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Isolation Of Genes Associated With Bone Metastasis Of Human Prostate Carcinoma And Targeted Gene Repression By Antisense Gene Transfection

Posted on:2008-09-05Degree:DoctorType:Dissertation
Country:ChinaCandidate:H LiaoFull Text:PDF
GTID:1114360272966760Subject:Surgery
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Object: To select human prostate carcinoma subpopulations with different metastatic potential to bone in nude mice by establishing animal models of bone metastasis, study on theirs biological characteristics and the difference of gene expression profile between them were investigated by human tumor metastasis microarray; to screen out genes associated with bone metastasis of human prostate carcinoma based on the preliminary screening by microarray. To study the biological effects of human CXCR4 gene antisense eukaryotic expression vectors on the high bone metastatic potential subline T3B cells of human prostate carcinoma after transfection.Methods: Human prostate carcinoma cell line PC-3 were injected into the left cardiac ventricle of nude mice to generate metastases in vivo, then PC-3 cell subpopulations were screened out by serial recovery from bone or lymph node metastasis and inoculation in vitro. The gene expression profiles of 3 cell lines with different bone metastatic potential were analyzed by using microarray, which contained 113 genes associated with human tumor metastasis. Total RNA of two sublines(T3B and P24) and their maternal cell line(PC-3) with different bone metastatic potential were extracted, reserve transcripted to cDNA, and then cRNA were synthesized and labeled with Biotin-16-UTP. The targets were mixed together and hybridized with the genes on Human tumor metastasis microarray. The chemiluminescent array image were captured by using X-ray film and array scanner. The data of each microarray was analyzed by software GEArray Expression Analysis. The genes which were found differently expressed between T3B and P24, PC-3 significantly by microarray were detected by reverse transcription-PCR and Western blot. The hCXCR4 gene cDNA was amplified and cloned into the eukaryotic expressing vector pIRES2-EGFP in transdirection using DNA recombinant technology. The recombinant vector was confirmed by double digestion with restriction endonucleases BamHâ… and EcoRâ… and DNA sequencing analysis. Human CXCR4 gene antisense eukaryotic expression vector was transfected into the high bone metastatic potential subline T3B cells. The expression of CXCR4 gene mRNA and protein were examined with RT-PCR and Western blot, respectively. The invasion ability of T3B cells was determined by Transwell in vitro, and the bone metastatic potential of T3B cells was examined by animal model of bone metastasis in vivo.Results: Different bone metastasic potential subpopulations of PC-3 was obtained by selection in vivo and denominated T3B and P24. Compared to P24(1/10, 10%) and parental PC-3(6/15, 40%), T3B showed a higher metastatic potential to bone(8/10, 80%) in vivo, and presented significantly increasing in the adhesion and invasion ability in vitro(P<0.01), but no significant difference in the soft-agar colony formation rate, tumorigenicity and tumor growth rate(P>0.05). There were 24 differently expressed genes between T3B and PC-3; 24 significant genes were found differently expressed between P24 and PC-3; and there were 22 differently expressed genes between T3B and P24. Many of the differently expressed genes belonged to cell adhesion, matrix degradation, Cell growth and proliferation, which might play an important role in bone metastasis of human prostate carcinoma. Compared to P24 and PC-3, CXCR4 and MMP3 were expressed highly in the high bone metastatic potential subline T3B using RT-PCR and Western blot (P<0.01). Antisense eukaryotic expressing vectors of hCXCR4 gene was successfully constructed and transfected into T3B cells. RT-PCR and Western blot analysis revealed that the expression level of CXCR4 gene mRNA and protein were down-regulated (P <0.05). The invasion ability in vitro and bone metastatic potentiality in vivo of T3B cells was remarkably decreased after transfection compared with the control groups.Conclusion: The animal models of human prostate cancer metastasis to bone could be established in a short period by injected PC-3 cells directly into the left cardiac ventricle of nude mice, and subpopulations with different metastatic potential to bone were obtained in nude mice models. Microarray is a high throughput, sensitive and efficient biomolecular technology to analyze genetic information of human prostate carcinoma with different bone metastatic potential. It provides an approach to investigate the mechanism of bone metastasis of human prostate carcinoma. The CXCR4 gene may become a new targeted site for inhibition human prostate carcinoma metastasis to bone.
Keywords/Search Tags:Prostate carcinoma, Bone metastasis, Microarray, Antisense, Transfection
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