The Study On Influence Of Tumor Tissue-Specific CXCR4-siRNA In Prostate Cancer Metastasis To Bone

⒈The effect of CXCR4/SDF-1 pathway on prostate cancer targeted metastasisObjective To investigate preliminarily the role of CXCR4/SDF-1 in prostate cancer (PCa) targeted metastasis. Methods CXCR4 mRNA and protein expression in two PCa cancer cell lines, LNCaP and PC-3m, were examined by reverse transcript-PCR,western bloting and prostate cancer tissue was detected by immunohistochemistry. SDF-1 mRNA expressions in human bone, lymph node, lung, brain and liver tissues were determined by RT-PCR and SDF-1 secretary level in the culture medium of primary human osteoblasts (HOB) was detected by in ELISA. The effect of exogenous recombinant SDF-1 on PCa cells adhesion, transendothelial migration and invasion, and the effect of anti-CXCR antibody in this procedure were observed using an in vitro Transwell and Matrigel models.Results Both CXCR4 mRNA and protein expressed in LNCaP and PC-3m. CXCR4 protein expressed highly in prostate cancer tissues. Meanwhile, SDF-1 mRNA expressed in divers human tissues and semi-quantitative analysis illustrated that most highly in lymph node and bone. It is also demonstrated that SDF-1 were detected in the culture medium of HOB cells. In addition, pretreatment of exogenous SDF-1 enhanced significantly the in vitro adhesion, invasion and migration of both cell lines with a dose-dependent manner, and 50% primary osteoblast conditioned medium collected at 72h increased as well in vitro PCa cell invasion. Such performances could be blocked by administration of anti-CXCR4 antibody.Conclusion PCa Cells highly express functional CXCR4 and the most frequent metastasis organs intensively express SDF-1. SDF-1 can obviously increase the in vitro adhesion, migration and invasion of PCa cells while anti-CXCR4 antibody can block such effect. CXCR4/SDF-1 pathway may therefore play a of very importance role in PCa targeted metastasis. ⒉The Inhibitory Effects of siRNA Expression Vector on CXCR4 Expression of Prostate carcinoma cell linesObjective To investigate the inhibitory effects of RNAi (RNA interference, RNAi) expression vector on CXCR4 expression of Prostate carcinoma cell lines. Methods CXCR4 targeting siRNA gene was inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP and siRNA expression vectors were constructed to be aimed directly at CXCR4 gene. The recombinants were transfected into Prostate carcinoma cell line, PC-3m and LNCaP, with liposomes.The expression of CXCR4 was detected by RT-PCR and Western Blot. Results Expression vectors could reduced the expressions of CXCR4 mRNA and protein in the PC-3m and LNCaP cells. Compared with NC, the former, the ratio of inhibition of the expression of CXCR4 mRNA was 87.81±10.20%,56.10±9.32% in 24th hour and 48th hour, the latter,was 56.93±8.78%,49.24±11.23% , respectively. The ratio of inhibitory of the expression of CXCR4 protein was 64.71±6.68%,58.66±11.56% in 24th hour respectively . Conclusion The RNAi expression vectors containing CXCR4-siRNA sequence can effectively inhibit the expression of CXCR4 gene, which lays a foundation for post study.⒊Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by hTERT promoter and its biological effects on hTERT+ prostate cancer cells in vitroObjective: To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human telomerase reserve transcriptase gene promoter and investigate CXCR4 expression in hTERT+ carcinoma cells. Methods: To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP.U6 promoter was replaced by hTERT promoter. Then, the recombinant EGFP-hTERT-siCXCR4 fragment was sub-cloned into PLXSN and EGFP was replaced by ERFP.Then the recombinant was evaluated by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected respectively into PC-3m,LNCaP,MCF7,MRC5 cell. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western Blot. The effect of exogenous recombinant SDF-1 on adhesion, transendothelial migration and invasion of PCa cells interferenced by CXCR4-siRNA were observed using an in vitro Transwell and Matrigel models. Results: The expression of CXCR4 mRNA and protein in MCF7,PC-3m and LNCaP were reduced by the recombinant PLXSN-DsRED2-hTERT-siCXCR4 constructed successfully. The inhibitory rates of the expression of CXCR4 mRNA and protein were(88.31±9.20)%,(87.20±10.34)%,(76.40±9.77)% and (82.60±7.26)%,(80.23±4.54)%,(84.09±3.37)% at 48th hour respectively. CXCR4 mRNA and protein can not be inhibited by PLXSN-hTERT-siCXCR4 in MRC5 cell. The ability of exogenous SDF-1 enhancing adhesion, invasion and migration of prostate cancer cells in vitro descended. Conclusion: The downstream CXCR4-siRNA controlled by hTERT promoter in retrovirus system can be expressed selectively in hTERT+ carcinoma cells, but not in hTERT- cells, which showed the obvious targeting character. Knocking down CXCR4 gene can inhibit significantly PCa adhesion, invasion and migration of prostate cancer cells in vitro.4. The study on inhibition of prostate carcinoma metastasis to bone by using retrovirus-mediated CXCR4-siRNA driven by hTERT promoter in vivoObjective: To establish a stable prostatic carcinoma osseous metastasis NOD/SCID mouse model expressing enhanced red fluorescent protein and investigate effect of prostate carcinoma metastasis to bone by inhibiting CXCR4 expression. Methods: 6-8 monthsold, male mice were selected and implanted with human adult bone (HAB) by surgical implantation subcutaneously. Then about 2-3 weeks later, human prostatic carcinoma cell lines PC-3m and LNCaP, transfected by retroviral containing ERFP gene and interference sequence, were harvested and injected into the NOD/SCID mice through the lateral tail vein.2 weeks after injection, all NOD/SCID mice were started to be observed under the whole-body fluorescent imaging system to look for the sites with the expression of ERFP. Then, to 2 prostate carcinoma cell line, ratio of osseous metastasis,humid weight,volume and time of surviving were contrasted between interferenced group and uninterferenced group. Tissue slices of metastasic tumor used HE dyeing were detected. Results: To 2 prostate cancer cell lines PC-3m,LNCaP interferenced by CXCR4-siRNA,ratio of osseous metastasis,humid weight,volume were all lower than those of uninterferebnced prostate cancer lines, but time of surviving was longer and histodifferentiation degree of osseous metastasic tumor was superer. Conclusion: A stable prostate cancer osseous metastasis model expressing enhanced red fluorescent protein (ERFP) observed dynamically in vitro in a noninvasive way was established. Knocking down expression of CXCR4 gene can inhibit prostate cancer metastasis to bone and inhibit cancer cell maligdifferentiation in vivo.