摘要
背景:一些研究表明,CD133是包括胰腺癌在内的多种肿瘤细胞的干细胞标记,与癌症的预后不良有关。miR-15家族参与凋亡、细胞分化与周期调控、应激等重要细胞功能活动的调节,具有潜在的干预价值。目的:探讨miR-15家族对CD133^+胰腺癌干样细胞迁移和侵袭的影响。方法:首先构建高迁移性胰腺癌细胞亚系CD133^+Capan-1M9,经鉴定具有干样细胞特征,在此基础上通过慢病毒转导的方式构建过表达miR-15a、miR-15b和miR-15c的CD133^+Capan-1M9细胞系,同时将稳定转染NC-miRNA的细胞系作为阴性对照组。qRT-PCR检测过表达细胞系中miR-15a、miR-15b和miR-15c表达情况及上皮-间充质转化相关蛋白纤维连接蛋白、波形蛋白和N-神经钙黏素mRNA表达;MTT法检测过表达细胞系增殖能力和吉西他滨抗性;球体形成实验检测过表达细胞系自我更新能力;细胞迁移和侵袭实验检测过表达细胞系的迁移和侵袭能力;Western blot检测过表达细胞系中上皮-间充质转化相关蛋白纤维连接蛋白、波形蛋白和N-神经钙黏素表达。结果与结论:(1)与阴性对照组相比,过表达CD133^+Capan-1M9细胞系中miR-15a、miR-15b和miR-15c表达量分别升高了8.3,10,9.5倍,而纤维连接蛋白、波形蛋白和N-神经钙黏素mR NA和蛋白表达也显著升高(P<0.05);(2)过表达CD133^+Capan-1M9细胞系增殖能力与阴性对照组相比差异无显著性意义(P>0.05),而对吉西他滨的抗性显著增强(P<0.05);(3)与阴性对照组相比,过表达CD133^+Capan-1M9细胞系的球体数量和体积没有显著变化(P>0.05);(4)与阴性对照组相比,过表达CD133^+Capan-1M9细胞系的细胞迁移和侵袭能力显著增强(P<0.05);(5)miR-15家族能够调控上皮-间质转化进而促进CD133^+胰腺癌干样细胞的迁移与侵袭。
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.
作者
蔡尚党
蔡满地
娄宁
郭艳萍
殷涛
Cai Shang-dang;Cai Man-di;Lou Ning;Guo Yan-ping;Yin Tao(Department of Anorectal,Henan Traditional Chinese Medicine Hospital,Zhengzhou 450002,Henan Province,China;Department of Clinical Medicine,Medical College of Henan University,Kaifeng 475000,Henan Province,China;ICU,Sun Yat-sen University Cancer Center,Guangzhou 510000,Guangdong Province,China;Department of Pathology,Henan Provincial People＇s Hospital,Zhengzhou 450008,Henan Province,China;Pancreatic Disease Research Institute,Wuhan Union Hospital of Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430022,Hubei Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第21期3316-3321,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81372665)~~
