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王华帅,谢彪,罗敏,何永恒.天马颗粒通过FLI-1/Klotho通路对结肠癌细胞HCT116增殖抑制的作用机制研究[J].湖南中医药大学学报,2022,42(10):1656-1662[点击复制] |
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| 天马颗粒通过FLI-1/Klotho通路对结肠癌细胞HCT116增殖抑制的作用机制研究 |
| 王华帅,谢彪,罗敏,何永恒 |
| (湖南中医药大学, 湖南 长沙 410208;湖南省中医药研究院附属医院肛肠科, 湖南 长沙 410006;湖南中医药大学第二附属医院肛肠科, 湖南 长沙 410005) |
| 摘要: |
| 目的 研究天马颗粒通过FLI-1/Klotho途径对结肠癌细胞（HCT116）增殖抑制的作用机制。方法 将HCT116细胞分为blank组、shRNA组、shTMKL组、TMKL组。blank组为空白对照，shRNA组为HCT116细胞瞬时转染FLI-1 shRNA，shTMKL组为shRNA组基础上加入天马颗粒血清干预，TMKL组为天马颗粒血清干预HCT116细胞。各组细胞培育24 h后，进行后续实验检测。qPCR检测各组细胞FLI-1 mRNA和Klotho mRNA表达；Western blot法检测各组细胞FLI-1蛋白和Klotho蛋白相对表达；流式细胞术检测各组细胞周期及凋亡，并计算S期细胞比值（S-phase fraction, SPF)；CCK-8法检测各组细胞增殖。结果 与blank组相比，shRNA组、shTMKL组FLI-1、Klotho蛋白及mRNA表达量降低（P<0.05），TMKL组FLI-1、Klotho蛋白及mRNA表达量升高（P<0.05）；与shRNA组相比，shTMKL组FLI-1、Klotho蛋白及mRNA表达量差异无统计学意义（P>0.05），TMKL组FLI-1、Klotho蛋白及mRNA表达量升高（P<0.05）；与shTMKL组相比，TMKL组FLI-1、Klotho蛋白及mRNA表达量升高（P<0.05）。与blank组相比，shRNA组HCT116细胞增殖能力增强（P<0.05）、SPF值升高（P<0.05）、凋亡率下降（P<0.05），shTMKL组、TMKL组HCT116细胞增殖能力受到抑制（P<0.05）、SPF值降低（P<0.05）、凋亡率升高（P<0.05）；与shRNA组相比，shTMKL组、TMKL组HCT116细胞增殖能力受到抑制（P<0.05）、SPF值降低（P<0.05）、凋亡率升高（P<0.05）；与shTMKL组相比，TMKL组HCT116细胞增殖能力受到抑制（P<0.05）、SPF值降低（P<0.05）、凋亡率升高（P<0.05）。结论 天马颗粒可通过FLI-1基因调控Klotho蛋白表达，进而抑制HCT116细胞增殖、诱导凋亡。 |
| 关键词: 天马颗粒 FLI-1/Klotho通路 结肠癌 细胞增殖 质粒转染 中药血清 机制研究 |
| DOI：10.3969/j.issn.1674-070X.2022.10.011 |
| 投稿时间：2022-04-28 |
| 基金项目:国家自然科学基金青年项目（81704089）；湖南省自然科学基金重点项目（2021JJ30419）；湖南省中医药管理局科研基金重点项目（2021017）；湖南中医药大学中医学一流学科开放基金项目(2022ZYX06)。 |
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| Mechanism of Tianma Granule inhibiting the proliferation of HCT116 (colon cancer cell) through FLI-1/Klotho pathway |
| WANG Huashuai,XIE Biao,LUO Min,HE Yongheng |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Department of Anorectal Surgery, The Affiliated Hospital of Hunan Academy of Chinese Medicine, Changsha, Hunan 410006, China;Department of Anorectal Surgery, The Second Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To study the mechanism of Tianma Granule inhibiting the proliferation of HCT116 (colon cancer cell) through FLI-1/Klotho pathway. Methods HCT116 cells were randomly divided into blank group, shRNA group, shTMKL group and TMKL group. Blank group served as the control group. In shRNA group, HCT116 cells were transiently transfected with FLI-1 shRNA. In shTMKL group, Tianma Granule serum was added on the basis of shRNA. And in TMKL group, Tianma Granule serum was added to intervene HCT116. The cells in each group were cultured for 24 hours. And then, the follow-up experiments were carried out. We detected the expression of FLI-1 mRNA and Klotho mRNA in each group by qPCR, the relative expression of FLI-1 protein and Klotho protein by Western blot, the cell cycle and apoptosisby flow cytometry, calculated the S-phase fraction (SPF) and tested the cell proliferation in each group by CCK8. Results Compared with blank group, the expression of FLI-1 protein, Klotho protein and mRNA in the shRNA group and shTMKL group decreased (P<0.05), while the expression of FLI-1 protein, Klotho protein and mRNA in TMKL group increased (P<0.05). Compared with shRNA group, there was no significant difference in the expression of FLI-1 protein, Klotho protein and mRNA in shTMKL group (P>0.05), but the expression of FLI-1 protein, Klotho protein and mRNA in TMKL group increased (P<0.05). Compared with shTMKL group, the expression of FLI-1 protein, Klotho protein and mRNA in TMKL group increased (P<0.05). Compared with the blank group, shRNA group showed increase in proliferation rate of HCT116 cells (P<0.05) and SPF value (P<0.05), but decrease in the apoptosis rate (P<0.05). The proliferation of HCT116 cells in shTMKL group and TMKL group was inhibited (P<0.05), with decrease in SPF value (P<0.05), and increase in apoptosis rate (P<0.05); compared with shRNA group, the proliferation of HCT116 cells in shTMKL group and TMKL group was inhibited (P<0.05), with decrease in the value of SPF (P<0.05), and increase in the apoptosis rate (P<0.05); compared with shTMKL group, the proliferation of HCT116 cells in TMKL group was inhibited (P<0.05), with decrease in the SPF value (P<0.05) and increase in the apoptosis rate (P<0.05). Conclusion Tianma Granule may regulate expression of Klotho protein through FLI-1 gene, and then inhibit proliferation and induce apoptosis of HCT116 cells. |
| Key words: Tianma Granule FLI-1/Klotho pathway colon cancer proliferation plasmid transfection Chinese medicine serum mechanism research |
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