Inhibition of glucosylceramide synthase expression and reversal of multidrug resistance in human breast cancer cells by shRNA / 基础医学与临床

ABSTRACT

Objective To construct the plasmid containing short hairpin RNA (shRNA) of GCS in order to suppress the expression of GCS and to reverse the multidrug resistance in drug-resistant breast cancer cells. Methods Two reverse repeated motifs of sequence targeting GCS with 6 bp spacer were designed and synthesized in vitro,then they were inserted into the plasmid pSUPER. The recombinant plasmids were transfected into human breast cancer cells. GCS expression was detected by Western blot and immunocytochemistry. caspase-3 expression and its activity were assessed using Western blot and colorimetry,flow cytometry was performed to analyze the ratio of apoptosis. Results MTT method was used to evaluate the 50% inhibition concentration. Enzyme digestion analysis and DNA sequencing confirmed that the recombinant plasmids were successfully constructed. GCS protein content decreased 80%,82% respectively after transfection with recombinant plasmids. The positive rate of GCS expression reduced to 18.1%,16.7% respectively. caspase-3 expression and its activity were significantly higherand the apoptosis of cells increased dramatically. The drug resistance of breast cancer cells to antineoplastic drugs declined significantly. Conclusion shRNA can suppress GCS expression in human drug-resistant breast cancer cells effectively and restore the sensitivity to several antineoplastic drugs.