| In 1999, professor Zhou Jian-guang first cloned PC-1 gene. Previous studies revealed that, PC-1 expression was up-reguLated in C4-2 which was androgen-independent and metastatic prostate cancer cell lines, compared with androgen sensitive parent cell line LNCaP, and predominantly expressed in prostate tissue as well as that was reguLated by androgen. It suggested that PC-1 might play an important role in the transition from androgen-dependent to androgen-independent status and might be associated with prostate cancer progression.1.1kb promoter sequence was also cloned and it located in the upstream of mPC-1 gene. This promoter characterized relatively evident prostate-specific expression, it's transcriptional activity was much higher than prostate specific antigen PSA promoter and human PC-1 (hPC-1) 5kb promoter. Through recombination modification, two new promoters, 14-ERE and 23-ERE (In this study, it was named MPP), were gained to increase the transcriptional activity and improve the prostate-specific activity. Subsequently, 14-ERE and 23-ERE sequences were directly cloned into plasmid pGL3-Basic, the plasmids were named p14-ERE and p23-ERE. By dual luciferase assay, the result indicated that, p14-ERE and p23-ERE expressed in prostate and breast cancer cells, but their activities were higher in prostate cancer cells than that in breast cancer cells. Further study found that the activity of 14-ERE and 23-ERE was much higher than other promoters, such as PSA-61, hPC-1 5kb promoter. Moreover, the activity of 23-ERE was higher thanl4-ERE in prostate cancer cells and lower in breast cancer cells. Thus, 23-ERE displayed the obvious advantage of prostate-tissue specificity. So, in this study, 23-ERE (MPP) was chosen as the promoter of prostate-specific vectors of transgenic mouse.Transgenic mice models were important tools used for the investigation of the mechanism of initiation and development of prostate cancer, they were also the important way to study the in-vivo function of definite genes. Thus, based on the former molecuLar and cellular level study of PC-1 gene, we constructed transgenic expression vectors of PC-1 to investigate the in-vivo function and the possible effect of PC-1 in the initiation and development of prostate cancer. There were 4 kinds of vectors, pCAGGS-PC-1-XhoI,pCAGGS-PC-1-PC-1-NotI,pMPP-PC-1 and pMPP-PC-1-NdeI, divided into two types, such as non-tissue-specific and prostate-specific expression vectors. Among them, non-tissue-specific vectors based on pCAGGS plasmid, we produced pCAGGS-PC-1-XhoI and pCAGGS-PC-1-PC-1-NotI by inserting one PC-1 gene or two tandem PC-1 genes, respectively. Considering the convenience of microinjection, the above exogenous fragments were inserted the same Not I site, respectively. Two kinds of prostate-specific vectors, pMPP-PC-1 and pMPP-PC-1-NdeI, based on pIRES2-EGFP plasmid, and they both contained MPP promoter and one PC-1 gene, for more difference of them, to see appendix. Western Blot indicated that PC-1 protein might express in 293T cells transfected with pCAGGS-PC-1-XhoI and pCAGGS-PC-1-PC-1-NotI plasmids, respectively. Moreover, fluorescent microscope observation revealed that green fluorescent protein expressed both in LNCaP cells transfected with pMPP-PC-1 and pMPP-PC-1-NdeI plasmids; however, the expression of green fluorescent protein was stronger in LNCaP cells transfected with pMPP-PC-1-NdeI plasmid than pMPP-PC-1. So, pMPP-PC-1-NdeI was used for microinjection.Yeast two-hybrid assay was performed to indentify molecuLars interacted with PC-1, as a resuLt, Filamin A (FLNa) was isolated. Filamin A was an actin-binding protein, expressed widely in different types of cells. It was a homodimer of 280 kDa containing N-terminal actin-binding domain and 24 tandem repeats of 96 amino acids. Filamin A was a multifunctional protein, produced a marked effect in assemble of cytoskeleton, signal transduction and nucleic localization of specific protein, and so on. It was found that, Filamin A was able to interact with androgen receptor (AR) in prostate cancer cells, regulated the activity of AR signaling pathway. In the present study, shRNA vector of Filamin A gene was designed and the expression of Filamin A in C4-2 cell line was inhibited to investigate alteration of cell function after interfere of Filamin A by using MTT and plate colony forming assay. Thus, this study might shed novel light (or insight) on further study of biological significance of the interaction of Filamin A, PC-1 and AR in prostate cancer initiation and progression.
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