Effects of lentivirus-mediated knockdown of astrocyte elevated gene-1 on the biological behavior of uveal melanoma

Objective

To investigate the effects of lentivirus-mediated knockdown of astrocyte elevated gene-1 (AEG-1) on the biological behavior of uveal melanoma(UM).

Methods

MUM-2B and MUM-2C cell lines with different invasiveness and metastasis potential were cultured.Based on AEG-1 sequence that designed the effective (RNA interference) RNAi target sequence and the RNAi negative control scramble sequence, and then performed the preparation of the lentiviral vector and viral packaging.In these two kinds of cell lines, the cells infected by AEG-1-RNAi lentivirus were used as lentiviral infection group, the cells of the RNAi negative control of the blank lentivirus infected cells were used as negative control group, and the cells without any processing were used as normal control group.Real-time PCR and Western blot were used to detect the change of AEG-1 transcription and protein expression levels in these two kinds of cell lines’ groups, as well as by MTT method, Annexin V-APC method, cell invasion assay, and Transwell cell migration assay that to investigate the effect of lentivirus transfection induced knockdown of AEG-1 on the biological behavior of human UM cell lines.

Results

The successful preparation of RNAi lentivirus vector and its viral package were transfected to MUM-2B, MUM-2C cell lines of logarithmic growth phase, respectively.The relative expressions of AEG-1 mRNA in normal control groups, negative control groups and lentiviral infection groups were statistically significant (F=130.02, P<0.01; F=144.17, P<0.01). The relative expression of AEG-1 mRNA in lentiviral infection groups were lower than that in the negative control groups, and the AEG-1 gene knockout rates were 77.1% and 79.8%, respectively, the differences were statistically significant (both at P<0.01). There were no significant differences in the relative expression of AEG-1 mRNA between normal control groups and negative control groups (all at P>0.05). Western blot showed that no significant changes were found in the AEG-1 proteins expression levels between normal control groups and negative control groups in the two kinds of cell lines (F=146.17, P<0.01; F=156.79, P<0.01). The expression levels of AEG-1 protein in the lentiviral infection groups were significantly lower than that in negative control groups (all at P<0.01). The proliferation of both cells lines in lentiviral infection groups were significantly lower than that in negative control groups from the second day to the fifth day, the differences were statistically significant (t=5.78, 30.68, 23.99, 29.40, all at P<0.01; t=7.88, 7.09, 5.56, 6.60, all at P<0.01). In both cell lines, the apoptosis rate of lentiviral infection groups were significantly higher than those in the negative control groups, the differences were statistically significant (t=54.34, P<0.01; t=11.68, P<0.01). In both cell lines, the multiple of invasions in lentiviral infection groups were significantly lower than those in negative control groups, and the differences were statistically significant (t=16.04, P<0.01; t=13.98, P<0.01); In both cell lines, the multiple of transfer in lentiviral infection groups were significantly lower than those in negative control groups, and the differences were statistically significant (t=12.04, P<0.01; t=22.43, P<0.01).

Conclusions

Lentivirus transfection inducing knockdown of AEG-1 inhibits the transcription and protein expression level of AEG-1 in the melanoma cells, which changes the biological behaviors of UM, like slowing down cell proliferation, promoting apoptosis, and reducing the abilities of cells’ invasion and metastasis.