Construction of Wip1 gene recombinant lentiviral expression vector and its effects on breast cancer MCF-7 cell biological behavior / 重庆医学

ABSTRACT

Objective To construct the Wip1 gene recombinant lentiviral expression vector and to investigate its effects on breast cancer cell biological behaviors .Methods Wip1 gene short hairpin RNAs (shRNA) was transfected into breast cancer MCF-7 cells through lentiviral infection method .Wip1 mRNA and protein expressions before and after transfection were detected by u-sing qRT-PCR and Western blotting .The effects of Wip1-shRNA on the proliferation ,apoptosis ,cell cycle ,invasion and metastasis in MCF-7 cells were determined by using the MTT assay ,flow cytometry and transwell invasion test .Interfering RNA molecule p53dsRNA inhibiting p53 gene expression (p53 dsRNA) was screened ,and the effect of p53 inhibition on MCF-7 cells invasion and metastasis was analyzed .Results After transfection for 48 h ,the cellular fluorescence in the Wip1-shRNA group was stronger , while which in the NC-shRNA group was weaker .Cellular Wip1 mRNA and protein expressions in the Wip1 shRNA group were 0 .291 ± 0 .025 and 0 .203 ± 0 .021 respectively ,which in the NC-shRNA group were 0 .954 ± 0 .090 and 0 .963 ± 0 .092 respectively , the difference between the two groups was statistically significant (P<0 .05) .The cellular survival rate at various time points had statistical difference between the two groups (P<0 .05) .The early and late cell apoptosis number in the NC-shRNA group was less than that in the Wip1-shRNA group ,the difference was statistically significant (P<0 .05) .The cells numbers at phase G0 +G1 and phase S in the NC-shRNA group were 53 .5 ± 3 .6 and 27 .3 ± 1 .5 respectively ,which in the Wip-shRNA group were 72 .3 ± 5 .2 and 14 .6 ± 0 .8 respectively ,the difference was statistically significant(P<0 .05) .The Transwell invasion and metastasis results showed that the cell transmembrane number in the NC-shRNA group was more than that in the Wip1-shRNA group(P<0 .05) .The cellu-lar p53 mRNA and protein expression had statistical difference between the control group and p53dsRNA group(P<0 .05) .Conclu-sion RNA interference can effectively suppress Wip1 expression in MCF-7 cells .Wip1 may affect the proliferation ,apoptosis ,cell cycle ,invasion and metastasis of breast cancer cells by modulating protein expression .p53dsRNA increases the invasion and metas-tasis ability of breast cancer MCF-7 cells by interfering p53 gene down-regulation .