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Study on Relationship between TRAIL Expression and Multidrug Resistance Gene SIRT1

Author: CaoQiong
Tutor: JinGe
School: Zhengzhou University
Course: Biochemistry and Molecular Biology
Keywords: TRAIL SIRT1 Resistance Esophageal cancer Apoptosis
CLC: R735.1
Type: Master's thesis
Year: 2011
Downloads: 59
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Abstract

Esophageal (esophageal carcinoma) is a malignant esophageal epithelium, accounting for 2% of all malignancies of esophageal-prone areas in the world, an annual average of about 15 million people died, chemotherapy after surgery is the treatment of esophageal important methods. In recent years, because the drug does not regulate the use of genetic instability of tumor cells expressing heterogeneity, high mutation rate, as well as individual sensitivity to chemotherapy different reasons, resulting in patients with poor prognosis, tumors develop resistance is one of the main reasons for failure of chemotherapy and recurrence of the cancer patients after surgery, therefore, reduce the resistance of tumor cells to become clinical important problems to be solved. Silent information regulator factor 1 (silent information regulator type of SIRT1) is a human class Ⅲ histone deacetylase (histone deacetylases, HDACs) gene is located on 10q22.1, about 33 kb, post-translational protein molecular size of approximately 60 KD, with the enzyme activity of NAD-dependent deacetyl. DNA damage repair, cell cycle control, inhibition of apoptosis, plays an important role in resistance to oxidative stress and to prolong cell life, so SIRT1 is known as the \Studies show that The clinical tumor biopsy specimens and different sources of cells resistant cells are found high levels of expression of SIRTl can directly induce the expression of the multidrug resistance gene. Tumor necrosis factor-related apoptosis induction ligand (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL) was found by high sequence homology with FasL and TNF, is also referred to as Apo2L, positioning on human chromosome 3 3q26, the length of about 1769bp, a molecular weight of about 32.5KD, which encodes a 281 amino acid sequence. TRAIL its able to effectively inhibit and kill tumor cells, but the smaller role of apoptosis in normal cells and caused widespread concern. Studies have shown that of TRAIL may also play a role in conjunction with chemotherapy and radiotherapy, so as to achieve a reversal of tumor tolerance, the purpose of multi-resistant. But little research on the relationship between the role of TRAIL resistance gene SIRT1, has not been reported. This research constructed the eukaryotic expression vector pcDNA3.1 ()-of TRAIL methods, liposomal transfection of esophageal cancer cells to TRAIL expression in esophageal cancer cells to further study its apoptotic effect and to explore TRAIL effect on the role of tumor resistance gene SIRT1 lay the theoretical foundation for reducing tumor resistance. Objective: To construct the eukaryotic expression vector pcDNA3.1 ()-TRAIL, detect its expression in esophageal cancer cells and generate apoptosis, and to explore the effect of TRAIL on the role of tumor resistance gene SIRT1 for reversal of tumor cells multidrug resistance provide an experimental basis. Cut method: Hind Ⅲ and BamH Ⅰ digestion method from prokaryotic vector pCA13-TRAIL on the target gene TRAIL recovery of about 848bp size fragment and digested eukaryotic expression vector pcDNA3.1 (), and then with T4 ligase connect construct recombinant eukaryotic expression vector pcDNA3.1 ()-TRAIL and were identified by double digestion; liposomes recombinant plasmid transfected into human esophageal cancer cell EC9706, 48h later by RT-PCR and immune cells chemical methods were used to detect the expression of mRNA and protein levels in esophageal cancer cells; optical microscope and inverted phase contrast microscope to observe the general biology of the morphological changes of the apoptotic process; flow cytometry transfected with the recombinant plasmid 24h after 48 hours on the effect of apoptosis of esophageal cancer cells; effect of RT-PCR and immune cells chemical method to detect recombinant plasmid TRAIL resistance-associated gene SIRT1 mRNA and protein expression levels, investigate the role of TRAIL on tumor resistance gene SIRT1 . Results: The recombinant eukaryotic expression vector pcDNA3.1 ()-of TRAIL Construction and identification with Hind Ⅲ and BamH Ⅰ digestion pCA13-TRAIL plasmid recovered approximately 848bp size fragment, this fragment, while digestion of pcDNA3.1 () with T4 ligase connection, to construct the recombinant plasmid; obtained after double digestion fragments were about 5400bp and 800bp or so, indicating that the the TRAIL gene cloned into the expression vector pcDNA3.1 (). Recombinant plasmids using liposomes recombinant plasmid TRAIL gene expression in esophageal cancer cells transiently transfected esophageal cancer cells after 48 hours, the cells was extracted RNA, RT-PCR results showed that transfection of pcDNA3.1 ()-TRAIL group than not transfection group transfected with pcDNA3.1 () empty plasmid group expression significantly increased electrophoresis results by the software detects untransfected, transfected with empty plasmid group and transfection group of gray values ??with the corresponding internal reference ratios were 1.05 ± 0.33,1.05 ± 0.33 and 1.80 ± 0.35, the purpose of transfected genome TRAIL gene mRNA expression was significantly enhanced (P lt; 0.05); 48h after transfection using immunocytochemistry method was used to detect the expression of TRAIL in the cell membrane, cytoplasm and brown yellow transfected target gene group was significantly higher than the non-transfected group, after the software detects get the gray value 118.23 ± 0.46 and 78.19 ± 0.41, respectively, after transfection, the protein levels of TRAIL gene expression was significantly higher (P lt; 0.05 ). 3 TRAIL apoptosis of esophageal cancer cells by flow cytometry results show: the recombinant plasmid transfected 24h no apoptosis curve after 48h apoptosis curve of the cell cycle, compared with untransfected proliferation index was significantly reduced (P lt ; 0.05), reached a peak 48h TRAIL apoptosis gene transfection. 4 recombinant plasmid of recombinant plasmid liposome effect on the role of tumor resistance gene SIRT1 transiently transfected esophageal cancer cells after 48 hours, the cells was extracted RNA, RT-PCR showed that transfection of pcDNA3.1 ()-TRAIL than not transfected group, transfected pcDNA3.1 () empty plasmid group expression significantly reduce the electrophoresis results by the software detects untransfected, transfected with empty plasmid group and transfection group of gray values ??with the corresponding internal reference ratio is 1.79 ± 0.35,1.79 ± 0.35 and 1.06 ± 0.32, after transfection resistance gene SIRT1 mRNA expression was significantly inhibited (P lt; 0.05); 48h after transfection, immunocytochemistry method detection SIRT1 significantly in the nucleus, qualitative expression reduce almost no brown particles, significantly lower than the untransfected group, after the software detects get the gray value were 78.18 ± 0.42 and 118.24 ± 0.46, TRAIL could significantly inhibit the expression of the resistance gene SIRT1 protein levels (P lt ; 0.05). Conclusion: an experiment successfully constructed the eukaryotic expression vector pcDNA3.1 ()-TRAIL and confirmed the expression of TRAIL gene in human esophageal cancer EC9706 cells in the mRNA and protein levels. The two experiments confirmed that a the TRAIL gene can EC9706 cells produce apoptotic effects. Three experiments confirmed that the recombinant eukaryotic expression vector pcDNA3.1 ()-of TRAIL in TRAIL gene can inhibit tumor resistance genes SIRT1 expression at the mRNA and protein levels for further research TRAIL by SIRT1 gene regulating tumor resistance mechanisms experimental basis.

Study on Relationship between TRAIL Expression and Multidrug Resistance Gene SIRT1

Abstract

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