[Effects of ribonuclease inhibitor on apoptosis and invasion of human breast cancer MDA-MB-231 cells].

OBJECTIVE

RI gene is transfected into human breast cell line MDA-MB-231 which is relatively hypo-expression RI gene. To investigate how RI gene affect the cell apoptosis and invasion of MDA-MB-231.

METHODS

(1) A recombinate pLNCX-RI and an empty pLNCX were transferred into MDA-MB-231 cells by using Lipofectamin(TM) 2000. After transfection, positive clones were screened with G418 and expanded by culture. RT-PCR and Western blot methods were used to analyze expression of RI mRNA and protein in MDA-MB-231 cells before and after transfection. (2) Transwell test, FCM test were used to search for the effects of RI expression on transfected cells apoptosis and invasion. (3)To study preliminarily the related mechanism by which RI induced breast cancer cell apoptosis, the mRNA and protein expression of survivin in MDA-MB-231 cells were examined by RT-PCR and Western blot, and protein expression of Caspase-3 in MDA-MB-231cells were examined by Western blot. (4) To investigate preliminarily the mechanism by which RI inhibited breast cancer cell invasion, the mRNA and protein expression of CD24 in MDA-MB-231 cells were examined by RT-PCR and Western blot.

RESULTS

(1) The RI gene was transfected successfully to MDA-MB-231 by using LipofectamineTM2000, the subclone cell lines MDA-MB-231/pLNCX-RI which highly expressed RI were successfully selected. (2) Compared with MDA-MB-231 and MDA-MB-231/pLNCX cells, the results of FCM and transwell in MDA-MB-231/pLNCX-RI cells indicated: the percentage of cell apoptosis were obviously increased(P<0.01), penetrating membrane cells were decreased(P<0.01). The mRNA and protein expression of Survivin were degraded, and Caspase-3 was activated in MDA-MB-231/pLNCX-RI cells(P<0.01). The mRNA and protein expression of CD24 were degraded in MDA-MB-231/pLNCX-RI cells(P<0.01).

CONCLUSIONS

(1) Exogenous RI expression may promote apoptosis in human breast cells MDA-MB-231 by inhibiting the expression of Survivin and activating caspase-3.(2) Exogenous RI expression may inhibit invasion in MDA-MB-231 by inhibiting expression of CD24.