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Objective To explore the possible mechanism of emodin in inhibiting proliferation,migration,and invasion of AGS cells and in suppressing the expressions of YAP1 and FOXD1.Methods Normal gastric cell GES-1 and gastric cancer cell AGS were cultured with different concentrations of emodin.CCK8 test,scratch test and Transwell assay were used to verify changes in the biological phenotype of AGS cells.TCGA database was applied to analyze expressions of HK2,YAP1 and FOXD1 in gastric cancer tissues and normal gastric tissues.Western blotting method was used to detect the impacts of emodin on HK2,YAP1 and FOXD1 proteins in AGS cells.Exogenous pyruvic acid was added to verify the changes in YAP1 and FOXD1.Results The IC50 of emodin was significantly higher in GES-1 cells than in AGS cells(P<0.05).CCK8 proliferation test,scratch test,and Transwell assay showed that emodin significantly inhibited the biological abilities of AGS(P<0.05 for comparisons).Analysis on the TCGA bioinformatics database found that the expression of key enzymes HK2 in the glycolysis pathway and oncogenes YAP1 and FOXD1 was significantly higher in gastric cancer tissues than in normal gastric tissues(P<0.05 for comparisons).Emodin significantly inhibited the protein expressions of key glycolytic enzymes HK2 and oncogenes YAP1 and FOXD1(P<0.05 for comparisons).With supplement of exogenous glycolytic metabolite pyruvate,the protein expressions of oncogenes YAP1 and FOXD1 significantly increased(P<0.05 for comparisons).Conclusions Emodin has a significant pharmacological inhibitory effect on gastric cancer AGS cells,markedly suppressing their biological phenotype.Emodin not only significantly inhibits the key enzyme HK2 in glycolysis metabolism,but also the protein expressions of oncogenes YAP1 and FOXD1.With the addition of exogenous pyruvate to enhance the glycolytic metabolic pathway,the protein expressions of oncogenes YAP1 and FOXD1 significantly increased.The above results suggest a close association of YAP1 and FOXD1 with glycolytic metabolism.Emodin may inhibit oncogenes YAP1 and FOXD1 through the glycolytic metabolism of gastric cancer AGS cells.
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@#[摘 要] 目的：分析FOXD1在食管鳞状细胞癌（ESCC）组织中的表达及其与临床病理特征和患者预后的关系，探讨其对ESCC TE1细胞增殖、侵袭能力的影响及其对TGF-β1诱导TE1细胞EMT进程的影响。方法：采用qPCR和IHC法检测ESCC组织和细胞中FOXD1的表达，并分析其与临床病理特征和患者预后的关系；构建FOXD1敲减质粒并转染TE1细胞，检测其对TE1细胞增殖、侵袭能力的影响；用qPCR和WB法检测TGF-β1处理前后FOXD1及EMT相关基因和蛋白的表达变化及敲减FOXD1对EMT相关基因和蛋白表达的影响。结果：ESCC组织和细胞中FOXD1均呈高表达（均P<0.01），并与患者OS呈负相关；FOXD1表达水平、肿瘤TNM分期以及淋巴结转移均是影响ESCC患者预后的独立危险因素（均P<0.01）。TGF-β1可促进TE1细胞FOXD1的表达，并诱发其EMT进程（均P<0.05）；敲减FOXD1可抑制TE1细胞的增殖和侵袭能力，并可部分逆转由TGF-β1诱发的TE1细胞EMT进程。结论：FOXD1在ESCC组织及TE1细胞中呈高表达且是影响ESCC患者预后的独立危险因素，敲低FOXD1可显著抑制TE1细胞的增殖、侵袭及TGF-β1介导的EMT进程。
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Objective To study the effect of inhibiting the expression of FOXD1 gene on the radiosensitivity of colorectal cancer cells. Methods The expressions of FOXD1 mRNA and protein in human colorectal cancer tissues and cells were detected by Real-time PCR ( qRT-PCR) and Western blot. The colorectal cancer cell line HCT116 was irradiated with 0, 2, 4 and 6 Gy of X-rays. The expression of FOXD1 in each groups were detected by qRT-PCR and Western blot. HCT116 cells were transfected with FOXD1 siRNA and its negative control and termed as si-FOXD1 group and si-NC group. When these cells were irradiated with 4 Gy X-rays, they were termed as si-FOXD1+4 Gy group and si-NC+4 Gy group. Cell proliferation was detected with MTT method, cell survival fraction was measured with colony formation assay, and DNA-PK activity was detected by TECT DNA-PK kit. The siRNA-transfected colorectal cancer cells were inoculated into BALB/c nude mice to establish the xenograft model. After irradiation, the volume and quality of the subcutaneous transplanted tumors were measured every 5 days. Results The expression of FOXD1 mRNA and protein in colorectal cancer tissues was higher than that in adjacent normal tissues (t=5. 579, 4. 816, P<0. 05). The mRNA(t=5. 85-17. 62, P<0. 05)and protein(t=9. 04-11. 42, P<0. 05) expression of FOXD1 in different colorectal cancer cell lines was higher than that in colonic mucosa epithelial cell line NCM460. The expression of FOXD1 in colorectal cancer cells HCT116 was increased after radiation in a dose dependent manner(t=9. 13-44. 15, P<0. 05). Transfection of si-FOXD1 effectively inhibited the expression of FOXD1 (t=10. 51, P<0. 05), decreased proliferation (t=10. 41, P <0. 05), increased radiosensitivity with a radiosensitization ratio of 1. 797, and reduced the radiation-induced DNA-PK activity ( t = 6. 20, P < 0. 05 ) in colorectal cancer cells. After localized irradiation, the tumor volume and weight in nude mice transplanted with si-FOXD1 HCT116 cells were significantly smaller than those in HCT116 (t=11. 29, 3. 69, P<0. 05). Conclusions Knock-down of FOXD1 gene increases the radiosensitivity of colorectal cancer cells and inhibits the growth of colorectal cancer xenograft in nude mice, which provides a potential target gene in improving the effect of radiotherapy on colorectal cancer.