Human Prostate Specific Homeobox Gene NKX3.1 Inhibit Expression Of Vascular Endothelial Growth Facor A(VEGF) In Prostate Cacer Cell Line Lncap And PC3

Homeobox-containing genes are a family of regulatory genes that primarily play a crucial role during development. Recent evidence indicates that homeobox genes might also play a role in carcinogenesis. Homeobox genes are unlike 'classic' oncogenes or tumor-suppressor genes, their gain or lost of fuction does ot seem to be sufficient for tumorigenesis;rather than being the driving force for carcinogenesis Now there are also evidence suggest some homeobox-containing genes regulate agiogenesis.NKX3.1 is a prostatic specific homeobox-containing gene that expressed from the earliest stages of prostate formation to adulthood and is required for differentiation of the prostatic epithelium. Mice with deletion of one or both alleles of NKX3.1 develop prostatic Intraepithelial Neoplasia(PIN) indicating that NKX3.1 functions as an haploinsufficient tumor suppressor gene. Haploinsufficiency has been proposed as a mechaism by which the efficiency of tumorigenesis can be increased. Loss of a single allele of an haploinsufficient tumor suppressor is sufficient to confer some growth advantage on the mutated cell, thereby expanding the target popolation in which subsequent mutations can occur. Although NKX3.1 loss of function predisposes to but is not sufficient for development of prostate carcinoma, NKX3.1 can cooperate with other genes in suppression of prostate carcinogenesis. A majority of Nkx3.1(+/-);Pte^+/- mice greater than 1 years of age develop invasive adenocarcinoma, which is frequently accompanied by metastases to lymph nodes. This phenomenon suggest the idea that multiple mutation are necessary for the development of prostate cancer.Many factors have been priscribed as associated with prostate cancer110'11'12'13'14'15'16'. Somatic targets of genomic damage include NKX3.1, PTEN and p53 genes that may modulate the progression of prostate cancer. A model that Stages of progression to prostate cacer are correlated with loss of specific chromosome regions and candidate tumor suppressor genes such as NKX3.1, PTEN and P53 was established by Abate-Shen[17], but organogenesis, apoptosis and angiogenesis may also play critical roles in prostate cancer.Both loss of NKX3.1 expression and increasing angiogenesis are strongly associated with prostate cancer progression (18-19]5 and as some homeobox containing genes can regulate angiogenesis . We suppose NKX3.1 may also play a role in angiogenesis.We use two kinds of prostate cell line- Lancap and PC3 to demonstrate our hypothesis. Lncap is an androgen-depedent prostate cacer cell line and expresses NKX3.1, and PC3 is an androgen-independent prostate cancer cell line without expressing NKX3.1. We construct NKX3.1 gene into pcDNA3.1(+), a high efficient eukaryocyte expression vectors and transfect it in PC3, we use pcDNA3.1(+) as control. After G418 selection, we obtain a clone stable expression of NKX3.1. we analysis cell cycle and apoptosis by flow cytometry. We find no cell cycle change between NKX3.1 expressed PC3 and negative controls, but more apoptosis in NKX3.1 expressed PC3 than in controls, which suggest NKX3.1 may have a role in apoptosis. Then we use western blot analysis expression of VEGF, we use GAPDH as inner control. We find VEGF expression is inhibited in NKX3.1 expressed PC3. then we use RNA interference to knock down NLX3.1's expression in Lncap. We designed two target sequences and a negative sequences, we use RT-PCR to analysis NKX3.1 's mRNA level and also, GAPDH was used as inner control. One of our target sequence 's efficiency as high as 80%, then we use this target sequence to block NKX3.1's expression and then we analysis the expression of VEGF. After block of NKX3.1, expression of VEGF become high. This result is coincidence with our former result. In conclusion, NKX3.1 can inhibit expression of VEGF in prostate cancer cell line.We found that NKX3.1 can inhibit the expression of VEGF, but we can't explain the correct mechanism behind this phenomenon. We also don't know wether loss of NKX3.1 will induce angiogenic phenotype. As loss of NKX3.1 can lead to increase expression of VEGF, angiogenesis switch may open through this way.PSA maybe a critical molecular to understand the mechanism how NKX3.1 inhibit VEGF.NKX3.1 can inhibit expression of PSA by cooperate with prostate deriver Ets factor1201. PSA has antiangiogenic properties1211 through it's ability to inhibit endothelial cell proliferation, migration, and invasion.Elevation of PSA in prostate cancer maybe a defensive process to fight cancer progression. So It's coincidence with increasing expression of PSA and VEGF inNKX3.1 lost of function prostate cancer, though the machaism of NKX3.1 inhibit VEGF maybe not through expression of PSA.Based on the specificity of NKX3.1, the phenomenon that NKX3.1 inhibit expression of VEGF may provide us with significant clues. It suggest angiogenesis is an early event in carcinogenesis and angiogenesis maybe can divide into tissue specific angiogenesis and non-tissue specific angiogenesis. Tissue-specific angiogenesis may exist in initiation of carcinogenesis and is regulated by tissue specific genes such as NKX3.1. Non tissue specific angiogenesis may exist in late stage of carcinogenesis and is regulated by non tissue specific genes such as p53.For the more, We proposed a relationship between carcinogenesis, organogenesis, apoptosis and angiogenesis. For PTENN and P53, There are evidence for they are critical in both of apoptosis and angiogenesis1-22'2324-25\ Our finding demonstrate NKX3.1 may also candidate gene for regulate apoptosis and angiogenesis, Loss of NKX3.1, PTEN and P53 was thought as there major events in prostate cacer's initiation, progression and metastasis, and since all of them seem correlate with aopotosis and angiogenesis, then a network for carcinogenesis correlated with organogenesis, apoptosis and angiogenesis is established. A definitive molecular pathway of prostate cancer initiation and progression maybe not only a event accompanied by carcinogenesis, but also a event accompanied by organogenesis, apoptosis and carcinogenesis.