miR-15 family promotes the migration and invasion of CD133+ pancreatic cancer stem-like cells

doi:10.3969/j.issn.2095-4344.0897

Abstract

Abstract:

BACKGROUND: Some studies have shown that CD133 is a stem cell marker for a variety of tumor cells, such as pancreatic cancer, which is associated with a poor prognosis in cancer patients. miR-15 family is involved in cell apoptosis, differentiation, cycle regulation and stress, and has potential value as an intervention.
OBJECTIVE: To investigate the effects of miR-15 family on the migration and invasion of CD133⁺ pancreatic cancer stem-like cells.
METHODS: First, a highly migrating pancreatic cancer cell line, Capan-1M9, was constructed, and then CD133⁺Capan-1M9 cell lines that overexpressed miR-15a, miR-15b and miR-15c were constructed by lentivirus transduction. NC-miRNA cell lines were taken as negative controls. Expression of miR-15a, miR-15b and miR-15c in the recombinant cell lines and epithelial-mesenchymal transition (EMT)-related proteins, fibronectin, vimentin and N-cadherin mRNA were determined by qRT-PCR. MTT assay was used to detect the proliferation and gemcitabine resistance of recombinant cell lines. Spheroid formation test was used to detect the self-renewal ability of recombinant cell lines. Migration and invasion ability of the recombinant cell lines were detected by cell migration and invasion assay. EMT-related proteins fibronectin, vimentin and N-cadherin were detected by western blot.
RESULTS AND CONCLUSION: qPCR results showed that the expression of miR-15a, miR-15b and miR-15c in the CD133⁺ Capan-1M9 cell lines increased by 8.3, 10 and 9.5 times compared with the negative control group, and the expression of fibronectin, vimentin and N-cadherin mRNA was also significantly increased (P < 0.05). There was no significant difference in cell proliferation between the recombinant cell lines and the negative control group (P > 0.05), but the gemcitabine resistance was significantly increased in the recombinant cell lines as detected by the MTT (P < 0.05). The number and volume of spheres in the recombinant cell lines did not change significantly compared with the negative control group (P > 0.05). The cell migration and invasion abilities of the recombinant cell line were significantly enhanced compared with the negative control group (P < 0.05). Our experimental findings suggest that the miR-15 family can promote migration and invasion of CD133⁺ pancreatic cancer stem-like cells via EMT regulation.

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: Pancreatic Neoplasms, MicroRNAs, Neoplastic Stem Cells, Cell Movement, Tissue Engineering

CLC Number:

R394.2

Cai Shang-dang, Cai Man-di, Lou Ning, Guo Yan-ping, Yin Tao. miR-15 family promotes the migration and invasion of CD133⁺ pancreatic cancer stem-like cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3316-3321.

Figures/Tables 2

2.1 过表达miR-15a、miR-15b和miR-15c的细胞系通过慢病毒转导的方式建立过表达miR-15a、miR-15b和miR-15c的CD133+Capan-1M9细胞系，同时转导随机序列的细胞系作为对照，使用FACSAria分析GFP表达阳性的CD133+Capan-1M9细胞比例，GFP阳性细胞比例超过90%(图1A)，qPCR检测结果表明，构建的3种细胞系中miR-15a、miR-15b和miR-15c的表达量显著升高(图1B)。 2.2 过表达miR-15不影响CD133+Capan-1M9细胞增殖和球体形成为检测过表达miR-15a、miR-15b和miR-15c对CD133+Capan-1M9细胞增殖的影响，使用MTT法检测细胞增殖情况。结果表明，与对照组细胞相比，过表达miR-15a、miR-15b和miR-15c不影响细胞增殖(图2A)。通过球体形成实验检测肿瘤干细胞的自我更新能力，结果表明，与对照组相比，miR-15a、miR-15b和miR-15c过表达细胞中球体数量和大小没有显著差异(图2B和D)，表明miR-15a、miR-15b和miR-15c对CD133+Capan-1M9的球体形成没有显著影响。检测miR-15a、miR-15b和miR-15c对细胞吉西他滨化疗抗性的影响。结果表明，miR-15a、miR-15b和miR-15c过表达细胞系均对吉西他滨产生抗性(图2C)。miR-15a、miR-15b和miR-15c能够促进CD133+Capan-1M9细胞的吉西他滨抗性。 2.3 miR-15a、miR-15b和miR-15c促进CD133+ Capan-1M9细胞迁移和侵袭过表达miR-15a、miR-15b和miR-15c均显著促进了CD133+Capan-1M9细胞的迁移和侵袭(图3A和B)。 2.4 miR-15家族促进间充质标记表达荧光定量RT-PCR检测结果表明，miR-15a、miR-15b和miR-15c过表达CD133+Capan-1M9细胞系中，间质标记纤维连接蛋白、波形蛋白和N-神经钙黏素mRNA表达均显著增加(图4A)。Western blot检测结果表明，miR-15a、miR-15b和miR-15c过表达CD133+Capan-1M9细胞系中纤维连接蛋白、波形蛋白和N-神经钙黏素表达量均增加(图4B)。上述结果表明，miR-15a、miR-15b和miR-15c促进CD133+ Capan-1M9细胞迁移和侵袭与间质表型之间有相关性。"

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