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The Feasibility and Mechanism of Survivin Regulation of Transcription Level by P21^CIP1 Overexpression in HepG2 Hepatoblastoma Cell

Author: XiongJuan
Tutor: HuLiHua
School: Huazhong University of Science and Technology
Course: Clinical Immunology
Keywords: p21cip1 gene Transfection Green Fluorescent Protein Liver tumors survivin Regulation of gene expression HepG2 cells Adriamycin Proliferation
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 27
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Abstract

Construction of the first part of the eukaryotic expression vector pEGFP-C2-p21 and p21 ^{cip1 gene stably transfected HepG2 cell line Objective: Construction and identification of eukaryotic expression vector pEGFP-C2-p21, and The recombinant plasmid was transfected into human hepatoblastoma cell line HepG2 screened positive cell clones. Methods: p21 ^{cip1 Full-length cDNA fragment was cloned into the eukaryotic expression vector pEGFP-C2 double digestion, colony PCR and sequencing to construct the recombinant plasmid containing the target gene p21 pEGFP-C2-p21 . PEGFP-C2-p21 plasmid liposome-mediated transfection of HepG2 cells, fluorescence microscopy transfection efficiency in HepG2 cells, and screened to obtain the integration of the p21-positive cells cloned with G418 medium, and total RNA was extracted from the cells to be with. Results: (1) build a complete pEGFP-C2-p21 plasmid vector that stably expressed and filter characteristics, and sequencing results are fully consistent with the expected design. (2) after transfection visible pEGFP-C2-p21 positive cells expressing green fluorescent protein, and G418 selection to obtain a sufficient number of positive clones containing wild-type p21 in HepG2 cells by fluorescence microscopy examination. Conclusion: successfully constructed the eukaryotic expression vector pEGFP-C2-p21, screening high expression of p21 the HepG2 positive clones cells, available for follow-up research use. The second part of p21 ^{cip1 hepatoblastoma cell survivin transcription and regulation mechanisms of purpose: to study the cyclin-dependent kinase suppressor protein cip1/waf1 (referred to as p21) on human hepatoma cells inhibition of survivin transcription regulation, and to explore the appropriate mechanism. Methods: doxorubicin treatment liver neuroblastoma cell lines HepG2 and also liposome plasmid pEGFP-C2-p21 import HepG2 and were screened by G418; real-time PCR quantitative detection of p21, p53 and survivin mRNA expression by flow cytometry surgery, reverse transcriptase PCR analysis of changes in the cell cycle after transfection, E2F-1 and p300 mRNA changes. Results: (1) doxorubicin treatment induced increased expression of p53 and p21 gene and is associated with the reduction of the expression of survivin within 24 h; (2) stable transfection significantly increased p21 mRNA expression in HepG2 group, HepG2 -C2 group of 2100.11 times and 980.89 times; survivin mRNA levels were also significantly lowered, respectively 0.54% and 0.59% of the control group; p53 mRNA expression had no significant effect. (3) transfection of p21, a cell cycle arrest in G0/G1 phase (F = 31.59, P lt; 0.01); transcription factor E2F-1 The p300 mRNA content has reduced by (F E2F-1 = 125.28 P lt; 0.05 F p300 = 46.01, P lt; 0.01). Conclusions: p21 may inhibit survivin transcription factor E2F-1 and p300, thereby inhibiting survivin expression in HepG2 cells and cause G1 arrest. The third part of Adriamycin of p21 ^{cip1 gene on human hepatoblastoma cell line HepG2 proliferation of purpose: Adriamycin class traditional chemotherapy drugs in the treatment of hepatoblastoma, but in recent years its poor clinical efficacy, currently with the gene therapy to improve the efficacy of doxorubicin, a new trend of development of the treatment of hepatoblastoma. This study investigated the p21 gene with doxorubicin combination of human hepatoblastoma HepG2 cell proliferation. METHODS: The experiment points transfected group and combined treatment group. Transfection group was the control group, pEGFP-C2 transfected with the control group, P21 transfected group; combined treatment group was the control group, the drug alone group, P21 transfected group and P21 transfected with the drug combination group. Methyl thiazolyl tetrazolium blue (MTT) assay after transfection of HepG2 cell growth trend and the inhibition of cell proliferation status of joint processing; fluorescence quantitative PCR (real-time quantitative polymerase chain reaction) detection of p21 mRNA expression after transfection survivin mRNA level changes in the level of joint processing. RESULTS: After transfection, p21 transfection group and two control groups (P lt; 0.01); identified p21 mRNA expression increased significantly slower than the growth rate of 3 and 4 days, the blank control group 2100.11 times (P lt; 0.05). Blank group for control in 3 ^{5 day, a joint group proliferation inhibition rate the separate dosing group and p21 transfection group was significantly higher than (3 days: 43.92% vs. 32.97%, 35.77% , P lt; 0.01; 4 days: 59.86% vs.39.35%, 40.96%, P lt; 0.01; 5 days: 51.81% vs.33.91%, 10.68%, P lt; 0.01);, and 1 ~ 4 d within with a combined time of prolonged inhibitory effect enhanced (r = 0.91, P lt; 0.05), and in the 4th day more significant (Q = 1.07). PCR showed that survivin mRNA level of the combined group was significantly lower than the separate dosing groups; transfected with p21 group, the combined effects only difference was statistically significant (P lt; 0.01) at 48 h. Conclusion: p21 within a certain time range can be enhanced doxorubicin inhibited the proliferation of HepG2 cells, reducing the intracellular expression of survivin mRNA.}}}}}

The Feasibility and Mechanism of Survivin Regulation of Transcription Level by P21CIP1 Overexpression in HepG2 Hepatoblastoma Cell

Abstract

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The Feasibility and Mechanism of Survivin Regulation of Transcription Level by P21^CIP1 Overexpression in HepG2 Hepatoblastoma Cell