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The Regulatory Mechanisms of PCAN1 Gene Expression by NKX3.1 in Prostate Cancer LNCaP Cells

Author: LiuWenWen
Tutor: JiangAnLi
School: Shandong University
Course: Biochemistry and Molecular Biology
Keywords: Homeobox gene NKX3.1 PCAN1 Promoter Cis-acting elements Trans-acting factors
CLC: R737.25
Type: Master's thesis
Year: 2008
Downloads: 25
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Abstract

NKX3.1 is a homeobox gene by androgen regulation of prostate cell-specific expression. Human NKX3.1 gene is located on chromosome 8p21 gene length of approximately 4.2 kb, encoding 234 amino acids, and its product is a nuclear transcription factor on the structure and function, inhibit prostate epithelial growth and maintain differentiation. Study found that about 80% of prostate cancer exists in the 8p21 region heterozygous deletion showed that NKX3.1 in prostate cancer may play a role as a tumor suppressor gene, and its loss of expression is not only closely related to the initiation and progress of prostate cancer and determine the tissue specificity of tumor. Prostate cancer gene 1 (PCAN1) is a newly discovered prostate-specific gene expression. In prostate cancer cell lines, only LNCaP cells weak expression PCAN1 gene, several other poorly differentiated highly metastatic cell line (PC-3, DU145) detection of less than PCAN1 gene expression. PCAN1 located on chromosome 4q21 heterozygous deletion of this region in prostate tumors often approximately 35% of prostate tumor specimens PCAN1 gene mutation, suggesting that may PCAN1 genes as tumor suppressor genes play a role in prostate tumor. The NKX3.1 and PCAN1 is closely related to prostate epithelial differentiation and prostate cancer with prostate-specific gene expression in cell differentiation and tumorigenesis exact mechanism of its regulation of gene expression mechanism is not yet fully clear, need to be in-depth study. NKX3.1 gene NKX3.1 cDNA expression plasmid transfection raised the the LNCaP cells PCAN1 on prostate cancer gene expression. The purpose of this project is to promoter - reporter gene levels and the level of gene transcription NKX3.1 role of prostate cancer cells PCAN1 gene expression, and to explore its regulatory mechanism. In this experiment, (1) First build NKX3.1 cDNA eukaryotic expression vector, transfection prostate cancer LNCaP and PC-3 expression efficiency of the expression vector by RT-PCR and Western blotting. (2) build NKX3.1siRNA eukaryotic expression vector transfected LNCaP cells, using RT-PCR and Western blotting NKX3.1 interfere with the efficiency of silence and screening identification stably transfected LNCaP cell lines. (3) the use of PCR amplification PCAN / gene 5 'upstream 2.6 kb promoter fragment (32 bp -2 598 bp) build PCAN12.6 kb promoter - luciferase reporter plasmid and to detect PCAN1 promoter activity. (4) PCAN1 promoter - luciferase reporter gene plasmid with NKX3.1 eukaryotic expression vector were co-transfected into LNCaP cells, dual luciferase reporter gene system and were detected by RT-PCR NKX3.1 the PCAN1 promoter activity and mRNA expression. (5) changes in NKX3.1 is interference silent LNCaP cells PCAN1 mRNA expression was detected by RT-PCR. (6) MatInspector 2.2 software analysis of cloned PCAN1 2.6 kb fragment, cis-acting elements that may exist on the fragment, which has five potential NKX3.1 binding sites the (Line After NKX3.1 Binding Site NBS) through staining immunoprecipitation (ChromatinImmunoprecipitation ChIP), dual luciferase reporter gene system, electrophoretic mobility shift assay (Electrophoretic Mobility Shift Assay EMSA) to detect 5 NKX3.1 binding sites (NBSs) the the binding activity of the transcription factor NKX3.1 . (7) identified two the NBS components with NKX3.1 binding activity, and further build the two components of the in-frame deletion plasmid, NKX3.1 cDNA eukaryotic expression vector of transmission of LNCaP cells, dual luciferase report the gene systems analysis further confirmed that the the NKX3.1 to promoter activity PCANl gene regulation. The results show that: (1) successfully built NKX3.1 cDNA eukaryotic expression vector pcDNA3.1-NKX3.1, PC-3 and LNCaP cells were transiently transfected effective expression. (2) Construction of NKX3.1 siRNA eukaryotic expression vector pRNAT-in Line After NKX3.1 RNAi1 in stably transfected LNCaP cell lines can effectively silence NKX3.1 expression. (3) cloning a 2.6 kb fragment of human PCAN1 gene upstream build the 2.6-kb fragment promoter - luciferase reporter plasmid strong promoter activity in prostate cancer LNCaP. (4) in LNCaP cells, total transfected pcDNA3.1-NKX3.1 expression enhanced PCAN1 promoter activity and PCAN1mRNA in, NKX3.1 expression is interfering RNA silencing, can PCAN1 mRNA expression decreased. (5) Identification of 2 PCAN1 gene upstream NKX3.1 binding elements (NBS1:-1848bp ~-1836bp, NBS3:-803bp ~-791bp), and further confirmed the two elements involved in NKX3.1 gene expression on PCAN1 forward role. Conclusion: The results of this study show that The Line After NKX3.1 with PCAN1 gene upstream of the two functional NKX3.1 binding element binding, positive regulation PCAN1 gene expression. The results of this study laid a foundation for further study the of NKX3.1 and PCAN1 in the prostate epithelial differentiation and prostate cancer.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Male genitalia tumors > Prostate cancer
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