MiRNA-7 Replacement Effect on Proliferation and Tarceva-Sensitivity in U373-MG Cell Line

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.

Methods: The effect of miRNA-7 on EGFR expression was examined using RT-qPCR and western blotting. Trypan blue and MTT assays were performed to explore the effect of treatments on cell growth and survival, respectively. The combination index analysis was used to evaluate the interaction between drugs. Apoptosis was measured by ELISA cell death assay.

Results: We showed that miRNA-7 markedly inhibited the expression of EGFR and decreased the growth of glioblastoma cells, relative to blank control and negative control miRNA (p < 0.05). Introduction of miRNA-7 synergistically increased the sensitivity of the U373-MG cells to erlotinib. Results of apoptosis assay demonstrated that miRNA-7 can trigger apoptosis and enhance the erlotinib-mediated apoptosis.

Conclusions: Our results show that miRNA-7 plays a critical role in the growth, survival and sensitivity of the U373-MG cells to erlotinib by targeting EGFR. Thus, miRNA-7 replacement therapy can become an effective therapeutic procedure in glioblastoma.

Keywords: Apoptosis; EGFR; Glioblastoma; MiRNA-7; erlotinib.

Figure 1

RT-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 2

EGFR 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 3

Growth 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 4

Synergistic 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

Figure 5

Effect 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