miRNA-302b在结肠癌HT-29细胞“干性”维持中的作用

朱蓉; 鲁生念; 李伶俐; 俆永素; 赵逵

doi:10.3971/j.issn.1000-8578.2017.17.0420

miRNA-302b在结肠癌HT-29细胞“干性”维持中的作用

Effect of miRNA-302b on "Stemness" Maintaining of Colon Cancer HT-29 Cells

摘要

摘要:
目的探讨miRNA-302b在结肠癌HT-29细胞“干性”维持中的作用及可能机制。
方法体外培养人结肠癌细胞株HT-29，转染miRNA-302b模拟物（miRNA-302b mimics），同时以未转染组（control）和阴性模拟物转染组（negative control-mimics, NC-mimics）作为对照，采用实时定量反转录聚合酶链反应法（real-time PCR）检测HT-29细胞结肠癌“干性”标志物Ascl2、Lgr5、Oct4和Sox2 mRNA的表达情况；采用蛋白印迹法（Western blot）检测以上“干性”标志蛋白表达的变化；采用克隆形成实验及干细胞成球实验分别观察HT-29细胞克隆形成和细胞球形成能力的变化。
结果 Real-time PCR结果示，miR-302b mimics转染HT-29细胞48 h，“干性”标志物Ascl2、Lgr5、Oct4和Sox2 mRNA表达一致性上调（P < 0.05）；Western blot结果示Ascl2、Lgr5、Oct4和Sox2蛋白表达也上调（P < 0.05）。克隆形成实验及干细胞成球实验结果示，miR-302b mimics转染后HT-29细胞克隆形成和细胞球形成能力增强，与control组和NC-mimics组相比差异均有统计学意义（P < 0.05）。
结论 miRNA-302b可通过上调“干性”标志物Ascl2、Lgr5、Oct4和Sox2的表达促进HT-29细胞的克隆形成和细胞球形成能力，从而在结肠癌“干性”维持中发挥重要作用。

Abstract:
Objective To investigate the effects and possible mechanisms of miRNA-302b on "stemness" maintaining of colon cancer HT-29 cells.
Methods Human colon cancer cell line HT-29 was cultivated in vitro, and miRNA-302b mimics were transfected into HT-29 cells, while the non-transfection group (control) and negative control-mimics transfection group (NC-mimics) were taken as controls. Real-time PCR was used to detect mRNA expressions of colon cancer "stemness" markers (Ascl2, Lgr5, Oct4 and Sox2). Western blot was used to detect their protein expressions. Colony formation experiment was used to detect the colony formation ability of HT-29 cells, and stem cell sphere formation experiment was used to detect the sphere formation ability.
Results Real-time PCR showed that the mRNA expressions of "stemness" markers (Ascl2, Lgr5, Oct4 and Sox2) were up-regulated 48h after miR-302b mimics transfection into HT-29 cells (P < 0.05); Western blot showed that the protein expressions were also up-regulated (P < 0.05). Colony formation and sphere formation experiment showed that the colony and sphere formation abilities of HT-29 cells in miRNA-302b mimics transfection group were enhanced, compared with control group and NC-mimics group (P < 0.05).
Conclusion miRNA-302b enhances the colony and sphere formation abilities of HT-29 cells, through upregulating the expressions of "stemness" markers (Ascl2, Lgr5, Oct4 and Sox2), thus, miR-302b plays an important role in "stemness" maintenance of colon cancer.

HTML全文

参考文献(16)

施引文献

资源附件(0)