| Literature DB >> 32592373 |
Vahab Alamdari-Palangi1,2, Zahra Karami3, Hadi Karami2,4, Maryam Baazm5.
Abstract
BACKGROUND: Deregulation of the EGFR signaling pathway activity has been shown to can be effective in resistance to EGFR-TKIs, such as Tarceva (erlotinib), in glioblastoma cells. In addition, reports have shown that the reduction of miRNA-7 expression levels is associated with an increase in the expression of EGFR. Here, we evaluated the effect of miRNA-7 on EGFR expression and sensitivity of the U373-MG glioblastoma to erlotinib.Entities:
Keywords: Apoptosis; EGFR; Glioblastoma; MiRNA-7; erlotinib
Year: 2020 PMID: 32592373 PMCID: PMC7568905 DOI: 10.31557/APJCP.2020.21.6.1747
Source DB: PubMed Journal: Asian Pac J Cancer Prev ISSN: 1513-7368
Figure 1RT-qPCR Analyses of EGFR mRNA in U373-MG Cells. To measure the expression of EGFR in glioblastoma cells, the U373-MG cells were transfected with negative control (NC) miRNA and miRNA-7 for 24 and 48 h. Relative EGFR mRNA expression was quantified by RT-qPCR using 2 - (∆∆Ct) method and β-actin as an internal control. Data are presented as mean ± SD of three independent experiments. *p < 0.05; #p > 0.05 versus blank control
Figure 2EGFR Protein Expression Levels in Glioblastoma Cells Transfected with miRNAs. Representative western blots of EGFR and β-actin proteins after 24 (A) and 48 (B). The effect of miRNA-7 on expression levels of EGFR was quantified using densitometry and normalized to the respective β-actin (C). The results are expressed as mean±SD of the results of three independent experiments. *p < 0.05; #p > 0.05 versus blank control
Figure 3Growth Inhibition of U373-MG Cells Transfected with miRNA-7. The cells were transfected with negative control (NC) miRNA and miRNA-7 for 24-120 h. The cell growth rate was analyzed by trypan blue exclusion assay at the end of each period. The results are represented as mean ± SD (n=3). *p < 0.05 versus blank control or NC miRNA
Figure 4Synergistic Effect between miRNA-7 and Erlotinib in Human U373-MGCells. Twenty-four (A and B) and forty-eight (C and D) hours effect of miRNA-7 on the sensitivity of the glioblastoma cells to erlotinib were measured by MTT assay as described in the method section. The cell survival curves were plotted using GraphPad software. The results are expressed as mean ± SD of three independent experiments. Combination index (CI) versus fractional effect (Fa) was plotted using the Chou-Talalay method and CalcuSyn software. A horizontal dashed line shows CI of 1
IC50 of Erlotinib Alone and in Combination with miRNAs in U373-MG Cells, after 24 and 48 h of Treatment
| Treatment | IC50 (µM) | |
|---|---|---|
| 24 h | 48 h | |
| Erlotinib | 46.27 ± 1.18 | 31.92 ± 2.20 |
| NC miRNA and erlotinib | 43.61 ± 1.10# | 29.59 ± 0.80# |
| miRNA-7 and erlotinib | 20.30 ± 1.25* | 14.48 ± 1.45* |
IC50 were determined by sigmoidal dose-response model using GraphPad software. Results expressed as the mean ± SD (n=3). *p < 0.05 versus corresponding erlotinib group; #p > 0.05 relative to the corresponding erlotinib.
CI Analysis of Combination Treatment in U373-MG Cells
| Erlotinib concentration (µM) | 24 h | 48 h | ||||
|---|---|---|---|---|---|---|
| Fa | CI | Combined effect | Fa | CI | Combined effect | |
| 2.5 | 0.16 | 0.94 | S | 0.21 | 0.92 | S |
| 5 | 0.18 | 0.93 | S | 0.26 | 0.9 | S |
| 10 | 0.25 | 0.90 | S | 0.29 | 0.87 | S |
| 20 | 0.47 | 0.88 | S | 0.51 | 0.85 | S |
| 40 | 0.69 | 0.83 | S | 0.79 | 0.82 | S |
| 80 | 0.91 | 0.80 | S | 0.96 | 0.80 | S |
| 160 | 0.98 | 0.79 | S | 1.00 | 0.75 | S |
The effects of miRNA-7 and erlotinib combination treatments were determined with the combination index (CI) analysis based upon the Chou-Talalay method. A CI values less than 1, equal to 1, or greater than 1 indicates synergistic (S), additive, or antagonistic interaction, respectively.
Figure 5Effect of miRNA-7 on Erlotinib-Mediated Apoptosis in U373-MG Cells. The cells were treated with miRNA-7 (50 nM), negative control (NC) miRNA (50 nM) and erlotinib (IC50 doses of 24 and 48 h), alone and in combination. After 24 and 48 h, apoptosis was measured by cell death ELISA assay. The data are expressed as mean ± SD (n=3). *p < 0.05 relative to blank control; #p < 0.05 relative to miRNA-7 or erlotinib alone