ABSTRACT
AIM: To investigate the effect of microRNA-mediated exogenous Id3 gene silencing on proliferation and apoptosis of human lung adenocarcinoma A549 cells in vitro. METHODS: A recombinant miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-Id3, pcDNA/miRId3) which targets human Id3 gene was constructed. After 24 h of transfection, the transfection efficiency was monitored by inverted fluorescence microscopy.EGFP expression efficiency in A549 cells was analyzed by flow cytometry (FCM).Id3 expression vector pEGFP/Id3 and pcDNA/miRId3 were cotransfected into A549 cells by liposome-mediated method. After 24 h of transfection, the transfection efficiency was monitored by inverted fluorescence microscopy.Semi-quantitative RT-PCR and Western blot were used for identifying Id3 mRNA and protein expression respectively in A549 cells after transfection. Cell proliferation rate and apoptosis ratio were evaluated by MTT assay and Annexin V/7-ADD staining followed by FCM to observe the down-regulatory effect of Id3 expression by miRNA-mediated RNA interference (RNAi). RESULTS: pcDNA/miRId3 and pEGFP/Id3 were successfully transfected into A549 cells. RT-PCR and Western blot results showed that after 24 h of cotransfection of pEGFP/Id3 and pcDNA/miRId3 in A549 cells, the exogenous expression of Id3 both at mRNA and protein levels were significantly reduced compared with the pEGFP/Id3 group. MTT assay and Annexin V/7-AAD staining showed that after 24 h of transfection with pEGFP/Id3, the proliferation rates were significantly reduced and apoptotic cell ratios were significantly higher than those of pEGFP-transfected cells.Whereas there were not any significant differences in proliferation rates or apoptotic cell ratios between pcDNA/miRId3+pEGFP/Id3 cotransfected group and pEGFP or miRNA negative controls. CONCLUSION: Exogenous expression of Id3 in A549 cells could inhibit proliferation and induce apoptosis of A549 cells. Cotransfection of pcDNA/miRId3 and pEGFP/Id3 into A549 could reverse the Id3-induced proliferation inhibition and apoptosis. Construction and application of Id3-targeting miRNA expression vector may build some foundations for investigation the mechanisms of Id3-induced proliferation inhibition and apoptosis in A549 cells.
