Effects Of The Ubiquitin Ligase Pirh2 In Inhibiting Cell Growth Of Lung Cancer

The ubiqutin-proteasom pathway (UPP) was found to be a new channel of proteins metabolism in recent years. It could induce multiple kinds of proteins to be degraded and plays a critical role in cells transformation, tumor genesis, neural fading diseases, immune responses and inflammation reaction. In this way, ubiquitin polypeptides (usually more than four ubiquitin) are covalently attached one by one to lysine residues of target protein by the concerted action of at least three enzymes: ubiqutin-activating enzymes (E1), ubiqutin- conjugating enzymes (E2) and ubiqutin-protein ligase (E3). Once the substrate-E3 complex was successfully forms, substrate proteins are targeted for rapid proteolysis by the 26S proteasome.Pirh2 (p53 induced RING-H2 protein) was a new member of ubiqutin-protein ligase family, containing 261 amino acids. Its generation was induced by wild type p53, but on the contrary, pirh2 could mediate p53 to be degraded through UPP, therefore making the typical negative-feedback loop. The wild-type pirh2 is an unstable protein with a short half-life and is a target substrate for RING domain-dependent proteasomal destruction. Accordingly, the tat-interactive protein of 60 kDa(TIP60)was necessary to bind pirh2 at N-terminal amino acids and stabilize it. The data was proved that pirh2 highly expresses in mastocarcinoma, prostatic carcinoma and bronchogenic carcinoma (mainly located in endochylema), but not being detected in normal tissues or expresses with a relatively low level. In these tumors tissues, the pirh2 plays an important role in the wild-type p53 proteolysis, so aggravating cancer genesis. However, scientists have detected the high level of pirh2 in the osteogenic sarcoma cell line Saos2 and yeast fungus which do not express p53. This phenomenon mightily implies that there must be some proteins besides p53 interacting pirh2. So, this study firstly adopts immunohistochemistry method to detect the expression of pirh2 in lung cancer specimens and then use techniques of short hairpin RNA interfering to investigate effects on A549 (one kind of lung adencarcinoma cell line) cell's proliferation, apoptosis and distribution of cell cycle. At last, we constructed a eukaryotic expression plasmid carrying the full DNA coding sequence of pirh2 to invest its impact on the level of p27 (Cyclin dependent kinase inhitior, CKI) RNA and protein. The experiment will supply new strategy or for gene therapy and explore the potential substrate of pirh2. Following three parts constitute this research project:Partâ… Detection the Expression of Pirh2 and P27^Kip1Objective This study was to explore the expression of pirh2 and p27Kip1 in lung cancer tissue and cell line A549 and their effects on the genesis and progression of the tumor. In addition, statistical method was used to analysis potential relationship between them. Methods 53 cases of lung cancer and 17 cases of tumor-surrounding lung tissues during the years 1998²004 were collected at the department of pathology, Wuhan union hospital and made 4μm microtome section. Immunohistochemistry was applied to detect the expression of pirh2 and p27Kip1 protein; differences of pirh2 or p27 Kip1 level among various pathological type, histological grades, lymphaden metastasis, and TNM were established with statistical method. Results Both of them were observed either in tumor specimen or cell line. However, pirh2 was located in endochylema whereas p27kip1 exists in nuclear. Compared with the tumor-surrounding tissues, the higher expression of pirh2 protein (79.2%) and the lower expression of p27Kip1 protein (22.6%) in lung cancer were observed. There were distinctly difference(P<0.05). Both of them were not associated with different tumor types, histological grades, lymphaden metastasis and TNM (P < 0.05). The expression of pirh2 and p27Kip1 protein was negative correlation in lung cancer. Conclusion The higher expression of pirh2 protein and lower expression of p27Kip1 protein may strengthen lung cancer information. According to the negative correlation, we can presume that p27Kip1 may be degraded by pirh2 at protein level through ubiquitin-proteasome pathway. Partâ…¡Effects of Pirh2 Gene Silence on Apoptosis and Cell Cycle of A549 Cells via Short Hairpin RNAObjective Oligonucleotide with ability of generating short (shRNA) was designed and cloned into RNA interfering vector, whose transcription was efficiently in cells, then the effects of shRNA on pirh2 expression in A549, cells proliferation ability, apoptosis and changes of cell cycle were investigated. To explore the role of A549 in the occurrence of lung cancer and the application of RNAi technique in human cell lines, which is helpful to look for a more effective way for therapy of lung cancer. Methods Two pieces of oligonucleotide chains targeting at postions 670～690 and 760～780 of pirh2 mRNA were synthesized and inserted into psiRNA-hH1-neo (one prokaryotic vector), which named as psiRNA-pirh2A and psiRNA-pirh2B. In addtiona, oligonucleotide randomly generated was set up as negative control named psiRNA-Con. The three shRNA plamids as well as psiRNA-hH1-neo were transfected into A549 cells respectively via Lipofectamine 2000. Cells stablely express shRNA were sieve out by G418. RNA and protein expression of pirh2 in A549 cells were detected by using realtime-PCR and western-blotting analysis respectively. After that, cells viability was analyzed by CCK-8 assay, apoptosis rate and changes of cell cycle were detected through fluorescence-activated cell sorting Calibur (FACS). Results Two shRNA plasmids were successfully constructed by us, which were helpful to investigate role of pirh2 in lung cancer. When psiRNA-pirh2A and psiRNA-pirh2B were transfected into A549 cells, it highly decreased pirh2 expressing. Accordingly, apoptosis rates of A549 cells transfected with this recombinant plasmid were elevated remarkably, cells viability was reduced, and cells number at G0/G1 phase was increased obviously compared with control group. Conclusion Pirh2 RNAi recombinants constructed by could effectively generate shRNA molecular in vitro. Results of this study suggest that pirh2 plays important roles in proliferation, progression and cell cycle control of cells, and inhibition pirh2 expression in lung cancer cells via efficient RNAi technique may provide a new way against cancer. Partâ…¢Deregulation the P27^Kip1 Protein Level by Pirh2 in Two Kinds of Cell LineObjective The aim of this study was to investigate the effects of pirh2 in the network of tumor-regulating. Methods Firstly, A549 and human umbilical vein endothelial cells were maintained in RPMI1640 and M199 medium respectively, then immunofluorescence technique was used to detected the expression of pirh2 and p27 Kip1. Subsequently, total RNA was isolated from A549 cells and the first strand cDNA of human pirh2 was generated by reverse transcription. The DNA sequence coding of pirh2 protein was obtained by PCR and was inserted into the polylinker region between EcoRâ… and BamHâ… sites of plasmid pIRES2-GFP, then named pIRES2-pirh2, which was subjected to identification by double digestion and DNA sequencing. The recombinant was transfected with Lipofectamine 2000. RNA and protein level of pirh2 orp27 Kip1 in these two kinds of cells were detected by realtime-PCR and western-blotting analysis respectively. Results Pirh2 and p27 all exists in A549 and umbilical vein endothelial cells, which is the precondition for the next investigation. Electrophoresis analysis showed the PCR product was about 750bp long, which was approximate to that we anticipated, 786bp. DNA sequencing results showed pirh2 coding gene was cloned into pIRES2-GFP correctly and there is no any mutations. P27kip1 protein level was lower than cells that not being transfected. Conclusion Active pirh2 protein was generated by recombinant pIRS2-pirh2 in vitro and down-regulated the level of p27kip1. This study supply the evidence that p27kip1 may be another target protein of pirh2 except for WT p53.