miR-34a inhibits the proliferation and invasion of colon cancer stem cells by regulating SIRT1 expression

doi:10.3969/j.issn.2095-4344.0521

Abstract

Abstract:

BACKGROUND: miR-34a has an antitumor effect and can regulate the expression of multiple target genes. Therefore, it may become a new target for the treatment of tumors.
OBJECTIVE: To investigate the effect of miR-34a and its target gene SIRT1 on the proliferation, apoptosis and invasion of colon cancer stem cells in vitro.
METHODS: Colon cancer stem cells were enriched from human colon cancer cell line HCT116 by serum-free suspension culture. Then, the percentage of CD44⁺/CD133⁺ cells was identified by flow cytometry method. Colon cancer stem cells in transfection and control groups were transfected with artificially synthesized miR-34a mimics and negative control sequences, respectively. Cells with no transfection were used as non-transfection group. Cell proliferation, apoptosis, and invasion were detected through cell counting kit-8 assay, cell apoptosis experiment, and cell invasion experiment, respectively. The expression of SIRT1 mRNA and protein was detected by qRT-PCR and western blot analysis, respectively.
RESULTS AND CONCLUSION: CD44⁺/CD133⁺ cells accounted for (78.3±6.7)% of the tumor stem cells enriched in the serum-free medium. The expression of miR-34a in miR-34a transfection group was higher than that of non-transfection group and miR-34a control group (P < 0.05). Compared with the non-transfection group and the miR-34a control group, the cell proliferation ability was significantly decreased (P < 0.05), the apoptotic rate increased significantly (P < 0.05), and the number of invasion cells decreased significantly (P < 0.05) in the miR-34a transfection group. The expression level of SIRT1 mRNA and protein in the miR-34a transfection group was significantly lower than that in the non-transfection group and the miR-34a control group (P < 0.05). These findings suggest that miR-34a may down-regulate the expression of SIRT1 to suppress cell growth and invasion and promote apoptosis in colon cancer stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Colonic Neoplasms, Neoplastic Stem Cells, MicroRNAs, Cell Proliferation, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Li He, Li Jun, Chen Xing-chao. miR-34a inhibits the proliferation and invasion of colon cancer stem cells by regulating SIRT1 expression[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2680-2685.

Figures/Tables 1

2.1 HCT116在无血清培养条件下形成的细胞球 HCT116结肠癌细胞接种于无血清培养基中，培养第2天后可观察到悬浮的细胞呈出芽式生长，第3天可见三维聚集体，第5天时已形成形态规则的球形，细胞之间连接紧密。所有的细胞球需要及时胰酶消化传代，第2代和第3代微球体形态更接近球形，生长旺盛，第5代之后，细胞球体数量减少，细胞之间连接较前疏松，且更加容易贴壁分化。流式细胞术分析发现，上述分离的肿瘤干细胞球中含有较高比例的CD44+/CD133+表型细胞，所占比例为(78.3±6.7)%。 2.2 miR-34a在结肠癌干细胞中表达下调采用qRT- PCR技术检测CD44+/CD133+结肠癌干细胞中miR-34a表达，以结肠癌细胞HCT116为对照。结果显示，miR-34a在CD44+/CD133+结肠癌干细胞中的表达水平显著低于结肠癌细胞，差异有显著性意义(P < 0.01)，见图1。 2.3 miR-34a转染效率采用脂质体转染法转染miR-34a模拟物后，qRT-PCR检测miR-34a转染效率，结果显示，miR-34a转染组的miR-34a表达量高于未转染组和miR-34a对照组，差异有显著性意义(P < 0.05)；而未转染组和miR-34a对照组的miR-34a表达量差异无显著性意义(P > 0.05)，见图2。 2.4 miR-34a过表达抑制CD44+/CD133+结肠癌干细胞的增殖采用CCK-8法检测转染后CD44+/CD133+结肠癌干细胞的增殖情况。结果显示，miR-34a转染组的细胞增殖能力明显低于未转染组和miR-34a对照组，差异有显著性意义(P < 0.05)，见图3。 2.5 miR-34a过表达促进CD44+/CD133+结肠癌干细胞的凋亡采用流式细胞技术检测转染后CD44+/CD133+结肠癌干细胞的凋亡情况，结果显示，miR-34a转染组的细胞凋亡率明显高于未转染组和miR-34a对照组，差异有显著性意义(P < 0.05)，见图4。 2.6 miR-34a过表达抑制CD44+/CD133+结肠癌干细胞的侵袭 Transwell小室实验结果显示，miR-34a转染组的侵袭细胞数明显低于未转染组和miR-34a对照组，差异有显著性意义(P < 0.05)，见图5。 2.7 miR-34a下调CD44+/CD133+结肠癌干细胞中SIRT1的表达水平采用qRT-PCR和Western blot法检测miR- 34a过表达后细胞中SIRT1 mRNA和蛋白的表达水平，结果显示，miR-34a转染组SIRT1 mRNA和蛋白的表达水平均明显低于未转染组和miR-34a对照组，差异有显著性意义(P < 0.05)，见图6。"

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