Influence Of Silencing Of Homeobox Gene NKX3.1 By RNA Interference On LNCaP Prostate Cancercell Lines

Prostate cancer is the most frequently diagnosed malignant tumor in male reproductive system in western countries. Prostate cancer is the first place in incidence of malignancy and the second leading cause of cance-related deaths in American males, just secend to lung cancer. With the growing population of the old and the signiaficnat development of diagnostic technology in China, there is a increasing proportion of inpatient. Most patients with early-state prostate cancer are asymptomatic, approximately 40% of prostate cancer have spread to distant sites when diagnosed at first.The majority of prostate cancer will progress from androgen dependence to independence if left untreated and always occurs in treated patients.Once this happens, the disease is difficult to control, and patient with advanced or androgen-independence cancer have short-term survival. Prostate cancer threaten seriously the health, longevity, and quality of life. The last decade has brought increased attention to and awareness of prostate cancer as a significant public health problem, as well as prevention and cure of prostate cancer facing urological surgeons. Since the discovery by Huggins in 1941 that androgen ablation benefited patients with prostate cancer,we have come to understand that androgens play a critical role in the development, progression,and treatment of prostate cancer.Finasteride, a clinically used 5a-reductase inhibitor, prevents the conversion of testosterone (T) to dihydrotestosterone(DHT) in the prostate, leading to a reduction of gland size. Finasteride is used in the treatment of BPH to relieve symptoms of outlet obstruction and reduce the risk of acute urinary retention. In the Prostate Cancer Prevention Trial involving 18882 participants, evidence showed that finasteride prevents or delays the appearance of prostate cancer during a seven-year study period, showing a promising drug that could prevent and treat prostate cancer. Evidence from studies in vitro shows finasteride reduces the proliferation of prostate cancer cells including androgen-insensitive cells PC3 and DU145, and androgen-sensitive cells LNCaP. To clarify the moleculormechanism, we identify a set of genes related to finasteride which reduce the proliferation of the LNCaP androgen sensitive prostate cancer cell line, including homeobox gene NKX3. 1 and tumor supression gene PTEN and so on. Homeobox gene NKX3.1 play a important role in the occurrenc and development of prostate cancer, loss of NKX3. 1 is strongly associated with precancerous lesion—prostatic intraepithelial neoplasia(PIN), and clinical materials shows that NKX3. 1 expression has the potential to be a promising new prognostic marker.To clarify the effects of NKX3. 1 in prostate cancer, we silence NKX3. 1 expression in androgen-sensitive prostate cancer cell line LNCaP which express homeobox gene NKX3.1 by RNA interference. We also invesgate the biological effcts of NKX3.1 silencing on LNCaP. Subsequently, we have successfully established the cell line LNCaP(-NKX3.1) which stably dose not express NKX3. 1. Comparing neoplasm-formation ability of LNCaP(-NKX3. 1) with LNCaP in vivo provides experimental evidence for its possible utilities as a tumor marker and gene-therapy target site in the future. PART I Gene expression change of prostate cancer cells LNCaP treated byfinasteride using cDNA microarrays OBJECTIVETo identify a set of genes related to finasteride which reduce the proliferation of the LNCaP androgen sensitive prostate cancer cell line. MATERIAL AND METHODSIn this study, we examined the effects of tesosterone and finasteride in cell growth of human prostate cancer cells, LNCaP. We compared gene expression of the LNCaP under conditions of finasteride treated and untreated, as well as condition of testosterone, on a cDNA microarray consisting of 96 human genes. RESULTSTestosterone enhanced cell proliferation of LNCaP, while higher concentration of Testosterone resulted in inhibited cell growth. Finasteride, however, exhibited inhibiitory actions in LNCaP cells. We identified 29 genes exhibited a probability of finasteride-responsive expression.Of these, 11 genes up-regulated while 18 genes down-regulated by finasteride treatment. Furthermore, we identified 9 up-regulated genesand 13 down-regulated genes in 22 genes with changed expression in LNCaPtreated by testosterone.CONCLUSIONSReducing proliferation of LNCaP by finasteride involves coorperation of many genes and pathways. PART II Construction of vector encoding NKX3.1 siRNA and effects ofNKX3.1 silencing OBJECTIVETo recombinate plasmid expression vector which encode NKX3. 1 siRNA. Silence the expression of NKX3.1 mRNA and NKX3.1 protein by trasfecting human androgen-sensitive prostate cancer cell line LNCaP with recombinated vector. MATERIAL AND METHODSWe designed three small DNA oligonucleotide inserts(about 70bp),based on the proprietary algorithm in the internet, which encoding two NKX3.1 siRNAs and a negtive siRNA. Small interfering RNA(siRNA) is 19~22nt double-stranded RNA which works by cleaving and destroying its cognate RNA. We prepared recombinated vectors with DNA inserts cloned into a commercially available plasmid vector pRNAT-U6. 1/Neo from Genscript company , preformed double-enzyme digest at the two enzyme-cut sites using restiction endonuclease BamHI and HindIII and DNA sequence anaysis. Subsequently, LNCaP cell at varying time points transfected with recombinated vectors were examined at the lever of NKX3. 1 mRNA and protein by real-time quantitative PCR and western-blot respectively. We successfully obtained LNCaP cells dose not expression NKX3. 1. RESULTSResults of double-enzyme digest and DNA sequence analysis verified the sequence of three DNA oligonucleotide inserts in recombinated plasmid vectors is correct. In LNCaP cells transfected with negtive siRNA vector siRNA-N , NKX3.1 expression was not changed. In LNCaP cells transfected with NKX3.1siRNA I and NKX3. lsiRNA II ,however,we displayed sequencespecific silengcing of homeobox gene NKX3.1 with down-regulation ofNKX3. 1 protein ande silencing of NKX3. 1 gene.CONCLUSIONSWe sucessfully recombinated plasmid vectors encoding NKX3. lsiRNA, and obtian human prostate cancer cell sublines:LNCaP(-NKX3. 1) in which NKX3. 1 expression silence, to investigate the effcts of NKX3. 1 expression silencing on LNCaP. PART III Biological effects of RNA interference on NKX3.1 expression inLNCaP cell and NKX3.1 silencing in vivo OBJECTIVETo examinine the effects on LNCaP cell by NKX3. 1 expression silencing, and to study neoplasm-formation ability of LNCaP(-NKX3. 1) II cell subline in experimental animal. MATERIAL AND METHODSWe compared effects on cell growth, morphology and cell cycles post and pre-silencing expression of homebox gene NKX3. I.We successfully establish the tumor burdened nude mice models of LNCaP, LNCaP(-NKX3. 1), LNCaP(-Neg) cells. Tumor xenografts volume were measured once a week, growth curves were drawn. Eight weeks later, nude mice with xenografts were executed, then tumors were taken out and tumor weight was measured, and hematoxylin and eosin staining and NKX3. 1 immunohistochemical staing were performed to examine tumor pathological changes. RESULTSMTT test indicated proliferation velocity of LNCaP(-NKX3.1) I and LNCaP(-NKX3. 1) II were higher than orther two control groups. S phase ratio increased in LNCaP(-NKX3.1) I and LNCaP(-NKX3.1) II cells, significant morphological phenotype changes were not discovered. LNCaP(-NKX3.1) tumor xenografts(positive group)velocity was larger than two control groups, and immunohistochemical assay results showed a negtive reaction in LNCaP (-NKX3. 1) xenografts and a more malignant phenotype of these tumor xenografts.