cancer cells. We also observed that SALL4 knockdown sensitized gastric cancer cells to cisplatin treatment (Supplementary Figure 2). Taken together, these results indicate that stable knockdown of SALL4 by shRNA inhibits the proliferation, migration and invasion of gastric cancer cells.Human gastric cancer cell line MGC80-3 and human embryonic kidney cell line 293 T were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high-glucose DMEM (Dulbecco's modified Eagle's medium; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in humidified air with 5% CO2. Cells have been regularly tested for Mycoplasma and were free of this contamination.This work was supported by the National Natural Science Foundation of China (81672416, 81572075, 81201660), the Natural Science Foundation of the Jiangsu Province (BK20141303), Jiangsu Province for Outstanding Sci-tech Innovation Team in Colleges and Universities (SJK2013-10), Jiangsu Province's Outstanding Medical Academic Leader and Sci-tech Innovation Team Program (LJ201117), Jiangsu Province's Major Project in Research and Development (BE2015667), the Key Research and Development Project of Zhenjiang (SH2015034), the Jiangsu Key Laboratory of Medical Science and Laboratory Medicine Project (JSKLM-2014-006).Supplementary Information accompanies this paper on the Oncogenesis website (http://www.nature.com/oncsis)
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