[Construction of glucosylceramide synthase-specific siRNA expression vector and its efficiency in reversal of drug resistance in breast carcinoma cells].
OBJECTIVE
To construct a glucosylceramide synthase (GCS)-specific small interfering RNA (siRNA) expression vector and to investigate the inhibitory effect of this siRNA on GCS expression and drug resistance in breast carcinoma cells.


METHODS
Two GCS gene-specific siRNAs were designed and cloned into the expression vector pSUPER to generate the plasmids pSUPER-GCS1 and pSUPER-GCS2. Human adriamycin (ADM)-resistant breast carcinoma cells of the line MCF-7/ADR and human adriamycin-sensitive breast carcinoma cells of the line MCF-7 were cultured and transfected with pSUPER-GCS1, pSUPER-GCS2, and blank vector pSUPER as controls. The expression of GCS mRNA was assayed by RT-PCR and the expression of GCS protein was observed by flow cytometry. The 50% inhibition concentration of ADM on MCF-7/ADR cells was evaluated by MTT method. Flow cytometry was performed to determine the ratio of apoptosis.


RESULTS
Double enzyme digestion analysis and DNA sequencing confirmed that pSUPER-GCS1 and pSUPER-GCS2 were successfully constructed. The GCS protein positive rate of the MCF-7/ADR cells 48 hours after transfection with pSUPER-GCS1 and pSUPER-GCS2 were 8.3% +/- 1.0% and 9.2% +/- 0.8% respectively, significantly lower than that before transfection (68.3% +/- 0.6%), with a inhibition rate of 89.4% and 88.5% respectively (both P < 0.01). Forty-eight hours after transfection with pSUPER-GCS1 and pSUPER-GCS2, the relative reversal rates of sensitivity to ADM of the MCF-7/ADR cells were 93.7% and 91.6%. Flow cytometry showed that the apoptotic rate of the MCF-7/ADR cells was 0.80 +/- 0.06 before transfection, 15.38 +/- 1.16 after transfection with pSUPER-GCS1 and 13.92 +/- 1.73 after transfection with pSUPER-GCS2 (both P < 0.05), and was 0.87 +/- 0.12 in the cells transfected with blank vector (P > 0.05).


CONCLUSIONS
A GCS-specific small interfering RNA expression vector has been constructed successfully that suppresses the GCS expression and reverses the multidrug resistance in breast carcinoma cells by increasing the ratio of apoptosis in drug-resistant cells.
Sun
Yan-lin
YL
Department of Pathology, Qilu Hospital, University of Shandong, Jinan 250012, China.
Zhou
Geng-yin
GY
Li
Kai-nan
KN
Li
Wen-tong
WT
Song
Xian-rang
XR
Gao
Peng
P
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D004317:Doxorubicin; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase