[shRNA-mediated insulin-like growth factor I receptor gene silencing inhibits cell proliferation, induces cell apoptosis, and suppresses tumor growth in non-small cell lung cancer: in vitro and in vivo experiments]

Abstract

Objective: To study the effects of RNA interference (RNAi)-mediated insulin-like growth factor I receptor (IGF-IR) gene silencing on human lung cancer cells.

Methods: Plasmids expressing IGF-IR shRNA1 and IGF-IR shRNA2 were constructed. Human non-small cell lung cancer cells of the line A549 were cultured and transfected with sequence-specific shRNA. RT-PCR was used to monitor the IGF-IR mRNA expression. Western blotting was used to detect the expression of IGF-IR, bcl-2 and caspase-3, associated with apoptosis, and IGF-IR signaling pathways-associated proteins, total and phospho-ERK1/2 and Akt. MTT assay and flow cytometry were used to examine the cell activity and cell cycle. Twelve nude mice were injected subcutaneously with A549 cells, 20 days later the mice were randomly divided into 3 groups to be injected into the tumor with IGF-IR, PBS, or blank plasmid respectively 4 times with the interval of 5 days. Five days after the 4th injection the mice were killed and the tumors were taken out. TUNNEL assay was used to detect the apoptotic cell in the tumor.

Results: RT-PCR showed that the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA1 was only 24% +/- 4% that of the A549 cells transfected with blank plasmid (P < 0.05); however, the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA2 was 78% +/- 5% that of the A549 cells transfected with blank plasmid (P > 0.05). The IGF-IR protein expression level of the A549 cells of the IGF-IR shRNA1 group was only 10.2% +/- 2.8% that of the A549 cells of the blank plasmid group (P < 0.05). Western blotting showed that the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA1 group were 46% +/- 6% and 156% +/- 8% those of the negative controls (both P < 0.05); however, the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA2 group were not different from those of the negative control cells. The Akt kinase and ERK phosphorylation levels of the A549 cells of the IGF-IR shRNA1 group were 10% and 36% +/- 3% those of the negative control cells respectively (both P < 0.05). Since 48h after the transfection the active cell number of the IGF-IR shRNA1 group was 64% +/- 7% that of the negative group (P < 0.05), and this decrease effect lasted to 72 h after (67% +/- 6% that of the negative cells, P < 0.05). 48 h after the transfection the percentage of cells at G(0)/G(1) phase of the IGF-IR shRNA1 group was 77.5%, significantly higher than that of the negative control group, and the percentages of the cells at S and G(2)/M phases of the IGF-IR shRNA1 group were 15.7% and 7.3% respectively, both significantly lower than those of the negative control group (23.0% and 29.9% respectively). Since the second injection the tumor size of the mice of IGF-IR shRNA group was 40% - 50% that of the PBS group (P < 0.05), and the tumor size of the mice of the PBS group was 90% that of the control group. TUNNEL assay showed that the number of apoptotic cells in the tumors of the IGF-IR shRNA1 group mice was 118 +/- 8/high power, significantly higher than that of the control group (70 +/- 9, P < 0.05).

Conclusion: RNAi technique effectively inhibits the expression of IGF-IR, thus decreasing the NSCLC cell proliferation inducing apoptosis and inhibiting the tumor growth.