miR-505 functions as a tumor suppressor in glioma by targeting insulin like growth factor 1 receptor expression

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Compelling evidence shows that there are causative links between miRNAs abnormal regulation and the development of cancer. miR-505 has been reported to be aberrant expression and functions as a tumor suppressor in many human cancers, but its roles and potential molecular mechanism in glioma remain unclear. Here, we found that the expression levels of miR-505 were down-regulated in glioma tissues and cell lines. Exogenous over-expression of miR-505 resulted in inhibited cell proliferation and invasion in glioma in vitro. Furthermore, dual luciferase reporter assay and western blot analysis confirmed that IGF1R (Insulin like growth factor 1 receptor) was a direct target gene of miR-505 in glioma. More importantly, over-expression of IGF1R rescued miR-505-mediated inhibition of cell proliferation and invasion in glioma in vitro. Taken together, our results suggest that miR-505 acts as a tumor suppressor in glioma via direct negative regulation of IGF1R, which may provide a novel therapeutic strategy.

Keywords: IGF1R; glioma; miR-505; miRNAs; suppressor.

Figure 1

Expression levels of miR-505 in glioma tissues and glioma cell lines. A. Quantitative real-time PCR analysis of miR-505 expression in glioma tissues and matched normal brain tissues. B. Expression levels of miR-505 in human glioma cell lines (U87 and U251) and a primary human fetal glial cell (PHFG). U6 was used as an internal control. *P<0.05.

Figure 2

Restoration of miR-505 inhibited cell growth in glioma in vitro. (A) The expression levels of miR-505 were detected in mimics or NC transfected cells by quantitative real-time PCR. Mimics: miR-505 mimics; NC: corresponding negative control. The U87 (B) and U251 (C) cells were transfected with the mimics or NC, and MTT assay was used to determine cell growth ability at 0, 24, 48 h and 72 h. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. *P<0.05.

Figure 3

Over-expression of miR-505 suppressed cell invasion in glioma in vitro. Invasion of U87 and U251 cells following mimics or NC transfection was analyzed by transwell assay. Representative photographs were shown (magnification, 200 ×). *P<0.05.

Figure 4

MiR-505 targeted IGF1R and negatively regulated its expression. A. The predicted miR-505 binding site on the IGF1R mRNA 3’-UTR are shown. B. U87 and U251 cells were transfected with the WT and MUT recombinant reporter vectors as well as the mimics or NC. C. Measurement of IGF1R mRNA and protein expression levels by quantitative real-time PCR and Western blot analysis. The endogenous expression levels of GAPDH was used an internal control. WT: wild-type; mutant: MUT; 3’-untranslated regions: 3’-UTR. *P<0.05.

Figure 5

Over-expression of IGF1R rescued the inhibitory effects of miR-505 on glioma cells. A. IGF1R expression on mRNA and protein levels in U87 and U251 cells transfected with pCDNA3.1+ IGF1R over-expression plasmid or pCDNA3.1+ empty vector was detected by quantitative real-time PCR and Western blot, respectively. B. Cell proliferation was determined in U87 and U251 cells transfected with mimics with pCDNA3.1+ IGF1R over-expression plasmid or pCDNA3.1+ empty vector. C. The invasion of U87 and U251 cells was detected by transwell assay after treated with mimics with pCDNA3.1+ IGF1R over-expression plasmid or pCDNA3.1+ empty vector. Representative photographs were shown (magnification, 200 ×). *P<0.05.