Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells
Yukti Choudhury, Felix Chang Tay, Dang Hoang Lam, Edwin Sandanaraj, Carol Tang, Beng-Ti Ang, Shu Wang
Yukti Choudhury, Felix Chang Tay, Dang Hoang Lam, Edwin Sandanaraj, Carol Tang, Beng-Ti Ang, Shu Wang
View: Text | PDF
Research Article

Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells

Abstract

In the human brain, microRNAs (miRNAs) from the microRNA-376 (miR-376) cluster undergo programmed “seed” sequence modifications by adenosine-to-inosine (A-to-I) editing. Emerging evidence suggests a link between impaired A-to-I editing and cancer, particularly in high-grade gliomas. We hypothesized that disruption of A-to-I editing alters expression of genes regulating glioma tumor phenotypes. By sequencing the miR-376 cluster, we show that the overall miRNA editing frequencies were reduced in human gliomas. Specifically in high-grade gliomas, miR-376a* accumulated entirely in an unedited form. Clinically, a significant correlation was found between accumulation of unedited miR-376a* and the extent of invasive tumor spread as measured by magnetic resonance imaging of patient brains. Using both in vitro and orthotopic xenograft mouse models, we demonstrated that the unedited miR-376a* promoted glioma cell migration and invasion, while the edited miR-376a* suppressed these features. The effects of the unedited miR-376a* were mediated by its sequence-dependent ability to target RAP2A and concomitant inability to target AMFR. Thus, the tumor-dependent introduction of a single base difference in the miR-376a* sequence dramatically alters the selection of its target genes and redirects its function from inhibiting to promoting glioma cell invasion. These findings uncover a new mechanism of miRNA deregulation and identify unedited miR-376a* as a potential therapeutic target in glioblastoma cells.

Authors

Yukti Choudhury, Felix Chang Tay, Dang Hoang Lam, Edwin Sandanaraj, Carol Tang, Beng-Ti Ang, Shu Wang

×

Figure 6

Unedited miR-376a* promotes aggressive glioma growth in vivo.

Options: View larger image (or click on image) Download as PowerPoint
Unedited miR-376a* promotes aggressive glioma growth in vivo. (A) Orthot...
(A) Orthotopic gliomas formed by stably transfected U87 cells in mouse brain. Representative images from two cell clones each for U87/376a*A and U87/376a*G are shown. Top: Arrows indicate tumors; N, necrosis. Scale bars: 2 mm. Bottom: Tumor boundaries at higher magnification. Black arrowheads: peritumoral vascular structures for clone D5; disseminated tumor satellites for clone D3. Scale bars: 200 μm. (B) Immunohistochemical staining for proliferation marker Ki-67. Left: Higher Ki-67 signals at tumor edge demarcated by white dotted line for U87/376a*A tumors. BV, blood vessel. Scale bars: 100 μm. Right: Ki-67 signal quantification using ImageJ. Relative levels at tumor edge and core are indicated (n = 8–12). (C) Expression of factors involved in glioma invasion and angiogenesis in orthotopic tumors measured by qRT-PCR (n = 3). Left: Upstream growth factor signaling factors. Right: Factors promoting glioma cell invasion and angiogenesis. (D) Kaplan-Meier survival analysis after intracranial implantation of glioma cells. U87/376a*A clone D5 and U87/376a*G clone 2A2 stably express miR-376a*. 1M1 and 2M1 cells were enriched from U87 cells by ELM assay as in Figure 3. (E) Median survival of animals in D. Statistical analysis was done using log-rank test (n = 10). Error bars indicate SD. *P < 0.05, ***P < 0.001 by t test. In E, error bars represent SEM; **P < 0.01 and ***P < 0.001 vs. U87/control; ††P < 0.01 and †††P < 0.001 vs. U87/376a*G; ‡P < 0.001 vs. 1M1.