miR-219-5p Inhibits Receptor Tyrosine Kinase Pathway by Targeting EGFR in Glioblastoma

<div><p>Glioblastoma is one of the common types of primary brain tumors with a median survival of 12–15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3′-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3′-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.</p></div

Overexpression of miR-219-5p in glioma cell lines decreased proliferation, anchorage independent growth and migration of glioma cells.

<p><b>A.</b> U138, U343 and U251 cells were transfected with negative control pre-miRs (P-neg) or pre-miRs for miR-219-5p (P-219) and plated in triplicates in 24-well plates. The cell number was counted by using hemocytometer at indicated time points. Average cell number ± SD was plotted. p value was calculated by Students' t test in MS excel. * represents significant p value. For U138, the p values were 0.042, 0.006, 0.003 and 0.003 for Day 2, Day 3, Day 4 and Day 5 respectively; for U343, the p values were 0.06, 0.004, and 0.003 for Day 3, Day 4 and Day 5 respectively; for U251, the p values were 0.009, 0.01 for Day 4 and Day 5 respectively. <b>B.</b> U343 cells were transfected with negative control pre-miRs (P-neg) or pre-miRs for miR-219-5p (P-219) and 300 cells were plated in triplicates in 12-well plates. After 3 weeks of plating, the colonies were stained with crystal violet, photographed (top) and counted (bottom). Average colony number ± SD was plotted. p value was calculated by Students' t test in MS excel. <b>C.</b> U138 and U251 cells were transfected with negative control pre-miRs (P-neg) or pre-miRs for miR-219-5p (P-219). After 36 hrs of transfection, cells were re-plated on the soft agar plates in triplicates. After 3 weeks, the soft agar colonies were counted under microscope. Average colony number ± SD was plotted. p value was calculated by Students' t test in MS excel <b>D.</b> U138 cells were transfected with negative control pre-miRs (P-neg) or pre-miRs for miR-219-5p (P-219). After 72 hrs of transfection, cells were counted and plated for matrigel migration assay and after 18 hrs, cells were fixed, stained with crystal violet, photographed. <b>E.</b> Quantification of <b>D.</b> Average cell number ± SD was plotted. p value was calculated by Students' t test in MS excel.</p

Overexpression of miR-219-5p reduced the activities of MAPK and PI3K pathways.

<p>U138 and U251 cells were transfected with pre-miR-219-5p (P-219) or negative control pre-miRs (P-neg). After 72 hrs of pre-miR transfection, cell lysate was prepared and subjected to the western blotting for phospho-ERK 1/2 (Tyr202/Tyr204) and total ERK 1/2; phospho-Akt (Ser473), total Akt and phospho-4E-BP1 (Ser65).</p

miR-219-5p is downregulated in glioblastoma.

<p>Expression of miR-219-5p in malignant astrocytoma [anaplastic astrocytoma (AA) and glioblastoma (GBM)] as compared to the control normal brain tissue (Normal) is plotted as scatter plot. <b>A.</b> From the miRNA microarray data, miR-219-5p expression was found to be significantly downregulated in anaplastic astrocytoma (AA; n = 13) and glioblastoma (GBM; n = 26) with a p value of 0.0024 (from Kruskal-Wallis test in One-Way ANOVA). <b>B.</b> In the real-time qPCR validation set, miR-219-5p expression was found to be significantly downregulated in glioblastoma (GBM; n = 24) with a p value of 0.0098 (from Mann-Whitney test). <b>C.</b> In the TCGA dataset, miR-219-5p expression was found to be significantly downregulated glioblastoma (GBM; n = 492) with a p value of <0.0001 (from Mann-Whitney test).</p

EGFR protein levels negatively correlated with miR-219-5p expression levels in glioblastoma samples.

<p>TCGA glioblastoma samples were divided into Low 219 (n = 66) and High 219 (n = 69) groups keeping median value for miR-219-5p as cut off. The EGFR transcript (<b>A</b>), total EGFR protein levels (<b>B</b>) and the protein levels of phosphorylated EGFR at Tyr1068, Tyr1173 and Tyr992 (<b>C</b>) were plotted as scatter plot for each group. p value was calculated by Mann-Whitney test in GraphPad Prism software.</p

miR-219-5p targets EGFR.

<p><b>A.</b> Predicted binding site for miR-219-5p in the 3′-UTR of EGFR. EGFR-mut-UTR shows the sequence of the mutated 3′-UTR. <b>B.</b> U138, U343 and U251 cell lines were transfected with Pre-miR-219 (P-219) or negative control pre-miRs (P-neg). After 72 hrs of transfection, western blot for EGFR was performed using the total cell lysate. <b>C.</b> U343 and U251 cell lines were transfected with pre-miRs for miR-219-5p (P-219) or negative control pre-miRs (P-neg). After 36 hrs of pre-miR transfection, pGL3-EGFR-UTR reporter plasmid was transfected and after 48 hrs, luciferase activity was measured. Mean luciferase activity ± SD was plotted. p value was calculated by Students' t test in MS excel. <b>D.</b> U251 cell line was transfected with Pre-miR-219 (P-219) or negative control pre-miRs (P-neg). After 36 hrs of pre-miR transfection either wild type pGL3-EGFR-UTR (wt UTR) or two clones of mutant pGL3-EGFR-UTR (mut1 and mut2) reporter plasmid was transfected and after 48 hrs, luciferase activity was measured. Mean activity ± SD was plotted and p value was calculated by Students' T test in MS excel. NS represents Non-Significant p value.</p

A 16-Gene Signature Distinguishes Anaplastic Astrocytoma from Glioblastoma

<div><p>Anaplastic astrocytoma (AA; Grade III) and glioblastoma (GBM; Grade IV) are diffusely infiltrating tumors and are called malignant astrocytomas. The treatment regimen and prognosis are distinctly different between anaplastic astrocytoma and glioblastoma patients. Although histopathology based current grading system is well accepted and largely reproducible, intratumoral histologic variations often lead to difficulties in classification of malignant astrocytoma samples. In order to obtain a more robust molecular classifier, we analysed RT-qPCR expression data of 175 differentially regulated genes across astrocytoma using <i>P</i>rediction <i>A</i>nalysis of <i>M</i>icroarrays (PAM) and found the most discriminatory 16-gene expression signature for the classification of anaplastic astrocytoma and glioblastoma. The 16-gene signature obtained in the training set was validated in the test set with diagnostic accuracy of 89%. Additionally, validation of the 16-gene signature in <i>multiple</i> independent cohorts revealed that the signature predicted anaplastic astrocytoma and glioblastoma samples with accuracy rates of 99%, 88%, and 92% in TCGA, GSE1993 and GSE4422 datasets, respectively. The protein-protein interaction network and pathway analysis suggested that the 16-genes of the signature identified epithelial-mesenchymal transition (EMT) pathway as the most differentially regulated pathway in glioblastoma compared to anaplastic astrocytoma. In addition to identifying 16 gene classification signature, we also demonstrated that genes involved in epithelial-mesenchymal transition may play an important role in distinguishing glioblastoma from anaplastic astrocytoma.</p></div

KEGG pathway analysis showed the upregulation of Focal adhesion pathway in GBM.

<p>67 differentially expressed genes between AA and GBM from GSE1993 was subjected to network analysis in Pathway Express which identified the focal adhesion as the significantly differentially regulated pathway between AA and GBM. The input genes present in the network are represented in red and blue indicating the upregulation or downregulation respectively in GBM as compared to AA.</p

Expression of 16 genes selected from PAM in AA (n = 30) and GBM (n = 78) samples of the training set.

*<p>P value from student's T-test.</p