Gene Expression Profile Of Prostate Cancer PC-3 Cell Induced By 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 cancerogenesis.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.In human NKX3.1 expression is generally restricted to the prostate and it is androgen-regulated.Since NKX3.1 gene maps to the chromosomal region 8p21,a region with high loss of heterozygosity in about 90%human prostate cancer,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 no mutations in the NKX3.1 gene have been found in prostate tumor specimens.Its second allele is inactivated by some mechanisms other than mutations in the coding region.The strong association of NKX3.1 with prostate development and prostate cancer makes this gene an attractive molecular target for further study.The NKX3.1 homeobox gene provides an excellent model to explore the relationship between embryogenesis and oncogenesis.So far little is known about the regulatory mechanisms of NKX3.1 gene expression as well as its function.As a DNA-binding transcriptional factor,its downstream targeted genes are not well known. In the current study,to further investigate the function of NKX3.1 and elucidate its function mechanism,the PC-3 cells were stably transfected with pcDNA3.1-NKX3.1 expression vector.The change of gene expression profile induced by NKX3.1 was analyzed using gene chip assay.Then the differentially expressed genes identified by gene chip were confirmed using RT-PCR and western blotting both in PC-3 cells stably transfected with pcDNA3.1-NKX3.1 and LNCaP cells stably transfected with pRNAT-RNAi.Finally,according to the results of gene chip,we further investigated the function and regulatory mechanism of NKX3.1 on the gene expression of IGF1R and caspase3.Our studies first analyzed the gene expression profiles in PC-3 cells induced by NKX3.1 and indicated that NKX3.1 might exert its functions by regulating the expression of relative genes.So,our study will be helpful for elucidating the molecular mechanism of NKX3.1 in the development and progression of prostate cancer.Meanwhile it will provide new clues for prostate cancer diagnosis and clinical therapy.PART ONE GENE EXPRESSION PROFILES IN THE PROSTATE CANCER PC-3 CELLS INDUCED BY NKX3.1【Objective】To explore the change of gene expression in the PC-3 cells induced by NKX3.1 and identify target genes downstream of NKX3.1 for better understanding the roles of NKX3.1 in prostate cancer【Methods and results】1.In the previous experiments we have successfully constructed NKX3.1 expression vector pcDNA3.1-NKX3.1.Using FuGENE HD transfection reagent,PC-3 cells were transfected with pcDNA3.1-NKX3.1.Through G418 screening and infinite diluteness culture,the stable clones PC-3(+) were isolated using cloning cylinders.The cells transfected with pcDNA3.1(+) vector served as control.Then the expression of NKX3.1 in PC-3(+) cells was detected by RT-PCR and Western blotting.The results showed that the clones 1,2,3,4,5,8 and 9 in 9 selected clones presented a comparable increased expression of NKX3.1,but the control clones showed no detectable expression of NKX3.1.2.MTT colorimetric assay and colony formation assay were performed to study the effect of NKX3.1 on the proliferation of PC-3 cells.Matrigel invasion assay was conducted to test the effect of NKX3.1 on the ability of cell invasion.As a result,we found that the cells displayed a decreased growth rate in clone 1,clone 8 and clone 9, expecially in clone 8.Their proliferation was inhibited by～50%compared to the control.Moreover,the colony-forming capacity of PC-3(+) cells in clone 1,clone 8 and clone 9 was weakened with～21.5%reduction averagely relative to the control. The cells in clone 1(63±11),clone 8(55±17) and clone 9(51±12) passed through the membrane to a much lesser extent than the control PC-3-pcDNA3.1 cells(144±13) did(P <0.05).3.Total RNA was respectively extracted from PC-3(+) cells and control cells.A gene chip containing 22,000 human genes was used to identify the gene expression differences.The results showed that there were 1,953 genes showing more than a two-fold difference in expression,with 1013 genes being up-regulated and 940 genes down-regulated.139 genes of the differentially expressed genes showed an over ten-fold change,of which 128 genes decreased and 11 genes increased.Subsequent ontological analysis revealed that a large proportion of the classified genes were related to cell growth/proliferation,cell signal/communication,cell adhesion/invasion and response to stimulus.4.The eukaryotic expression plasmid of siRNA targeting NKX3.1 was constructed and transfected into LNCaP cells.Through G418 screening and infinite diluteness culture,the stable clones LNCaP(-) were isolated using cloning cylinders.The cells transfected with a negative shRNA vector served as control.The expression of bcl-2, sp1,sod2,cyclinE,Tusc3,P27,Cdk6 and AMACR,randomly selected genes from chip data,were validated by RT-PCR and western blotting in PC-3(+)/PC-3 cells and LNCaP(-)/LNCaP cells.The results were consistent with the chip data.【Conclusion】1.NKX3.1 could be expressed stably in PC-3(+) cells.Forced expression of NKX3.1 inhibited cell proliferation and invasion of PC-3 cells.2.The gene expression profile changes in PC-3 cells induced by NKX3.1 indicated that NKX3.1 might exert its functions by regulating the expression of relative genes. NKX3.1 might be one of regulators involved in cell signaling,cell communication and adhesion regulators.These results provided a new insight in the study of the relationship between NKX3.1 and prostate cancer.PART TWO THE STIMULATIVE EFFECT OF NKX3.1 ON CASPASE 3 GENE AND APOPTOSIS OF PROSTATE CANCER CELLS【Objective】To study the effect of NKX3.1 on caspase3 gene expression and investigate its preliminary regulatory mechanism【Methods and results】1.The expression of caspase3 was detected by RT-PCR and Western blotting both in PC-3 cells and PC-3(+) cells.The results showed that the expression of caspase 3 was much higher in PC-3(+) cells than that in PC-3 cells.2.PC-3 and PC-3(+) cells were respectively treated with 10ng/ml TNF-αand 10μg/ml cycloheximide(CHX) for 6h.The activity of caspase3 was detected using caspase3 activity test kit.The cell apoptosis was evaluated by FACS.The results showed that with treatment of TNF-αand CHX,the activity of caspase 3 increased 2.4 times in PC-3(+) cells but 1.4 times in PC-3 cells.The apoptotic rate of PC-3(+) was 8.2%while the apoptotic rate of PC-3 cells was 3.14%.3.According to the sequence of caspase3 gene in Genbank,a pair of primer was designed for PCR.3764 bp-promoter of caspase3(-2880 bp/+884 bp) gene was amplified by PCR and inserted into pGL3-basic vector to form 3764 bp promoter-luciferase reporter plasmid(pGL3-pcas) that proved to be right by the restriction enzyme digestion and DNA sequencing.4.pGL3-pcas was transfected into PC-3 or cotransfected with pcDNA3.1/ pcDNA3.1-NKX3.1 using FuGENE HD,and then the activity of 3764 bp-promoter of caspase3 gene and the effect of NKX3.1 on the promoter activity were tested by luciferase reporter assay.The results showed that the 3764 bp-fragment presented an obvious reporter activity.Cotransfection with pcDNA3.1-NKX3.1 increased the promoter activity by 3 times.5.According to MatInspector2.2 software analysis,there are three potential NKX3.1 binding sites(NBS1～3) in the 3764bp region.EMSA(electrophoretic mobility shift assay) and ChIP(Chromatin immunoprecitation) were performed to investigate the binding activities of these NBSs with NKX3.1 transcription factor in vitro and in vivo.Finnally,the NBS1～3 sequences were synthesized and inserted into upstream of SV40 promoter in pGL3-promoter to form pGL3-NBSs-promoter.Then the pGL3-NBS-promoter was cotransfected with pcDNA3.1/ pcDNA3.1-NKX3.1 into PC-3 cells and the activity of SV40 promoter was tested by luciferase reporter assay.The results showed that NKX3.1 had specific binding activity with the NBS3 sequence.With the insertion of NBS3 into the upstream of SV40 promoter,the promoter activity increased 1.86 times.But the insertion of NBS1～2 into the SV40 promoter had no effect on the promoter activity.【Conclusion】1.NKX3.1 could increase the expression of caspase3 in PC-3 cells at transcription level,meanwhil it could promote its activity and induction of cell apoptosis.2.One functional NKX3.1 binding site(NBS3) located at -718 bp to -732 bp upstream of the caspase3 gene was involved in the positive regulation of caspas3 gene by NKX3.1. PART THREE THE INHIBITORY EFFECT OF NKX3.1 ON INSULIN GROWTH FACTOR 1 RECEPTOR(IGF1R) AND PROLIFERATION OF PROSTATE CANCER CELLS【Objective】To study the effect of NKX3.1 on IGF1R gene expression and investigate its preliminary regulatory mechanism【Methods and results】1.The expression of IGF1R was detected by RT-PCR and Western blotting both in PC-3 cells and PC-3(+) cells.The results showed that the expression of IGF1R was much higher in PC-3(+) cells than that in PC-3 cells.2.PC-3 and PC-3(+) cells were respectively treated with 100ng/ml IGF1 for 24 h. MTT assay was used to analyze the effect on cell growth.The cell cycle analysis was evaluated by FACS.The results showed that 100ng/ml IGF1 stimulated cell proliferation by 55.1%in PC-3 cells but only 17.1%in PC-3(+) cells. Stimulation of the cells with IGF1 for24 h decreased the ratio of cells in G₀-G₁ phase from 68.18%to 56.85%for PC-3 cells and from 92.4%to 85.51%for PC-3(+) cells,respectively.3.According to the sequence of IGF1R gene in Genbank,a pair of primer was designed for PCR.3304 bp-promoter of IGF1R gene(-3259 bp/+45 bp) was amplified by PCR and inserted into pGL3-basic vector to form 3304bp promoter-luciferase reporter plasmid(pGL3-pIGF1R) that proved to be right by the restriction enzyme digestion and DNA sequencing.4.pGL3-pIGF1R was transfected into PC-3 cells or cotransfected with pcDNA3.1/ pcDNA3.1-NKX3.1 using FuGENE HD,and then the activity of 3304 bp-promoter of IGF1R gene and the effect of NKX3.1 on the promoter activity were tested by luciferase reporter assay.The results showed that the 3304 bp-fragment presented an obvious reporter activity.Cotransfection with pcDNA3.1-NKX3.1 inhibited the promoter activity by 50%.5.According to MatInspector2.2 software analysis,there are two potential NKX3.1 binding sites(NBS1～2) in the 3304 bp region.EMSA and ChIP were performed to investigate the binding activities of these NBSs with NKX3.1 transcription factor in vitro and in vivo.Finnally,the NBS1～2 sequence were synthesized and inserted into upstream of SV40 promoter in pGL3-promoter to form pGL3-NBSs-promoter.Then the pGL3-NBSs-promoter was cotransfected with pcDNA3.1/ pcDNA3.1-NKX3.1 into PC-3 cells to test NBSs' effects on SV40 promoters by luciferase reporter assay.The EMSA and ChIP results showed that NKX3.1 couldn't bind with NBS1～2.The insertion of NBS1～2 into the SV40 promoter had no effect on the promoter activity.【Conclusion】1.NKX3.1 could down-regulate the expression of IGF1R in PC-3 cells and inhibited IGF-1 induced cell proliferation.2.NKX3.1 had no binding activity to NBS1～2 upstream of IGF1R gene. Down-regulation of IGF1R gene by NKX3.1 was not mediated by NKX3.1 binding directly with NBSs in the upstream of IGF1R gene.It might be indirect. The mechanism need to further study.