Application of target up-regulation PAR-4 gene small ribonucleic acid (RNA) in preparing bladder cancer resisting drugs
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A bladder cancer, gene technology, used in DNA/RNA fragments, gene therapy, antitumor drugs, etc.
Inactive Publication Date: 2012-08-22
ZHEJIANG UNIV
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More importantly, intratumoral injection of adenovirus overexpressing PAR-4 resulted in tumor apoptosis and growth inhibition in both subcutaneous ectopic and orthotopic prostate cancer mouse models
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Embodiment 1
[0020] Example 1 dsRNAs upregulate the expression of PAR-4
[0021] Experimental program:
[0022] 1. Cell line: Human bladder cancer cell line T24, 5637 (Shanghai Cell Institute)
[0023] 2. Synthesis of dsRNAs: dsRNAs were chemically synthesized by Shanghai Jikai Biotechnology Co., Ltd.
[0024] 3. Experimental grouping: 50nM dsPAR-4 group, control dsRNA (dsCon), non-homologous to known human genome sequence.
[0025] dsControl: sense strand, 5'-ACU ACU GAG UGA CAG UAG A[dT][dT]-3' (SEQ ID NO:7)
[0026] Antisense strand, 5'-UCU ACU GUC ACU CAG UAG U[dT][dT]- 3' (SEQ ID NO:8)
[0027] Blank control group (Mock, only added liposomes).
[0028] 4. Cell culture and transfection:
[0029] The cells were cultured in RPMI1640 medium containing 10% inactivated newborn bovine serum, placed in a 37oC cell culture box with a CO2 content of 5%. The day before transfection, the cells were subcultured and planted in culture plates at a density of about 30-40%. dsRNAs are ...
Embodiment 2
[0048] Example 2 The therapeutic effect of dsPAR-4-435 up-regulating PAR-4 on bladder cancer cells
[0049] Experimental program:
[0050] 1. Cell line: same as Example 1.
[0051] 2. dsRNAs synthesis: Same as Example 1.
[0052] 3. Experimental grouping: with embodiment 1.
[0053] 4. Cell culture and transfection: the same as in Example 1.
[0054] 5. Tetrazolium salt (MTT) colorimetric method
[0055] Bladder cancer T24 cells in the logarithmic growth phase were taken, digested with trypsin to make a single cell suspension, and the cells were planted in a 96-well culture plate at a density of 2000-5000 cells per well (in order to ensure the accuracy of the results, in the experiment Before measuring the cell attachment rate, doubling time and growth curve of cells with different inoculated numbers, then determine the inoculated number of cells in each well to ensure that the cells will not be overfilled when the culture is terminated), and the culture plate is placed at...
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Abstract
The invention provides application of target up-regulation PAR-4 gene small ribonucleic acid (RNA) in preparing bladder cancer resisting drugs. The nucleotide sequence of the target up-regulation PAR-4 gene small RNA is composed of six positive sense strands and negative sense strands of 21 nucleotides. The 3' end of each strand is provided with two nucleotides in suspension mode (generally dTdT, and the 19 nucleotides in the middle are paired.). The target up-regulation PAR-4 gene small RNA comprises sa RNA of different sequences. By means of expression of target up-regulation PAR-4 genes in the bladder cancer cells, the small RNA can restrain tumor cell activity, induces cell apoptosis, and further can be applied to preparation of bladder cancer resisting drugs. The application of target up-regulation PAR-4 gene small RNA in preparing the bladder cancer resisting drugs is simple in operation of preparing the small RNA, low in cost, small in using amount, capable of achieving good activation effect when the transfection concentration is 50nM and accurate in activation action. The mRNA and protein level of the target genes are both improved. In addition, the sa RNA can induce genes to express without damaging completeness of gene groups, thereby being safe to use.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of small activating RNA targeted to upregulate PAR-4 gene in the preparation of anti-bladder cancer drugs. Background technique [0002] In 2006, Li et al. reported that dsRNA (double-stranded RNA, double-stranded RNA) targeting the gene promoter region can cause sequence-specific gene transcription activation, and named this phenomenon as RNA-induced gene activation (RNA activation, RNAa). , and such dsRNAs are called small activating RNAs (small activating RNAs, saRNAs). RNA activation of gene expression is a common phenomenon in mammalian cells, and its mechanism may be that short double-stranded RNA binds to the antisense transcription product of the target gene, and carries out epigenetic modification on the promoter region (histone acetylation, demethylation, etc.) etc.), and further recruit transcription factors to the promoter region of the gene, thereby enhanc...
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