RNAi-mediated Silencing Of Type 1 Insulin-like Growth Factor Receptor Inhibited Growth And Metastasis Of Human Lung Cancer Cell A549 In Vitro And In Xenograft Nude Mice

Part â…  : Knockdown the Expression of Type 1 Insulin-like Growth Factor Receptor (IGF-1R) by Small Interfering RNAObjective: To construct the siRNA expression vector targeting IGF-1R and to evaluate its ability to downregulate the expression of IGF-1R in A549 human lung cancer cells.Methods: Two siRNA expression vectors, pIGF-1R-siRNA1, pIGF-1R-siRNA2 targeting IGF-1R, were constructed using pENTR/U6 vector, and a vector targeting luciferase gene, pcontrol-siRNA was constructed as control. The levels of mRNA and protein expressions were determined by RT-PCR and Western blot analysis after recombinant plasmid pIGF-1R-siRNA1, pIGF-1R-siRNA2 and pcontrol-siRNA were transfected into A549 cells 48 h, respectively.Results: After 48 h transfection, level of IGF-1R mRNA was significantlydecreased in IGF-1R-siRNA1-A549 cells [(20.1 ±3.4)% of that in pcontrol-siRNA group] compared with control-siRNA cells. The level of protein expression of IGF-1R in A549-IGF-1R-siRNA1 cells was decreased by (90.8±4.9)%. Similar but less profound knockdown of IGF-1R expression at both mRNA and protein levels were induced by pIGF-1R-siRNA2.Conclusion: IGF-1R specific siRNA can significantly and specifically down-regulate the expression of IGF-1R.Part â…¡: Down-regulation of Type 1 Insulin-like Growth Factor Receptor (IGF-1R) Expression by Small Interfering RNA Inhibited A549 Human Lung Cancer Cell Proliferation and Increased Apoptotic Response in vitroObjective: To evaluate the effect of IGF-1R silence on the proliferation, apoptosis and apoptosis-related signaling pathways of A549 human lung cancer cells.Methods: The cell cultures were measured for cell proliferation levels at different time points (at 0h, 24h, 48h and 72h after transfection) using MTT assay. At 48h after transfection, cell cycles analysis were performed on a flow cytometer, the apoptosis of tumor cells was analyzed by Apoptotic DNA ladder detection to analyze the internucleosomal DNA cleavage. The signal molecules of signaling pathways (Erk1/2 and Akt) were measured by Western blot analysis after IGF-1R specific siRNA plasmids or control siRNA expressing plasmids were transfected into A549 cells.Results: The results of MTT assay indicated that the proliferation level of A549 cells were no different at 24h, but obviously decreased at 48h and were maintained for 72h after transfection with pIGF-1R-siRNA1. The viable cell percentages at 48h and 72 h were (64.1 ± 6.7)% and (67.2 ± 6.4)% of the negative control, respectively. pIGF-1R-siRNA1 transfection induced an increase in G₀/G₁ phase cells (77.53%) as compared with the control (47.23%), and decreased in S phase (15.7%) and G₂/M phase (7.32%) cells as compared with the control (23.02% and 29.94%, respectively). It was shown that down-regulation of IGF-1R obviously enhanced apoptotic response to 3% ethanol of A549 cells by using apoptotic DNA ladder detection. There were no significant difference in total Erk1/2 and Akt levels between control and IGF-1R siRNA-transfected cells. However, the phospho-Erk1/2 and phosphor-Akt were reduced significantly in A549 cells after transfection with the pIGF-1R-siRNA1, and their levels were (19.52 ± 3.2)% of t-Erk1/2 and 9.94% of t-Akt, respectively.Conclusion: IGF-1R silence greatly inhibited proliferation of human lung cancer and obviously increased apoptotic response.Partâ…¢: Suppression of Type 1 Insulin-like Growth Factor Receptor (IGF-1R) Expression by Small Interfering RNA Inhibited A549 Human Lung Cancer Cell Invasion in vitro and Metastasis in Xenograft Nude Mice. Objective: To evaluate the effect of IGF-1R silence on the invasion, metastasis and invasion-related signaling pathway of A549 human lung cancercells.Methods: At 48h after transfection, MMP-2, MM-9 and u-PA expression levels were measured by RT-PCR, Western blot analysis, gelatinase zymography and casein-plasminogen zymography. Migration assay and invasion assay were performed at 48h after transfection. The signal molecule of signaling pathway (Akt) was measured by Western blot analysis after pIGF-1R-siRNA or pcontrol-siRNA were transfected into A549 cells. To further elucidate the effect of IGF-1R silence on the inhibition of metastatic potential, a lung metastasis model was established using nude mice which would received a tail vein injection of pIGF-1R-siRNA or pcontrol-siRNA as treatment twice per week (0.5μg/g body weight). The expression of hHPRT and indices of metastatic foci shown on the lung surface and under a dissecting microscope were measured.Results: The results showed that MMP-2, MMP-9 and u-PA expression at mRNA, protein levels and the activities were significantly decreased in IGF-1R-siRNA1 group compared with those in control-siRNA group. The phospho-Akt level displayed a significant reduction in IGF-1R-siRNAl group compared with the control after IGF-1R silence. A549 cells transfected with pIGF-1R-siRNA1 significantly reduced migration and invasion by (66.1±7.6)% and (64.4±5.4)%, respectively. The presence of metastatic foci was significantly decreased in the IGF-1R-siRNA1 group compared with the control group. These results were confirmed by the slight expression of hHPRT in the IGF-1R-siRNA1 group.Conclusion: These findings confirmed the prediction that IGF-1R silencing by siRNA significantly inhibited invasion, metastasis and signaling pathway of A549 human lung cancer cell.