Biomedical subjects
Publications and source records attributed to A V Vladimirova.
Inhibition of p-glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB-8-5 cells by synthetic 21-bp double-stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.
Suppression of c-myc protooncogene expression in KB-3-1 cells by siRNA was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA was prepared by in vitro transcription with T7 RNA polymerase on short dsDNA-templates. It was found that incubation of KB-3-1 cells in the presence of 75 nM siRNA results in decrease of the c-myc mRNA level down to 5% of the level in the control cells and significant decline of KB-3-1 cell proliferation rate. Using 200 nM siRNA four-fold decrease of KB-3-1 cells proliferation rate was observed and this effect was stable at least 96 h after transfection.
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Interaction of transcription factors with functional regions of gene mdr1 promoter has been studied. Binding of transcription factors to double-stranded (ds) oligonucleotides, mimicking four important regulatory regions before and after treatment of KB and K-562 cell lines with doxorubicin, cytarabine, and vinblastine was analysed by gel-shift assay. Gene induction resulted in enhancement of the main complex A(A1) proteins formation on the all four regions in both the cell lines. Inhibition of each complex formation by an excess of non-radiolabelled ds oligonucleotides suggests the possibility of regions interaction during promoter activation. Another indication of interaction of regulatory regions during activation is overlapping minor complexes sets. The results suggest that the four studied regulatory regions are important for mdr1 gene activation. Corresponding oligonucleotides mimicking these regions can be employed as inhibitors of gene transcription that bind specific transcriptional factors.
Small interfering RNAs (siRNA) provide a powerful approach for sequence-specific silencing of gene expression. In the present study we investigated inhibition of c-myc gene expression by siRNAs targeted to the sequence 1452-1470 b. in third exon of c-myc mRNA and to homologous regions in second exons of c-myc (697-715 b.) and N-myc (302-320 b.) mRNAs. siRNAs were prepared enzymatically according to the scheme, including dsDNA-templates preparation using Klenow fragment, separate in vitro transcription of each RNA strand with subsequent hybridization and removal of leader sequences by T1 RNase. Investigation of c-myc gene silencing by siRNAs revealed that enzymatically prepared siRNAs induce stronger inhibition of c-myc expression, than siRNA with the same sequence prepared by chemical synthesis. It was found that down-regulation of c-myc gene expression by investigated siRNAs results in efficient inhibition and even complete arrest of carcinoma cell proliferation, moreover, the extend of growth inhibition correlates with the level of siRNA-mediated reduction of c-myc mRNA.