ABSTRACT
AIM:To investigate the effect of small interfering RNA ( siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24.METHODS:The siRNA against ABCE1 was constructed and transfected into the T24 cells with LipofectamineTM 2000.The expression of ABCE1 was detected by RT-PCR and Western blot.Flow cytometry was used to detect the cell cycle.The effects of ABCE1 gene silencing on prolifera-tion, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion as-say, respectively.RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA.The cell cycle was arrested at G0/G1 phase and the cell number in S phase was decreased in the T24 cells.Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly.CONCLUSION:Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.
ABSTRACT
ATP-binding cassette protein E (ABCE1) has been annotated as an Rnase L inhibitor in eukaryotes. Previous study showed that the overexpression of ABCE1 was related with the occurrence and clinical stage of lung adenocarcinoma. As an initial investigation into the novel functions of ABCE1, siRNA-expressing vectors targeting sites of the ABCE1 gene were constructed from RNAi-Ready pSIREN-DNR-DsRed-Express vector. Cultured 95-D and NCI-H446 lung carcinoma cells were transfected with the siRNA-expressing vectors using FuGENE 6 and transfection efficiency was determined by using fluorescence microscopy. The expression level of ABCE1 protein was determined by Western blot and immunofluorescence staining. Cell viability was determined by MTT, cell cycle was analysed by flow cytometry.The apoptotic rate was observed by ELISA. Fluorescence microscopy showed a satisfactory transfection efficiency which was about 42.70%. Cell viability and the growth fraction were markedly suppressed, whereas the apoptosis was significantly increased in SiRNA-95-D and SiRNA-NCI-H446 cells than controls(P< 0.05). It can be concluded that the siRNA targeting ABCE1 gene shows a dramatic inhibitory effect on RNA transcription and protein expression and a promoting effect on the apoptosis in 95-D/NCI-H446 cells, which offers a reliable base for the further in vivo experiment.