ShRNA-mediated Gene Silencing Of IGF-IR Inhibits Cell Proliferation, Induces Cell Apoptosis, And Suppresses Tumor Growth In Non-small Cell Lung Cancer

Objective: To study the effects of RNA interference (RNAi)-mediated IGF-IR gene silencing on lung cancer cell line A549 in vivo and in vitro. Methods: two plasmids expressing shRNA for IGF-IR under the control of the human U6 promoter: IGF-IR-specific shRNA1 and 2 were constructed, and an unrelated shRNA was used as negative control. NSCLC cell lines A549 were transfected with sequence-specific shRNA as well as control. Quantitative reverse-transcriptase PCR and Western blot were used to monitor the reduction in the production of IGF-IR. Apoptosis-associated genes: bcl-2 and caspase-3, IGF-IR signaling pathways-associated proteins: total and phospho-ERK1/2 and Akt levels were detected by Western blot. MTT assay, flow cytometry and DNA ladder in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of IGF-IR silencing on tumor cell growth and proliferation. Results: Our data showed transfection of NSCLC cells with shRNA resulted in sequence specific silencing of IGF-IR with 78.9% in IGF-IR mRNA transcription and 89.8% in protein production in A549 cells. The decrease in IGF-IR levels caused significant growth inhibition, i.e.: reduction of bcl-2 level, activation of caspase-3, blunt Akt kinase and serum-induced Erk kinase activation. The decrease in IGF-IR levels caused significant growth inhibition, decreased the viability of A549 cells both at 48 h and at 72 h, increased cell apoptosis induced by 3% ethanol, and retained 77.5% A549 cells in the G0/G1 phase. Furthermore, shRNA specific for IGF-IR inhibited tumor growth in vivo in size by more than 50% and in increased apoptosis by more than 60%. Conclusions: A new therapeutic effect of sequence specific suppression of IGF-IR gene expression by RNAi, enabling inhibition of NSCLC cell proliferation and growth both in vitro and in vivo were demonstrated, which support the therapeutic potential of RNAi as a method for gene therapy in treating lung cancer, and the IGF-IR inhibition inducing apoptosis may be relate to the inhibition of ERK and Akt pathways activity.