| As one of the most common malignant tumors, breast carcinoma has high prevalence and mortality rate all over the world. The young tendency is increasingly obvious. At present the treatment of breast cancer is still faced with the problem of conventional treatment failure and tumor recurrence.Bmi-1gene, an important member of the polycomb gene family, plays an important role in cell permanent biochemical and cell senescence. In recent years, a large number of studies show that, Bmi-1is involved in the regulation of tumor cells and stem cell self-renewal and differentiation, in a variety of human tumors, including lung cancer, ovarian cancer, acute myeloid leukemia, nasopharyngeal cancer, breast cancer, and high expression in neuroblastoma, suggesting that Bmi-1may play an important role in the occurrence and development of cancer.Telomerase is to extend telomere elongation by reverse transcription DNA synthetase, telomerase can used its RNA as template to synthesize the lost of DNA in cell division, maintaining telomere length stability. Telomerase activity is mainly regulated at the transcriptional level, and human telomerase reverse transcriptase (hTERT) be a rate limiting factor in the regulation of telomerase activity, was consistent with the expression of telomerase.Telomeres shorten with each successive cell division in normal human cells whereas, in tumors, they are continuously elongated by human telomerase reverse transcriptase (hTERT). Telomerase is overexpressed in80-95%of cancers and is present in very low levels or is almost undetectable in normal cells. Because telomerase plays a pivotal role in cancer cell growth it may serve as an ideal target for anticancer therapeutics. Comprehensive domestic and foreign research present situation, at present, the use of siRNA interference gene therapy may be one of the effective means for future treatment of breast cancer in the most promising. In order to solve the low efficiency of small interference RNA transfection, domestic and foreign scholars have done many attempt and research. At present, a new vector:Lipid-polycation-Nucleic acid (LPN) has been widely concerned. Nanoparticle carrier is the new transport system for peptide, protein, oligonucleotide and therapeutic genes and other biological macromolecules in recent years.It has the advantages of high efficiency, non immunogenic, safe, cheap, biodegradable, stability and other gene carriers do not have the advantage, it is expected to become the application of non viral vector potential gene transfer and gene therapy. Polycation vector can be condensed nucleic acid molecules formed nanoparticles, and polyethylenimine (PEI) is the most widely used polycation vector in recent years. PEI with cationic charge rich, can be condensed nucleic acid into particles of nanometer size, in favor of therapeutic genes into cells. Liposome used to package PEI/DNA (RNA) compression body made of LPN, structure of LPN is similar to a virus, containing a genetic material core and a lipid shell. That will effectively protect the core of nucleic acid and cationic polymer from direct action of nucleases, LPN lipid layer may facilitate integration with the cell membrane, and by endocytosis or phagocytosis of the cells carrying the gene introduced into the cells. To further improve the transfection efficiency.This experiment will further validate the nano mediated transport system silencing Bmi-1and hTERT gene on human breast carcinoma cell line MCF-7, and observed the changes of the Bmi-1and hTERT protein levels, and observed the proliferation and apoptosis of MCF-7cells. Western Blot are used to analyse expression of Bmi-1and hTERT protein of MCF-7cells in each groups. Real time-PCR detected the expression of Bmi-lmRNA and hTERTmRNA of MCF-7cells in each groups. The MTT assay was used to detect on the proliferation of MCF-7cells in each groups. Flow cytometry were used to detect on the apoptosis of MCF-7cells in each groups. Objective:To investigate the interfering RNA mediated by nano transport system of hTERT and Bmi-1, lay the theoretical foundation of proliferation and apoptosis of human breast carcinoma cell line MCF-7.Materials and methods1Liposome transfection human breast carcinoma cell line MCF-7screening interfering Bmi-1and hTERT gene fragments of siRNA and the optimal concentrationHuman breast carcinoma cell line MCF-7in5%CO2,37℃incubator, at Liposome-mediated transfection of human breast carcinoma cell line MCF-7, screening the optimal concentration of screening and the use of Real time-PCR screening silence Bmi-1and hTERT siRNA interference best clips.2Preparation LPN nanoparticle carrier and using MTT assay detected its cytotoxicity1) The preparation of LPN nanoparticles carrier.2) Atomic force microscopy for detection of LPN nanoparticle carrier form.3) Gel retardation assay LPN nanoparticle carrier capabilities and anti-enzyme liposome encapsulation.4) Confocal microscopy to detect cell uptake of LPN nanoparticles.5) MTT assay detected its cell toxicity.3Interfering RNA silencing Bmi-1gene and hTERT gene mediated by nano delivery system to inhibit the proliferation of human breast carcinoma cell line MCF-7and induce its apoptosis.1) Take human breast carcinoma cell line MCF-7were divided into seven groups:control group, PEI group, Bmi-1siRNA group (G1), hTERTsiRNA group (G2), Bmi-1siRNA LPN transfected group (G3), hTERTsiRNA LPN turn transfection group (G4), hTERT+Bmi-1siRNALPN transfected group (G5). Using western blot technique to detect Bmi-1and hTERT protein expression in each group.2) Using Real time-PCR technique to detect the expression of Bmi-1mRNA and hTERT mRNA in each group.3) Using MTT assay to detect cell growth inhibition rate in each group.4) Using flow cytometry to detect the changes of cell apoptosis in each group.4Statistical analysis:Using the SPSS17.0statistical software, at an inspection level of α=0.05.Results1.Real time-PCR Screening Bmi-1siRNA and hTERT siRNA best specific fragments. On the mRNA level, Bmi-1siRNA-2transfected lowest levels of Bmi-1expression (P<0.05), follow-up experiments using Bmi-1siRNA-2. hTERT siRNA-3transfected hTERT expression levels of the lowest (P<0.05), follow-up experiments using hTERT siRNA-3.2.The morphology, size and potential measured zeta, their intake of the cell experiments of LPN nanoparticle carrier have reached the experimental requirements. Using MTT to detect the toxicity of LPN to transfected MCF-7after48h showed that: when the concentration of siRNA transfection efficiency15pmol best and least toxic.3.Western Blot protein expression was detected in Bmi-1and hTERT results showed:hTERT+Bmi-1siRNALPN transfected group inhibit the expression of the most significant (P<0.01).4.Real time-PCR technique to detect the expression of Bmi-1mRNA and hTERT mRNA showed:hTERT+Bmi-1siRNALPN transfected group inhibit the expression is the most significant (P<0.01).5.MTT assay24h,48h,72h inhibition rate of each group results showed: hTERT+Bmi-1siRNALPN transfected group inhibit the expression is the most significant (P<0.01).6.Changes in flow cytometry results showed:hTERT+Bmi-1siRNALPN transfected group inhibit the expression is the most significant (P<0.01). ConclusionBmi-1-siRNA and hTERT-siRNA by mediated nano delivery systems can effectively suppress human breast carcinoma cell line MCF-7proliferation and induce its apoptosis. |
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