| Objectives1. To use Ml RNA with guide sequence (Ml-GS RNA) and RNA interference (RNAi) to cleave BCR-ABL mRNA and reduce oncoprotein expression in chronic myelogenous leukemia (CML) cell line K562 cells.2. To determine the apoptosis effects of K562 induced by Ml-GS RNA and RNAi.Methods1. Study on cleavage effect of Ml-GS RNA on artificial substrate in vitro and construction of eukaryotic expression vector.pTK.117 was used as PCR template to produce the DNA sequence of Ml RNA with guide sequence (GS), in which GS was complement to fusion region of BCR-ABL mRNA. The product of PCR was purified and subcloned to pUC19 to make pGS210. The part of fusion region of BCR-ABL sequence was artificially synthesized and subcloned to pGEM-7zf(+) to construct pBCR-ABL. pGS210 and pBCR-ABL were identified by restriction analysis and DNA sequencing. The plasmids were transcribed in vitro and mixed, and the control was also used in the test. Autoradiography was used to determine the cleavage effect of the Ml-GS RNA. After the template of Ml-GS RNA being subcloned to pNAV-1, pAVGS4 was constructed. The recombinant was amplified by transforming to JM109. Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmids.2. The changes of BCR-ABL mRNA and expression of pGS210 in K562 cells after pAVGS4 was transfected into the Cells.pAVGS4 and pNAV-1 (without Ml-GS RNA temple regarded as the control) was mediated by X-tremeGENE Q2 to transfect K562 cell line. The Cells were collected 24, 48, 72 and 96 hours after transfecion. Total RNA from the cell was extracted by Trizol and measured by ultraviolet spectrophotometer. It was used as the template for RT-PCR. Human p-actin was used as internal reference standard. The RT-PCR products were analyzed by agarose electrophoresis and gray scale to reflect the change of BCR-ABL mRNA in differenttime after transfection. After collecting pAVGS4-transfected cell and the control in 48 and 96 hours after transfecion, total protein was extracted and quantified. Reduction of p210 was determined by Western blotting.3. Measuring apoptdsis effects of K562 cell induced by M1 -GS RNApAVGS4 transfected cells and the control were collected and analyzed in 24, 48, 72 and 96 hours after transfection. We analyzed apoptosis effects by three three ways, including TUNEL-FITC, Annexin V- FITC+ PI staining and electron microscope in different times.4. Study on construction of RNA expression vector containing siRNA template and its effect on K562 cellsWe chose BCR-ABL mRNA fusion region as cleavage site for small interfering RNA (siRNA), designing two oligonucleotide chains as siRNA template. The oligonucleotide chains were annealed and subcloned to pSilencerl.O-U6. After the recombinant constructed, pBCR6 was confirmed by restriction analysis and sequencing. Then pBCR was amplified and purified for transfection. Forty-eight and 72 hours after transfection by pBCR6 and pSilencerl.O-U6 (the control), apoptosis of K562 cell were determined by flow cytometer using TUNEL-FITC and Annexin V- FITC+ PI.Results1. pGS210 and pBCR-ABL were confirmeed by restriction enzyme anlysis and sequencing. Both of pGS210 and pBCR-ABL were transcribed and cleavage test was performed in vitro, then autoradiography showed that cleavage efficacy is 28.6% when Ml-GS RNA: substrate = 1:5 in Mol and 78.2% when Ml-GS RNA: substrate = 1:1 in Mol. No cleavage effect is seen in the control. pAVGS4 was constructed and identified by restriction analysis and sequencing.2. RT-PCR based on transfected cells in different times showed that amount of BCR-ABL mRNA begins to decrease in 24 hour after transfection. In Forty- eight and 72 hours later, the amount of BCR-ABL mRNA reduces to 9.2% and 2.5% comparing to that of the control at the same time. Western blotting showed that the expression of p210 in the pAVGS4 group reduce to 10.4% of the control in 48 hours after transfection, and 6.7% of the control 96 hours after transfection.3. Apoptosis determined by Annexin V+...
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