miR-139 Functions as An Antioncomir to Repress Glioma Progression Through Targeting IGF-1 R, AMY-1, and PGC-1β

Abstract

Gliomas are the most common primary malignant brain tumor with poor prognosis, characterized by a highly heterogeneous cell population, extensive proliferation, and migration. A lot of molecular mechanisms regulate gliomas development and invasion, including abnormal expression of oncogenes and variation of epigenetic modification. MicroRNAs could affect cell growth and functions. Several reports have demonstrated that miR-139 plays multifunctions in kinds of solid tumors through different pathways. However, the antitumor mechanisms of this miR-139 are not unveiled in detail. In this study, we not only validated the low expression level of miR-139 in glioma tissues and cell lines but also detected the effect of miR-139 on modulating gliomas proliferation and invasion both in vitro and in vivo. We identified insulin-like growth factor 1 receptor, associate of Myc 1, and peroxisome proliferator-activated receptor γ coactivator 1β as direct targets of miR-139 and the levels of them were all inversely correlated with miR-139 in gliomas. Insulin like growth factor 1 receptor promoted gliomas invasion through Akt signaling and increased proliferation in the peroxisome proliferator-activated receptor γ coactivator 1β-dependent way. Associate of Myc 1 also facilitated gliomas progression by activating c-Myc pathway. Overexpression of the target genes could retrieve the antitumor function of miR-139, respectively, in different degrees. The nude mice transplantation tumor experiment displayed that glioma cells stably expressed miR-139 growth much slower in vivo than the negative control cells. Taken together, these findings suggested miR-139 acted as a favorable factor against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new evidenced prognostic marker and therapeutic target for gliomas.

Keywords: AMY-1; IGF-1 R; PGC-1β; glioma; miR-139; tumor progression.

Figure 1.

miR-139 expression in glioma tissues and cell lines. A, The total RNA was extracted from the normal and glioma tissues excised from patients undergoing surgery. The miR-139 expression levels were detected by reverse transcription polymerase chain reaction (RT-PCR; normal: n = 10; gliomas: n = 24). B, The gliomas were divided into 2 groups according to the World Health Organization (WHO) guidelines. miR-139 expression was compared in gliomas with different grades (grade I/II: n = 12; grade III/IV: n = 12). C, miR-139 expression was compared in brain cells and different glioma cell lines (n = 6). D, The expression of insulin-like growth factor type 1 receptor (IGF-1 R), associate of Myc-1 (AMY-1), and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) in normal brain tissues and different grades glioma tissues. E, The linear regression relationship between the expression of miR-139 and its target genes. F, The expression of IGF-1 R, AMY-1, and PGC-1β in brain cells and glioma cell lines. Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 2.

miR-139 repressed gliomas proliferation. A and B, U251 and U87MG cells were seeded in 96-well plates after transfecting miR-139 or control oligonucleotide. The cell proliferation was evaluated at 24, 48, 72, and 96 hours (n = 5). C and D, U251 and U87MG cells were overexpressing miR-139 as above and collected for further flow cytometry analysis to detect the cell cycle (n = 4). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 3.

miR-139 repressed gliomas migration and invasion. A and B, U87MG and U251 cells were transfected miR-139 or control. The wound-healing assay was performed to evaluate the migration ability (n = 4). C and D, U87MG and U251 cells were treated as above, and the invasion was detected by using transwell assay (n = 4). E, Total RNA was extracted from U87MG and U251 cells transfected miR-139 or control. The expression of epithelial-mesenchymal transition (EMT markers was tested (n = 4). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 4.

miR-139 reduced the expression of its target genes. A, Sequence of the 3′ untranslated region (3′ UTR) of insulin-like growth factor type 1 receptor (IGF-1 R), associate of Myc-1 (AMY-1), and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) matched with the recognition site of miR-139. The seed sequence was marked in red color. B and C, Protein levels of IGF-1 R, AMY-1, and PGC-1β were regulated by miR-139 both in U87MG and U251 cells (n = 4). D, pGL3-promoter vector with the 3′ UTR of IGF-1 R, AMY-1, and PGC-1β (gray box), the mutant 3′ UTR regions (black box) or the control (white box) were co-transfected into U87MG cells with an miR-139 mimic or control mimic respectively. Twenty-four hours after transfection, luciferase activity was determined (n = 5). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 5.

miR-139 inhibited Akt, MAPK, and c-Myc signaling through insulin-like growth factor type 1 receptor (IGF-1 R) and associate of Myc-1 (AMY-1). A and B, U87MG and U251 cells were transfected with IGF-1 R or miR-139 and empty vector or scramble as negative controls. The protein expression of downstream signaling was detected (n = 4). C, Insulin-like growth factor type 1 receptor was transfected into miR-139 overexpressing U87MG cells and the total and phosphorylation level of Akt and MAPK were detected (n = 3). D, The glioma cells were overexpressing IGF-1 R and added with Akt inhibitor at the same time. The peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) expression was detected (n = 4). E, The glioma cells were transfected with AMY-1 or miR-139 and the expression level of c-Myc signaling molecules was evaluated (n = 4). F, AMY-1 was transfected into miR-139 overexpressing U87MG cells and the RNA levels of cell division cycle 25 homologue A (cdc25A) and p27 were detected (n = 3). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 6.

miR-139 repressed glioma cells proliferation through insulin-like growth factor type 1 receptor (IGF-1 R), associate of Myc-1 (AMY-1), and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β). A, The knock down efficiency of synthetic RNAs (siRNAs) for IGF-1 R, AMY-1, and PGC-1β (n = 4). B, The glioma cells were transfected with effective siRNAs of IGF-1 R, associate of Myc-1 (AMY-1), and PGC-1β and detected the proliferation by methyl thiazolyl tetrazolium (MTT; n = 5). C, The glioma cells were transfected IGF-1 R, associate of Myc-1 (AMY-1), and PGC-1β in the condition of miR-139 overexpressed. The MTT assay was performed to tested proliferation of glioma cells (n = 5). D, The glioma cells were transfected siRNA of IGF-1 R with PGC-1β together and tested the proliferation (n = 5). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 7.

miR-139 reduced glioma cells invasion through IGF-1 R and associate of Myc-1 (AMY-1). A and B, U87MG and U251 cells were transfected the synthetic RNAs (siRNAs) for target genes of miR-139 and seeded into the transwell chambers. The cells numbers of infiltrated gliomas were counted (n = 4). C and D, The different glioma cell lines that were transfected miR-139 were overexpressed IGF-1 R and associate of Myc-1 (AMY-1) to rescue. The migrated cell numbers were calculated (n = 4). Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.

Figure 8.

miR-139 plays an antitumor function and inhibit the Akt signaling in vivo. A, U87MG cells stably expressing miR-139 and negative control were injected subcutaneously in 2 groups, respectively. The phenotype of tumors was observed after 20 days (n = 5). B and C, The growth of tumors was monitored by measuring tumor size every 3 days from the 8th day, and the tumors’ weight was measured after the mice were killed. D and E, The RNA and protein were extracted from the tumors of different groups, and the expression of IGF-1 R, associate of Myc-1 (AMY-1), and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) were detected. F, The RNA levels of c-Myc pathway downstream genes were determined. G, The schematic diagram of miR-139 modulated gliomas progression. Bars represent means ± SD, *P < .05, **P < .01, ***P < .001.