The Primary Research On The Regulation Of Maspin Gene

Prostate cancer is one of the major life-threatening diseases in men. But its invasion and metastasis are poorly understood at the molecular level. Maspin is a member of serine protease inhibitor family with tumor suppressing activity for prostate cancers, playing an important role in tumor invasion and metastasis. Maspin is originally identified in normal mammary epithelium. It is down-regulated in primary prostatecancer cells and no detectable maspin expression in metastasic prostate cancer cells. Functional studies have demonstrated that Maspin inhibits tumor invasion and motility of human prostate cancer cells in vitro. Tumor growth and metastasis are repressed as well in the nude mice assay. Moreover, Maspin is an effective inhibitor of angiogenesis. When prostate cancer LNCaP cells were implanted into a xenograft mouse model, treatment with recombinant GST-Maspin inhibited tumor growth as well as vascularization. Maspin suppressed tumor progression not only at the step of invasion and motility, but also by regulating tumor cell apoptosis. Maspin overexpression in endothelial cells can actually induce endothelial cell apoptosis not only in vitro but also in vivo. The expression data and the functional studies of maspin gene indicated that maspin could be developed into a potent anticancer agent. Therefore, it is important to elucidate the mechanism underlying the regulation of maspin gene in prostate cancer cells.The maspin gene locates to human chromosome 18q21.3 and encodes a 3.0kb mRNA transcript and translates into a 42kDa protein locaized to the cell membrane and extracellular matrix (ECM) and nuclear of epithelial cells of multiple organs. Maspin cDNA was isolated from a library prepared from normal human mammary epithelial (76N) cells. The cDNA sequence contains 2584 nucleotides. Sequence analysis of cDNA and genes from normal and tumor cell lines show that the maspin gene sequence and structure are not altered in the tumor cells, indicating that maspin is possibly down-regulated or silenced rather than mutated in cancer cells. In this study, we cloned the luciferase reporter gene expression vector including 847bp (-764~+83) maspin promoter. Then we use nucleic acid mutation technique, reporter assays and electrophoretic mobility shift assay (EMSA) to define the elements regulating the maspin gene expression in the 847bp maspin promoter. Finally, based on the study of maspin expression regulation, we selected a drug influencing the signal factor (Spl and AR) of the upstream signal pathways involved in the regulation of maspin expression. Our results showed that there were a negative androgen-responsive element (ARE) in the region of (-265—278) and a positive Spl element in (+15~+-36). And androgen receptor (AR) can specifically bind to ARE to inhibit the maspin expression. And gum mastic can enhance maspin expression by inhibiting AR function and increasing Spl DNA binding activity.PART ONE: CLONING AND PRIMARY IDENTIFICATION OFMASPIN PROMOTERObjective: The aim of this part is to clone 847bp-promoter of maspin gene and to determine its promoter activity.Methods and results: According to the sequence of maspin gene in Genbank, a pair of primer was designed for PCR. 847bp-promoter of maspin gene was amplified by PCR and inserted into pGL,3-basic, a promoter-less luciferase reporter vector, to form 847bp promoter-luciferase reporter plasmid (pGL/3-847) that proved to be right by the restriction enzyme digestion and DNA sequencing. Prostate cancer cell line LNCaP was cotransfected with pGL3-847 and pRL-TK using lipofectamin? 2000, and then the expression of luciferase reporter driven by 847bp-promoter of maspin was tested by assay of luciferase activity. The results showed that the 847bp-fragment presented a strong promoter activity that was 2.7-fold stronger than that of pGL/3-control and 92-fold stronger than that of pGLa-basic. Maspin is closely related to the progression and metastasis of prostate cancer. To observe whether or not 847bp-promoter is regulated by androgen and its receptor, the transfected LNCaP cells with pGL3-847 vector and the co-transfected PC-3M cells with the androgen receptor (AR) expression vector and pGL/3-847 vector were treated by 10'8 M concentration of R1881 ( a synthetical androgen), and then checked for luciferase activity. The results showed that R1881 inhibited the activity of 847bp-promoter by 36% in LNCaP cells. The activity of 847bp promoter was decreased by 69% at 24 h after PC-3M cells were cotransfacted with pGL/3-847 vector and androgen receptor expression vector, and AR cooperated with R1881 inhibited the 847bp maspin promoter by 74%. While in PC-3M cells transfected with only pGL3-847bp vector, little effect was observed between the R1881 treatment and no treatment.Conclusion: 1) Cloned 847bp-promoter of maspin gene is spanning from +83bp to -764bp of maspin gene. 2) Cloned 847bp-promoter of maspin gene proved to be right in sequence compared with that in Genbank. 3) 847bp-promoter proved to have a strong promoter activity in LNCaP cell line. 4) 847bp-promoter was regulated by androgen and its receptor in certain extent.PART TWO: IDENTIFICATION OF ARE AND Spl ELEMENT IN THE MASPIN PROMOTERObjective: To identify DNA cis-acting elements within 847bp-promoter of maspin gene for understanding the regulatory mechanisms of its expression. Methods and results: 1) Transfac analysis showed that there were a ARE element in the region of (-278—265) of 847bp maspin promoter and a Spl element in (+15-+36). Then nucleic acid mutation technique was used to delete/mutate ARE and Spl element. And a series of deletions from 5' and 3' ends of 847bp-promoter were obtained by PCR using 847bp-promoter as the template and inserted into pGL/rbasic to form a series of deletion/mutants-luciferase. They were transfected into androgen-responsive and androgen-refractory prostate cancer cells LNCaP and PC-3M, respectively. The luciferase activities were detected to study the effect of deletion/mutation on the activity of maspin promoter. The results showed: In LNCaP cells, deletion from -278bp to -265bp, which removed ARE element, the activity of maspin promoter increased 2.1-fold; While in PC-3M cells, the deletion of ARE had little effect on the activity of maspin promoter. It is implicated that ARE element is a negative element in LNCaP cells. And in PC-3M cells the inhibitory effect of ARE element was impaired. The deletion from +15bp to +36bp, which removed Spl element, the activity increaed about 1.8-fold. It is implicated that Spl element maybe is a positive element in LNCaP and PC-3M cells. (2) Electrophoresis mobility shift assay (EMSA) was used to identify the binding proteins to ARE and Spl element. ARE and Spl element sequence was synthesized and labeled with digoxigenin by terminal transferase. The nucleic extracts were extracted and bound to the labeled probe of ARE and Spl element with unlabeled specific-competitive and unspecific-competitive oligonucleotides. The binding complexes run in polyacrylamide gel electrophoresis (PAGE) to observe electrophoresis mobility shift. The protein that bound to ARE and Spl element specifically was found in the nucleic extract of LNCaP cells. And the bands were confirmed to be a result of specific binding for ARE and Spl element, because the DNA-protein complex was competed out by unlabeled homologous sequence, not by heterogenous sequence. Moreover, a specific anti-AR antibody can block the binding of ARE to the protein in LNCaP cells. While no ARE-protein complexwas identified with PC-3M (AR") nuclear extracts. And Spl-protein complex was observed in PC-3M cells.Conclusion: 1) With deletion analysis, a ARE negative regulatory region was identified from -278bp to -265bp in the maspin promoter. 2) With deletion analysis, a Spl positive regulatory region was identified from +15bp to +36bp in the maspin promoter. 3) AR can bind to ARE specifically in the maspin promoter in LNCaP cells and no protein binding to ARE was observed in PC-3M cells. 4) Spl-protein complex were all identified with nuclear extracts of LNCaP and PC-3M cellsPART THREE : GUM MASTIC INCREASED MASPIN EXPRESSION BY INHIBITING AR FUNCTION ANDENHANCING Spl DNA BINDING ACTIVITYObjective: Take maspin gene as targeted gene, select the upstream factor influencing its expression, and further investigate the mechanism of action.Methods and results: (1) Androgen-dependent prostate cancer cell LNCaP cells were treated with different concentrations of epidermal growth factor (EGF), human papillomavirus (HPV), 17-P-estrogen, gum mastic, quercetin, 9-cis-retinoic acid, curcumin and 2 other extracted novelly, herbal compounds. After incubation of 48 h, MTT assay was carried out to detect the effects of different factors on the proliferation of LNCaP cells. And the effects of different factors on the maspin expression were assayed using western blot after incubation of 24 h. The results showed that gum mastic, quercetin and curcumin inhibited the proliferation of LNCaP cells. HPV inhibited the maspin expression. Gum mastic and curcumin increased the maspin expression. (2)Mainly focus on the effect of gum mastic on the maspin expression. Western blot and RT-PCR were used to detect the effect of gum mastic on maspin expression at the protein and transcriptional level. Transfection and reporter gene assay further tested the effect of gum mastic on the activity of 847bp maspin promoter. The results showed that gum mastic increased the maspin expression at the transcriptional and protein level, and gum mastic increased the activity of 847bp maspin promoter. (3) Further studied the potential action mechanisms of gum mastic on maspin expression. The effects of gum mastic on ARE DNA binding activity and Spl DNA binding activity in the maspin promoter werestudied by EMSA assay. And the effect of gum mastic on nuclear AR expression was detected by western blot. The results showed that gum mastic inhibited the nuclear AR expression and ARE DNA binding activity, and increased the Spl DNA binding activity. Conclusion: Gum mastic increased maspin expression by inhibiting AR function and enhancing Spl DNA binding activity...