| Along with the human mean lifetime's extension and the diagnosis technology's unceasing enhancement, the prostate gland cancer's disease incidence rate assumes unceasingly the trend of escalation. In the US, the prostate gland cancer disease incidence rate and the mortality rate are only inferior in the masculine malignant tumor in the lung cancer occupy second. Although in our country, the prostate gland cancer's disease incidence rate is lower than the Western country, but along with life style's change, the population aging as well as the prostate gland cancer histology tumor designated object like prostate specific membrane antigen(PSMA), cytokeratin34βE12(CK34βE12), P63, the Pca224 protein, human telomerase reverse transcrip tase(hTERT), differential disp lay code 3(DD3) and so on screening technology's widespread applications, the prostate gland cancer's disease incidence rate unceasingly is also rising, at present leapt to masculine urogenital system malignant tumor disease incidence rate third.Prostate cancer pathogenesis and the development of molecular biology is still unclear. Previous studies showed that the incidence of prostate cancer development was related to the inactivation of wild-type p53, p16, PTEN, mn23, NKX3.1, bcl-2, Rb and other tumor suppressor gene, abnormal expression of c-myc, c-met, ras, polymorphism of AR, PSA, VDR gene and DNA methylation processes. Recent studies have shown that the receptor of advanced glycation end products (RAGE) had also closely related to the incidence of prostate cancer.Akihiko Taguchi have reported that the interrupted interaction between RAGE and amphoterin could inhibit the growth and metastasis of tumor. Yet the role of RAGE in prostate cancer was discovered recently. In 2003, Kuniyasu have discovered the high level of co-expression between RAGE and amphoterin in tumor cells and stromal cells after the analysis of specimens in 40 postoperative patients with prostate cancer,especially in patients with metastasis. By further cytological test, they had presumed that RAGE-amphoterin system could promote the interaction between tumor cells and stromal cells which would accelerate the development and metastasis of prostate cancer throughout the process of androgen blockade. In 2005, Ishiguro and Hermani have also presumed that the interaction between RAGE and amphoterin, AGE, S100A8, S100A9 played an important role in the occurrence and development of prostate cancer. In 2008, Allmen have proved that the V-domain of RAGE was necessary for the interaction with its ligands to mediate the growth of prostate cancer. Further studies on RAGE are not only enable to reveal the pathogenesis of prostate cancer, but also have a positive meaning in prostate cancer prevention, diagnosis and treatment. However, the mechanism of RAGE in promoting the growth of prostate cancer is unclear.Retinoblastoma susceptibility gene (Rb gene) is large relatively, which locates on human chromosome 13 q14, and contains 27 exons, with the 4.7kb of the mRNA transcription product, coding Rb protein (928 amino acids)finally. Rb protein through regulation of cell cycle activity, a large number of studies has demonstrated that the combination of transcription factor E2F and the Proximal promoter element DNA promote the assembly of pre-start transcription complex (PIC),so that express the structural gene stability and promote cells to grow well. As a modified protein, there are two forms-phosphorylation and dephosphorylation in Rb protein, and its active form is dephosphorylation. The ffinity between dephosphorylation of Rb and E2F transcription factors lead to form a complex of their.Thus it blocks the combination of cis-acting elements and dephosphorylation of Rb, and then thwarts gene expression, the combination of the Rb protein with E2F can block the expression of some related gene,such as coding DNA polymerase, thymidine kinase, dihydrofolate reductase. These enzymes can not be synthesized, which blocks DNA biosynthesis and cell growth.In recent years, studies has showed that the cell cycle regulatory out of control is an important factor in the Canceration.Protein pRb was encoded by Rb gene,which is an important regulator in cell cycle.And it play a negative role in the cell cycle of G1 to S,which prevent uncontrolled growth cells from transformating to the neoplastic proliferation. We have found the mutation or deletion of Rb gene in kinds of human tumor, such as retinoblastoma, esophageal cancer, gastric cancer, breast cancer, colon cancer, prostate cancer, lung cancer and other tumors, which indicates that Rb gene is closely related with the happening of tumors. Rb can not only combine with E2F family, but also can combine with other proteins.So far, it has been found that there are more than 100 proteins interacting with the Rb protein,including transcription factors, cell cycle proteins, protein kinases, and so on..The Rb protein was initially discovered and named because of its role in retinoblastoma, but later researches has showed it is related with prostate cancer, bladder cancer, renal cancer, osteosarcoma, breast cancer, non-small cell lung cancer, glial quality of neuroblastoma and other tumors. Rb protein and related to the incidence of prostate cancer, but the specific mode of action and mechanism is unclear. Most studies have shown that Rb protein can inhibit the growth and proliferation of prostate cancer celles. There two major ways to inhibit tumor. Firstly, Rb protein inhibits the growth of prostate cancer cells through by affecting the cell cycle. Alpana Tyagi used silymarin (silibinin) to stimulate androgen-dependent prostate cancer cells LNCaP, which acted on the Rb protein serine sites to inhibit Rb protein phosphorylation. As a result, the Rb protein was combined with E2F protein,and E2F deactivated. E2F,a decision factor in cell cycle from G1 into S phase,which can induce a variety of cell growth-related gene expression. Therefore, the proliferation of prostate cancer cells were inhibited. Samir S. Taneja's research also has showed that Rb protein degradation contributed to the growth of LNCaP cells with the action of the male hormone, the degradation process involved ubiquitination and 26S proteasome. And the Rb phosphorylation induced by the cyclin A/Cdk2 and cyclin B/Cdk1 accelerated its degradation. Secondly, Rb protein can inhibit tumor cell activity by specifically combining with tumor-related proteins. Rb protein can specifically conbined with the three kinds of transformation of protein of DNA tumor virus (SV-40, adenovirus and human papilloma virus type 16),that is conbined with the SV-40 large T antigen, E1A and E7 bind\ and restrain the activity of tumor. Due to loss of exon 21 encoding 35 amino acids in mutant Rb protein of DU145 prostate cancer cells,it can not form complex with the SV-40 large T antigen and adenovirus E1A. In 1990, Bookstein, import the Rb gene into prostate cancer cells DU145, and found DU145 cell morphology, growth rate did not change with the normal expression of Rb protein,but tumorigenicity in nude mice had obviously been inhibited. Studies also showed that the Rb protein can be used as "coactivator" promote the development of prostate cancer. Shuyuan Yeh screened and verified that the Rb protein can combine with the androgen receptor protein specificly, and verified this combination can induce transcriptional activity of androgen receptor, which promotes the occurrence and development of prostate cancer. In conclusion, Rb protein plays an important role in the development of prostate cance. Depth study of the Rb protein is of great importance in exploring the mechanism of prostate cancer and looking for more effective prevention and treatment measures are meaningful.We have cloned a human RAGE intracellular domain of the prokaryotic expression vector, and express and purify human RAGE intracellular domain fusion protein successfully. We also obtained 25 species with a combination of the relationship between protein or peptide by the use of T7 phage display system, and then use the ELISA method for further validation of these proteins or peptides, and use BLAST software on GenBank sequences to search and analysis homology. Finally we obtained nine known proteins, and one of them was retinoblastoma protein. This study sought to further verify the interaction between RAGE and Rb, and then study its physiological significance, which reveals role of the RAGE in the development of prostate cancer and provides a new theoretical basis for the treatment and prevention of prostate cancerIn this study, we test the expression of RAGE in pathological specimens of prostate cancer and normal prostate tissue by immunohistochemistry. In 10 normal prostate tissue and prostate cancer could be detected in the expression of RAGE, and the expression of RAGE in prostate cancer significantly higher than in normal prostate tissue. Test results and in vitro cell culture test results are the same. Thus, we speculated that there are some correlation between RAGE protein and cell proliferation activity.Since immunohistochemistry can not be accurately quantified the expression of RAGE to further define the relationship between RAGE and prostate cancer, we used real time quantitative PCR,which have quantified the expression of RAGE in different prostatic tissues. The results showed that the expression of RAGE mRNA in prostate cancer tissue was 4.2 times more than in normal prostate tissue (P<0.01). In vivo, the expression abundance of mRNA and protein is not always consistent, and only proteins have biological functions. Western blot has showed a protein bands of molecular weight 46 000 in both normal and prostatic tissue, which is the expected size of human RAGE protein molecular. It have indicated the endogenous RAGE protein expression in prostatic tissue which were significantly higher in prostate cancer tissue than in normal prostate tissue. Results above indicate that the expression of RAGE was significantly different between prostate cancer and normal prostate tissues which was significantly higher in prostate cancer tissue, suggesting that RAGE may play an important role in the pathogenesis of prostate cancer.What is the main point of the highly expressed RAGE in the incidence and development of prostate cancer? MTT test was used to show the effect on prostate cancer cell proliferation. It showed that the interaction between RAGE and AGE-HSA could promote the proliferation of prostate cancer cells which was consistent with other reports, and indicated that RAGE indeed played an important role in development and progression of prostate cancer.In the previous literature reports, RAGE was mainly expressed in the cell membrane, while Rb mainly in the nucleus, would they overlap in prostate cancer cells? In our previous immunohistochemical study, we found that RAGE protein was expressed in both nucleus and cytoplasm rather than the specific expression in the cell membrane, while Rb protein mainly distributed in the nucleus, so we consumed that there could be an interaction between RAGE and Rb protein in the spatial distribution. In order to confirm our results, we repeated the immunohistochemistry test with commercial antibody excluding the impact of specific antibodies, which obtained similar results. Immunofluorescence was used to observe the distribution of RAGE in PC-3 cells to detect the distribution of RAGE in prostate cancer cells directly. The results show that RAGE in PC-3 cells distributed both in cell cytoplasm and nucleus, while in SW480 cells enriched in the membrane. Immunohistochemistry and immunofluorescence test results show that RAGE distributed in prostate cancer cells in the nucleus and cytoplasm widely, and this distribution has some specific. So there is overlap between the the distribution of RAGE and Rb, and there is the possibility of spatial interaction.Rb and RAGE antibodies was used for immunofluorescence experiments to observe the distribution of endogenous Rb and RAGE in PC-3 cells. The results showed that Rb is mainly distributed in the nucleus, while RAGE distributed in whole cell. There was colocalized phenomenon in the nucleus. This phenomenon showed that Rb and RAGE in the cell nucleus would form a complex which may exsist interactions between them.Proteins play an important role in cells, the interaction among proteins is a major component of a cellular biochemical reaction network, which is significant to the cellular regulation and its signal. Immunoprecipitation is a traditional method to detect the interaction among proteins based on the specificity of antibody and antigen. It is an effective method to determine the physiological interaction among proteins in intact cells. This method is commonly used in the determination of whether the two target proteins are binding in vivo, and to define a new role of specific protein. The main advantages are as followed:First,the proteins are in a natural state;Second, the interaction among proteins is conducted in a natural state to avoid the man-made impact; Third, it can be isolated protein complex from the natural state. To verify the existence of interaction between Rb and RAGE, we decided to hold co-immunoprecipitation experiments in vivo.First, we obtained plasmid pFAD102 carried the cDNA sequences ofRb gene from Professor Nick Dyson,Harvard University. In order to facilitate follow-up test experiments, pFAD102 plasmid was modified to add with a FLAG epitope tag at the 5'end of the Rb coding sequence by PCR which maked it into a CMV-FLAG-Rb plasmid. CMV-FLAG-RB plasmid sequence was proved completely correct by restriction enzyme digestion and sequencing analysis.Then CMV-FLAG-RB plasmid and pcDNA3-HA-RAGE plasmid were transfected into HEK293 cells according to the ratio of 1:1. Western blot was used to detect the expression of RAGE and Rb in 24h,36h,48h. It showed CMV-FLAG-RB and pcDNA3-HA-RAGE could express in HEK293 cells, Rb and RAGE had the most efficient co-expression in the 36 hours after transfection; Because the expression of RAGE is higher than Rb, so future experiments were hold after 36 hours with the transfection of CMV-FLAG-RB and pcDNA3-HA-RAGE in accordance with the ratio of 3:2.In summary, our preliminary study demonstrated the existence interaction between RAGE and Rb,but due to time constraints, immunoprecipitation in vivo can not present positive results, requiring further study to obtain the exact results. |
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