The Studies On Regulation Of Human Homeobox Gene NKX3.1

Prostate cancer is the most common male cancer and the second leading cause of male cancer deaths in the developed countries.In China more men are suffering from this disease in recent years.Prostate cancer has intimate relation to the homeobox gene NKX3.1.NKX3.1 is an androgen regulated prostate-specific homeobox gene that is thought to play the important roles in normal prostate development and tumorigenesis.In mice NKX3.1 is exclusively expressed in prostate epithelium and its targeted disruption leads to aberrations in prostate ductal morphogenesis and secretary protein production,and epithelial hyperplasia and dysplasia.Notably NKX3.1 mutant mice display the pathologic changes of prostatic intraepithelial neoplasia(PIN)that is the presumed precursor to prostate cancer in human,which implies that loss of NKX3.1 expression correlates with the initiation of prostate carcinogenesis.Human NKX3.1 gene maps to the chromosomal region 8p21,containing two extrons,one intron,short 5′-UTR and long 3′-UTR.The region with high loss of heterozygosity in about 80%human prostate cancer,so the gene has been proposed to have tumor suppressor function.Loss of NKX3.1 protein expression is closely related with the initiation of prostate carcinogenesis and with prostate tumor progression.But there are different opinions.Some research showed that NKX3.1 overexpressed in prostate cancer and the rate in metastatic region was higher than previous site.This indicated that NKX3.1 overexpression was related to cancer metastasis.The strong association of NKX3.1 with prostate development and prostate cancer makes this gene an attractive molecular target for further study.So far little is known about the regulatory mechanisms of NKX3.1 gene expression as well as relevant regulatory elements and factors.In the thesis we did some studies on the transcriptional regulation of human homeobox gene NKX3.1.We identified the regulatory elements in the intron and the 10 fragments in the 10 kb upstream region of NKX3.1 gene.A 44 bp-positive element was found from -6548～-6505 bp in the upstream of NKX3.1 gene and its binding protein was identified.It is not responsible for androgen stimulus and has no tissue-specific enhancer.We also found that Sp1 enhanced NKX3.1 expression by the direct interaction with Sp1 response elements in the NKX3.1 promoter.In the thesis we also did some studies on the effects of p53 and curcumin on NKX3.1 gene expression.We found that p53 and curcumim repressed androgen receptor-induced transactivation of NKX3.1 by inhibiting the expression of the AR and blocking AR-DNA binding activity.These will provide an insight into the regulatory mechanisms of NKX3.1 expression and a new explore about the effects of NKX3.1 on normal prostate development and cancerogenesis in further study.PART ONE Identification of the functional DNA elements in the intron and the 10 fragments in the 10kb upstream regions of human homeobox gene NKX3.1Objective:The aim of this part is to clone intron and the 10 fragments in the 10 kb upstream region of NKX3.1 gene and determine their effects on NKX3.1 promoter activity,to find the androgen response element and the tissue-specific enhancer and to identify DNA cis-acting elements and the binding proteins for further understanding the regulation mechanisms of NKX3.1 gene expression..Methods and results:1.During the previous experiments we have identified the basic promoter activity between -212 bp and +48 bp in the upstream of NKX3.1 gene. According to the sequence of NKX3.1 gene in Genbank,a 521 bp promoter-luciferase reporter plasmid(pGL₃-521)was constructed by PCR technology.A intron in NKX3.1 gene and 10 fragments in the 10 kb upstream region of NKX3.1 gene were amplified by PCR and inserted into the upstream of 521 bp NKX3.1 promoter in pGL₃-521 plasmid generating an intron-521 bp promoter-luciferase reporter plasmid (pGL₃in-521)and 10 fragments-521 bp promoter-luciferase reporter plasmids (pGL₃1-521～pGL₃10-521).2.Using lipofectamin^TM2000 prostate cancer cell line LNCaP was transfected with pGL₃in-521 or pGL₃1-521～pGL₃10-521 respectively, and with pGL₃-521 as the control.The effects of the intron or the 10 fragments in the upstream of NKX3.1 gene on the NKX3.1 promoter activity were tested by luciferase reporter assay.The results showed that the 7th fragment from -7681～-6483 bp increased the NKX3.1 promoter activity,which indicated this region might contain positive regulatory elements.3.NKX3.1 is an androgen-regulated gene.To observe whether the intron and the 10 fragments are regulated by androgen,the transfected LNCaP cell with them was treated with 10^-8M R1881,and pGL₃-PSA transfection of LNCaP cell was used as positive control.The lueiferase activities were analyzed.The results showed that the activity of PSA promoter was increased by R1881 obviously, while the activities of the intron and the 10 fragments were not increased.The results indicated that androgen-responsible sequence was not in the intron and the 10 fragments in the 10 kb upstream region of NKX3.1 gene.4.NKX3.1 is prostate-specific gene,to detect whether there exists prostate-specific enhancer in the 7th fragment,pGL₃7-521 or pGL₃-521 was transfected into several tumor cell lines and the luciferase activities were analyzed.The results showed that the 7th fragment increased the activity of NKX3.1 promoter in all cells,which indicated that the DNA elements that determined the tissue-specificity of NKX3.1 expression were not in this region.5.To find the positive regulatory DNA cis-acting elements within the 7th fragment and its binding protein,a series of 5′deletion mutants-luciferase reporter plasmids were constructed by 5' deletion mutant technology.All of them were transfected into LNCaP cells respectively to test the luciferase activities.The results showed that the deletion from -7681～-6658 bp did not influence the activity of pGL₃7-521,-6658～-6483 bp still increased the 521 bp NKX3.1 promoter activity obviously,which indicated the positive regulatory element maybe exist in this region. A refined 5′deletion mutants from -6658～-6483 bp were obtained and transfected into LNCaP cells.The results showed that deletion from -6548～-6505 bp made the pGL₃7-521 activity decrease obviously which indicated the 44 bp might be a positive regulatory element.The 44 bp-sequence was synthesized and inserted into the upstream of homologous or heterologous promoter to test its effects on these promoters.The results showed that 44 bp had positive regulatory effects on both homologous and heterologous promoters.To observe the effect of androgen on the 44 bp positive regulatory element,the transfected LNCaP was treated with 10^-8M R1881, the results showed that the 44 bp element was not androgen-responsible. Electrophoresis mobility shift assay(EMSA)was used to identify the binding protein to the 44 bp-sequence.The protein that bound to 44 bp-sequence specifically was found in the LNCaP nucleic extract.The result identified that the 44 bp-sequence was a functional positive regulatory element.Conclusion:1.We cloned a intron of NKX3.1 gene and 10 fragments in the 10 kb upstream region of NKX3.1 gene which spans from -10641～-474 bp of NKX3.1 gene. 2.The region between -7681～-6483 bp presented a positive regulation on NKX3.1 promoter but it is not tissue-specific.The intron and other 9 fragments could not increase the NKX3.1 promoter activity.3.Further research showed that there existed a functional positive regulatory element from -6548～-6505 bp upstream of NKX3.1 gene and a specific binding protein to the 44 bp element in nucleic extract of LNCaP cells.4.The intron and 10 fragments were not androgen-responsible. PART TWO The enhanced effect of Sp1 overexpression on human homeobox gene NKX3.1 and the identification of the functional Sp1 elementsObjective:To identify the relationship of Sp1 and NKX3.1 gene and the functional Sp1 elements within the NKX3.1 promoter for further understanding the regulatory mechanisms of NKX3.1 gene expression.Methods and results:1.LNCaP cells were transfected with different concentrations of pCMV-Sp1 and RT-PCR and Western blot assay were performed to investigate the effects of Sp1 overexpression on NKX3.1 expression.The results showed that the expressions of NKX3.1 mRNA and protein were up-regulated by Sp1 overexpression in a dose-dependent manner.2.In the previous experiments we have constructed the luciferase reporter plasmid pGL₃-521 which has the basic NKX3.1 promoter activity.Prostate cancer cell line LNCaP was cotransfected with pGL₃-521 and different concentrations of pCMV-Sp1,then the effects of Sp1 overexpression on the NKX3.1 promoter activity in pGL₃-521 were tested by luciferase reporter assay. The results showed that Sp1 increased 521 bp NKX3.1 promoter activity in a dose-dependent manner.3.Transfac showed that there were three Sp1 response elements in +29 bp～+43 bp,+9 bp～+23 bp and -60 bp～-46 bp on the upstream of the NKX3.1 gene,which were named as Sp1a,Sp1b and Sp1c.We deleted the Sp1 elements in the 521 bp and constructed a series of deletion mutants.LNCaP was transfected separately with pGL₃-521 and the deletion mutants,the luciferase activities were tested.The results showed the deletion of Sp1a and Sp1c decreased the NKX3.1 promoter activity in pGL₃-521,while the deletion of Splb did not influence the NKX3.1 promoter activity.These results meant that Sp1a and Sp1c might be functional elements.4.The Sp1a and Sp1c-sequences were synthesized separately and inserted into upstream of homologous or heterologous promoters respectively to test their effects.The results showed that Sp1a and Sp1c had significantly increased effects on NKX3.1 promoter,SV40 promoter and maspin promoter.5.EMSA was used to identify the specific binding proteins to the Sp1a and Sp1c-sequences.The results showed that the proteins that bound to Sp1a or Sp1c-sequence specifically existed in the LNCaP cell nucleic extracts.The result meant that Sp1a and Sp1c were functional Sp1 response elements.6.The DNA decoy technology was used to investigate the effects of the Sp1a and Sp1c elements further.Different amount of Sp1a and/or Sp1c elements decoy were transfected into LNCaP cells with pGL₃-521 or pGL₃-Sp1a-SV40 and pGL₃-Sp1c-SV40.The activities of luciferasc reporters showed that the promoter activities were inhibited by transfection of different excess amount of Sp1a and Sp1c elements decoy in dose-dependent manner.These results meant the Sp1a or Sp1c decoy could competitively inhibit the interaction of Sp1 transcription factor with Sp1a and Sp1c positive elements.Conclusion:1.Sp1 overexpression could increase NKX3.1 promoter activity and its expression in a dose-dependent manner.2.The Sp1 elements in +29～+43 bp and -60～-46 bp might be functional elements.3.The specific proteins that bound to Sp1 elements in +29～+43 bp and -60～-46 bp were found in the nucleic extracts of prostate cancer cell line LNCaP,Sp1a and Sp1c were functional Sp1 response elements.4.The effects of Sp1a and Sp1c positive elements could be inhibited by the Sp1 decoy DNA in a dose-dependent manner.PART THREE The inhibition and mechanisms of p53 and curcumin on androgen receptor mediated induction of homeobox gene NKX3.1Objective:To study the effects of p53 and curcumin on NKX3.1 gene expression for further understanding the gene regulation mechanisms.Methods and results:1.To determine whether NKX3.1 expression level changed with p53 or curcumin treatment in the androgen dependent prostate cancer cell LNCaP,in one hand LNCaP cells were transfected with different concentrations of pCMV-p53 wt by lipofectamin^TM2000 to increase the exogenous p53 or exposed to UV irradiation to induce endogenous p53 expression,in the other hand LNCaP cells were treated with different concentrations of curcumin.RT-PCR and Western blot assay were performed to investigate the effects of p53 or curcumin on NKX3.1 expression.The results showed that the expression of the NKX3.1 mRNA and protein dramatically decreased by exogenous or endogenous p53 or curcumin in a dose-dependent manner.2.p53 expression can lead to induction of the downstream target gene p21^waf1/tip1that is an inhibitor of the cell cycle and that causes cell growth arrest.To demonstrate whether NKX3.1 down-regulation is mediated by this p53-p21 pathway,LNCaP cells were transfected with pPSA-p21.RT-PCR and Westem blot assay were performed to investigate the effects of p21 on NKX3.1 expression.The results showed that p21 had no effect on NKX3.1 expression.It indicated that the NKX3.1 expression was not inhibited as a result of cell growth arrest mediated by p21. 3.NKX3.1 is an androgen-regulated gene.To investigate whether this inhibition of NKX3.1 expression by p53 is related to AR activity,LNCaP cells were cotransfected with pCMV-p53 wt and pSG5-hAR.RT-PCR and Western blot showed that p53 overexpression repressed androgen receptor-induced expression of the NKX3.1,while AR overexpression can relieve the inhibition of p53 on NKX3.1 expression.These results indicated that inhibition of NKX3.1 expression by p53 was mediated by AR.4. To investigate whether this inhibition of curcumin on NKX3.1 expression is related to AR activity,we treated the LNCaP cell with 10^-8M R1881 and the AR antagonist flutamide to detect if they can influence the expression of NKX3.1 protein in LNCaP cells exposed to curcumin.Western blot demonstrated that NKX3.1 protein expression was up-regulated by androgen and down-regulated by AR antagonist flutamide without curcumin treatment.When the LNCaP cells were exposed to curcumin,the NKX3.1 protein had a notable decrease,and R1881 no longer stimulated NKX3.1 protein expression significantly.Furthermore,curcumin and flutamide together could further decrease the NKX3.1 protein.The results suggested that the expression of NKX3.1 was partially dependent on the AR Signaling pathway,and curcumin could repress androgen and AR mediated induction of NK.X3.1 expression.5.To further elucidate the role of AR in p53 or curcumin induced NKX3.1 depression,LNCaP cells were cotransfected with the pGL₃-AR and pCMV-p53 wt or treated with curcumin, the luciferase activities were tested.The result showed that AR promoter activity was reduced by p53 overexpression or curcumin treatment in a dose-dependent manner. After LNCaP cells were transfected pCMV-p53 wt or treated with curcumin,the effects of p53 and curcumin on AR expression in LNCaP cells were detected directly. Both RT-PCR and Western blot analysis gave very similar results,indicating that AR expression significantly decreased in mRNA and protein levels by p53 or curcumin in a dose-dependent manner.6.To further investigate whether the function of the AR could be affected by p53 and curcumin.EMSA was used to determine the AR binding activity with ARE.It was performed using a DIG-labeled ARE probe and nuclear extracts from LNCaP cells that were transfected with different concentrations of pCMV-p53 wt or treated with curcumin.The results show that AR-ARE specific binding activity was dramatically inhibited with p53 and curcumin in a dosedependent manner.This indicated p53 and curcumin probably repressed androgeninduced expression of NKX3.1 by inhibiting the expression of the AR and blocking AR-DNA binding activity.Conclusion:1.p53 and curcumin inhibited the expression of NKX3.1 at the mRNA and protein levels in a dose-dependent manner.2.The inhibition of NKX3.1 expression was not the result of nonspecific effects of the cell growth arrest mediated by p21.3,p53 and curcumin repressed androgen receptor-regulated NKX3.1 by inhibiting the AR expression and blocking AR-DNA binding activity.