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Effects of the Ubiquitin Ligase Pirh2 in Inhibiting Cell Growth of Lung Cancer

Author: SuYuan
Tutor: BaiMing
School: Huazhong University of Science and Technology
Course: Internal Medicine
Keywords: Lung cancer Immunohistochemistry Immunofluorescence Ubiquitin Pirh2 P27Kip1 RNA interference Apoptosis Cell Cycle A549 cells Endothelial Cells Cloning
CLC: R734.2
Type: PhD thesis
Year: 2007
Downloads: 174
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Abstract

Ubiquitin - proteasome pathway (Ubiqutin-Proteasom pathway) is a protein found in recent years metabolic pathways, mediated by a variety of protein degradation, and cell transformation, tumor-faded lesions of the nervous system, the immune response have a close relationship and inflammation. Ubiquitin activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) collaborative, ubiquitin ligase (E3) the ubiquitin substrate proteins covalently bound, when the target protein is connected to several (usually more than 4 ) ubiquitin molecules, he incurs the proteasome 26S hydrolysis. Pirh2 (P53 induced RING-H2 protein) is a new member of the E3 family turn to play the function of the ubiquitin ligase induced by p53, the p53 poly-ubiquitination and proteasome, forming a typical negative feedback loop. The Pirh2 unstable free state itself can also be ubiquitination and proteasome, histone acetyltransferase TIP60 (Tat-interactive protein of 60 kDa) and its N-terminal to form complexes to stabilize. The Pirh2 found highly expressed in breast cancer, prostate cancer, and lung cancer (mainly localized in the cytoplasm), or low expression in normal tissues. However, there are scholars in the expression of p53-negative osteosarcoma cell line Saos2 and yeast as detected high levels of Pirh2 prompt must act on the substrate outside the p53. For this topic first Pirh2 was overexpressed in lung cancer detected by immunohistochemical method, then lowered its expression in lung adenocarcinoma cell line A549 cells by RNA interference was observed Pirh2 this new cancer gene in lung cancer role in cell growth, is designed to provide new ideas for the gene therapy. Pirh2 gene sequences carry a full eukaryotic expression vector finally build observation the Pirh2 of the cyclin-dependent kinase inhibitor the factor p27 RNA and protein levels affect the initial quest Pirh2 Pan biotinylated substrates other than p53. The test is divided into three parts: the first part of immunohistochemistry assay Pirh2 and P27kip1 of expression in lung cancer tissue collected in our hospital from 1998 to 2004 archive 53 cases of lung cancer and 17 cases of cancer tissue paraffin made 4μm slices Using immunohistochemistry (SABC) Pirh2 and p27kip1 in lung cancer and their expression levels in different histological type, degree of differentiation, metastasis and clinical stage were analyzed statistically. It was found, Pirh2 and p27 ^{Kip1 for protein expression in lung cancer, but the former is located in the cytoplasm, the expression rate was 79.2%, p27 ^{Kip1 protein located in the nucleus, expression was low, the difference was statistically significant (P lt; 0.01), compared to 22.6%, the two adjacent organizations. Statistical analysis suggests that the Pirh2 and p27 ^{Kip1 with the pathological type, histological grade, lymph node metastasis and TNM staging is there was no significant relationship (P GT; 0.05). However, our analysis of the correlation was found between, Pirh2 expression of lung cancer, p39 ^{Kip1 detection rate was significantly lower than the the Pirh2 negative specimens, there is a significant negative correlation. Thus, pirh2 in lung cancer high expression of p27 ^{Kip1 protein expression was low, negative correlation between the cue-mediated tumor suppressor protein p27 ^{Kip1 ubiquitin of degradation may be one way to the Pirh2 promote tumor formation. The second part of the short hairpin RNA down Pirh2 expression on the growth of A549 cells to further explore the role of Pirh2 in lung cancer, we constructed a short hairpin RNA (short hairpin RNA, shRNA), and observe its effect on A549 cells proliferation, apoptosis and cell cycle. The two synthetic array the Pirh2 genes (670 to 690 and 760 to 780) locus shRNA sequences cloned in psiRNA-hH1-neo plasmid named psiRNA-Pirh2A, and psiRNA-Pirh2B and design negative control (psiRNA- Con), respectively stably transfected into A549 cells. Test is divided into normal control group, psiRNA the-HH1 empty vector group, the negative control psiRNA-Con group and psiRNA-Pirh2A and psiRNA-Pirh2B Group. Realtime-PCR and Western blotting were used to detect the expression of Pirh2 RNA and protein levels change, the proliferative activity of cells in each group detected by CCK-8, flow cytometry apoptosis and cell cycle transition. It was found that this project to build two for of Pirh2 the RNA interference plasmid specifically inhibit the expression of A549 cells Pirh2; Accordingly, decreased cell proliferation, apoptosis rate increased, and is located in G0/G1 phase cells than the control group increased significantly, appear obvious cell cycle arrest. Therefore, pirh2 expected to become another target in the field of gene therapy of lung cancer, shRNA design for its success in the in vitro inhibition of growth of lung cancer cells, but in future studies to further explore the effect of in vivo tumor suppressor still need. The third part of the Pirh2 eukaryotic expression vector and its gene A549 P27 ^{kip1 To further explore the role of Pirh2 in tumor regulatory networks, the subject building carrying the entire coding sequence of the gene eukaryotic expression vector, and verify that the expression product on p27 RNA and protein levels. First routinely cultured lung adenocarcinoma cell line A549 and primary cultured human umbilical vein endothelial cells by immunofluorescence Pirh2 and p27 ^{Kip1 in which the expression. Total RNA was extracted from A549 cells, reverse transcribed into cDNA as a template, PCR amplification Pirh2 gene sequence, and connected to the eukaryotic expression vector pIRES2-GFP, named pIRES2-Pirh2. Were transfected into A549 cells and human umbilical vein endothelial cells, Realtime-PCR and Western blotting were used to detect the two cell lines p27 RNA and protein levels. The double digested product Electrophoresis found that Pirh2 gene coding sequence was successfully cloned into the eukaryotic expression plasmid pIRES2-GFP in the the pIRES2-Pirh2 name. DNA sequencing proved that the inserted gene without any mutation or deletion. Transfected A549 and umbilical vein endothelial cells, Pirh2 protein level increased in varying degrees, on the contrary, the p27 protein levels in a downward trend. It can be concluded, eukaryotic expression plasmid pIRES2-pirh2 in vitro biological activity of Pirh2 protein; p27 following p53 after another Pirh2 pan-biotinylated substrate.}}}}}}}}

Effects of the Ubiquitin Ligase Pirh2 in Inhibiting Cell Growth of Lung Cancer

Abstract

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