Functional assessment of miR-1291 in colon cancer cells

概要

[Background] MiR-1291 has an anti-tumor effect in a subset of human carcinomas including pancreas, kidney, esophagus, and prostate. However, its role in colorectal cancer (CRC) is unknown.

[Purpose] The purpose of this study is to assess the anti-tumor effects of miR-1291 in CRC.

[Materials and Methods] We explored the expression of miR-1291 in CRC cell lines, and CRC tissues and normal mucosa from 20 paired clinical tissues. In vitro experiments including cell viability, BrdU proliferation, invasion, cell mobility, colony formation capability and apoptosis experiments were used to assess the effects of transfection of miR-1291 on DLD-1, HT29, and HCT116 cells. TargetScan human, miRwalk, miRabel, and miRmap were used to indicate the binding target of miR-1291 in silico, and luciferase reporter assay was used to verify the direct binding. QRT-PCR, Western blot analysis and Flow cytometric analysis were used to explore the effects of miR-1291 on cancer stem cell (CSC) markers including doublecortin-like kinase 1 (DCLK1), BMI1 and CD133. Sphere formation assay was also used for analyzing the effects on cell stemness. The function of DCLK1 was verified with DCLK1 siRNAs and sh-DCLK1 HCT116 clones to knockdown DCLK1, and with overexpression of DCLK1. Western blot and Flow cytometric analysis were used to analyze the change of cell cycle. Finally, DLD-1 xenograft mouse model was used to explore the anti-tumor effect of miR-1291 in vivo.

[Results] We found that miR-1291 expression was significantly lower in CRC tissues than in normal mucosa. MiR-1291 significantly suppressed the viability, BrdU proliferation, invasion, cell mobility, colony formation capability and induced apoptosis in CRC cells. In silico analyses indicated that CSC marker DCLK1 is a potential target of miR-1291. A luciferase reporter assay showed that miR-1291 directly bound the 3’UTR sequence of DCLK1. Among 3 CRC cell lines, HCT116 is known to retain the most CSC-like properties and HCT116 cells express DCLK1. MiR-1291 suppressed DCLK1 expression at both the mRNA and protein levels in HCT116 cells and it suppressed CSC markers, BMI1 and CD133 as well as sphere formation ability. With DCLK1 siRNAs, we also explored and verified the inhibitory effects of miR-1291 on sphere formation, invasion, and mobility of HCT116 cells. And we verified that sh-DCLK1 clones displayed decreased sphere formation of HCT116 cells, compared to sh-negative control cells. Moreover miR-1291 induced CDK inhibitors p21WAF1/CIP1, p27KIP1 in DLD-1, HT29, and HCT116, and overexpression of DCLK1 in HCT116 cells conversely led to a decrease of p21WAF1/CIP1 and p27KIP1. Furthermore, intravenous administration of miR-1291 loaded on the super carbonate apatite delivery system significantly inhibited a tumor growth in the DLD-1 xenograft mouse model. The resultant tumors showed significant up-regulation of the p21WAF1/CIP1 and p27KIP1 protein with treatment of miR-1291.

[Conclusions] MiR-1291 has an anti-tumor effect by modulating multiple functions, including cancer stemness and cell cycle regulation. MiR-1291 could be a promising nucleic acid medicine against CRC.