| In the past two decades, the growing speed of prostatic carcinoma incidence rate in China is much higher than that of other tumors. Research of new tumor markers can not only elucidate etiopathogenesis of tumor, but also serve as the base for new diagnose and treatment technique. So the search for a new tumor marker always pioneers the tumor basic research including prostatic cancer.N-myc downstream regulated genes are a new gene family discovered in recent years, including NDRG1, NDRG2, NDRG3 and NDRG4.The NDRG family shares a conserved Ndr domain, and the domain similar to theα/β-hydrolase superfamiy. Phylogenetic analysis revealed that NDRG1 and -3belong to one subfamily, whereas NDRG2 and -4 belong to another.Up to now, functions of NDRG family have been studied preliminarily by scholars at home and abroad.NDRG1 is distributed primarily in the cytoplasm, but also in the cell membrane and in the nucleus. It appears to play a role in growth arrest and cell differentiation, possibly as a signaling protein shuttling between the cytoplasm and the nucleus. Various conditions including hypoxia and androgens induce NDRG1 expression. Two contradictory viewpoints are held towards the role of NDRG1 in tumors. Some believe that overexpression of NDRG1 in tumor cells decreases the proliferation rate, enhances differentiation, suppresses the metastasis of the cancer cells and decreases tumor-induced angiogenesis. The results suggested a role of NDRG1 as a candidate tumor-suppressor gene. Others report that NDRG1 is in positive correlation to tumor development, i.e., the occurrence, growth, and transfer of colorectal cancer can result in the enhancement of NDRG1 expression, and the high-level expression NDRG1 has relation to do with short term survival, advanced stage tumor, tumor size, and canalis haemalis invasion. NDRG2 in many human tissues are differential expression genes, that is, their expression amount in cancer tissues is manifestly lower than the corresponding normal tissues. Transfection of human glioblastoma U373 cells with NDRG2 markedly reduced proliferation of the glioblastomacells. It is generally believed that NDRG2 is a candidate tumor- suppressor gene.NDRG3 is expressed in many organs but the highest expression levels were found in testis and prostate in mice.It may play a role in mouse spermatogenesis as it was shown to be localized to the outer layers of the seminiferous epithelium.NDRG4 participate in neural cell differentiation and brain development. In brain tissue of patients with Alzheimer's disease the expression of NDRG4 was significantly lower than the health adult. Decreased NDRG4 expression of PC12 cell line by stable transfection of NDRG4 antisense expression vector can inhibit the axon growth induced by NGF.However,there is no report regarding the function of NDRG3,NDRG4 in tumors and their relationship with tumors.This experiment carry out research of NDRG3's expression and function in prostatic cancer by virtue of molecular biology such as MPSS,mmunohistochemistry, gene transfection and RANi ,so as to prove the biological functions of NDRG3 in prostatic cancer.Results:1 Result of MPSS technique shown that NDRG3 expression in 33 different human normal organs is tissue-specific with the highest expression detected in testis and prostate.2 RNA and protein of NDRG3 is expressed both by RT-PCR and Western blot analysis in all the five prostate cell lines (PC-3, DU145, CL-1, LNCaP and WPMY-1) and with the highest expression detected in prostatic cancer cell than stromal cells. 3 We successfully constructed pEGFP/NDRG3 green fluorescent protein and fusion expression vector . Transient transfected expression vector to PC-3,LNCaP and WPMY-1 prosatatic cell lines showed that NDRG3 fusion protein main was a cytoplasmic protein.4 NDRG3 expression was detected in 58.6% (41/70) of prostate-cancer specimens compared to 13.2% (5/38) of BPH specimens by immunohistochemistry in tissue array.There was significant difference between the two groups(P<0.05). The cellular distribution of NDRG3 were further analyzed in these specimens.NDRG3 expression was detected in the epithelial cells in BPH tissue, and no significant staining was observed in the stromal cells in BPH tissue (Fig. 4a). Among the 41 NDRG3-positive prostate cancer specimens, NDRG3 expression was detected in epithelial cells and stromal cells.Expression of NDRG3 was detected in 56.1%(23/41)of stromal cells.The results indicated that NDRG3 is overexpressed both in epithelium and stroma of prostate caner, compared to that in BPH samples. In addition, NDRG3 was main expressed in the cytoplasm of the epithelium cells in BPH and no obvious nuclear staining was detected . But obvious nuclear staining was detected in epithelial and stromal cells of prostate cancer specimens. However,we did not observed the relation of stain intensity with clinical index,such as Gleason grade,clinical stage and age of patient.5 Knockdown of NDRG3 expression in CL-1 cells reduced their growth rates and colony-formation abilitiesCL-1 cells were transfected with mixture of NDRG3-siRNA and GFP-siRNA respectively.After 72 hours transfection, the cells were harvested for western blotting analysis. Resut shown that NDRG3 expression was lowest in NDRG3-siRNA group than GFP-siRNA group.This result indicated NDRG3-siRNA knocked down most of the endogenous NDRG3 proteinThe effect of knocking down NDRG3 on cell-growth were examined by cell counting method . After 72 hours transfection, The growth rate (mean±SD)of CL-1 cells transfected with NDRG3-siRNA and GFP-siRNA (control) were 105.7%±0.14 and 188.67%±0.17,respectively(P<0.05).After incubation for additional 48 hours, the growth rate of the 2 groups of cells were 142%±0.19 and432.36%±0.28, respectively(P<0.05). The result indicated that knockdown of NDRG3expression in CL-1 cells reduced their growth.The clone formation effect of NDRG3-siRNA in CL-1 cell were observed.After two weeks incubation, the average number of colonies were 62.7±6,60.3±5 and 30.7±4.5, respectively,for Control,GFP-siRNA and NDRG3-siRNA group for three replicate experiments. Colony number in NDRG3-siRNA group were significantly lower than GFP-siRNA and NDRG3-siRNA group(P<0.05).Moreover, the average numbers of"big"colonies (diameters were larger than 1 mm)were 28.7±4.7, 27.6±4 and 7.7±3.1, respectively,for Control,GFP-siRNA and NDRG3-siRNA group for three replicate experiments. Colony number in NDRG3-siRNA group were significantly lower than GFP-siRNA and NDRG3-siRNA group(P<0.05)6 Overexpression of NDRG3 increased the growth rate , migratory Property, and nude mouse tumor formation of PC-3 cellsTo investigate the function of NDRG3 in prostate cancer cells, We successfully constructed pcDNA3.1/myc/His(2)B-NDRG3 expression vector .we established stably transfected NDRG3 clone (PN3, PN5 and PN10) and stably transfected empty vector clone ( PNK ) by transfecting the NDRG3 expression vector pcDNA3.1/myc/His(2)B-NDRG3 and empty vector into PC-3 cells.The levels of NDRG3 protein in the parental PC-3 cells, PNK, PN3,PN5 and PN10 were compared by western blot. All of these 5 cell lines expressed endogenous NDRG3 as shown by the 41 kD band. Exogenous Myc/His-NDRG3 fusion protein was observed in PN3,PN5and PN10 as a 44 kD band due to the addition of Myc/His tag, which is approximately 3 kD. The expression levels of the NDRG3 fusion protein were dramatically higher than the endogenous levels of NDRG3 protein in PN3, PN5 and PN10 cells. This result showed that the stable transfection cell lines of NDRG3 overexpression and their negative control had been established.To examine whether the expression of NDRG3 has an effect on PC-3 prostate cancer cell growth, the growth rates of aforesaid 5 cell lines were determined by counting the cells numbers. At Day 5, the growth rate were 480.8% ±0.35(mean±SD),445.3%±0.16, 740.0%±0.20, 796.7%±0.60 and 822.8%±0.30, respectively,for the parental PC-3 cells, PNK, PN3, PN5,and PN10 comparing to those at Day 0. The growth rates of all the clones that overexpress NDRG3 were higher than those of PNK and parental PC-3 cells (p < 0.05). There was no significant difference between the growth rates of PNK and parental PC-3 cells(P> 0.05). These data indicate that overexpression of NDRG3 promotes the proliferation of prostate cancer cells. In addition,overexpression NDRG3 also enhances the migratory properties of PC-3 cells in migration experiment.The role of NDRG3 in tumor growth of nude mouse were observed in vivo experiment. By Day 48,the tumor volumes were1.95±0.08 cm3, 1.438±0.41 cm3, 0.625±0.21 cm3,0.546±0.09 cm3,respectively,for the parental PN10,PN3, PC-3 cells, and PNK.The tumor volumes of PN3 and PN10 were significantly larger than those of PNK and parental PC-3 cells (p < 0.05). The data suggested that NDRG3 overexpression in PC-3 promotes tumor growth of nude mouse.7 The up-regulated and down-regulated genes of NDRG3 expressions were summarized by cDNA array technology, and this paved the theoretical base for the study of function of NDRG 3 in prostatic cancers. RT-PCR technology was applied to identity downstream gene expression under regulation of NDRG3 overexpression. Results show that Overexpression of NDRG3 in PC-3 cell up-regulated expression of FGFBP1 and chemotatic factors - CXCL1,CXCL3 and CXCL5, and meanwhile,Over-expression of NDRG3 in WPMY-1 cell also up-regulated expression of chemotatic factors CXCL3 and CXCL5. In addition, the expression of NDRG3 protein in LNCaP was upregulated significantly by Androgen analog R1881. NDRG3 overexpression inhibits mevastatin originated apoptosis in drug experiment.The research first reported that NDRG3 is an high expression and androgen controlling gene in prostate and its protein expression ratio were higer in prostatic cancer specimens than BPH specimens. Both in vivo and in vitro studies show that NDRG3 can enhance oncogenic potential of prostatic cancer cells,and analysis tumor enhancement of NDRG3 may be its relation with its suppressing of apoptosis, participateing of tumor angiogenesis. For this reason, NDRG3 as a target may provides assistance for prostate cancer therapy.
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