[Analysis on SSR information in transcriptome of Astragalus membranaceus var. mongholicus and its polymorphism].

In order to enrich the library of SSR and provide more powerful tools for molecular marker-assisted breeding in Astragalus membranaceus var. mongholicus, simple sequence repeats (SSR) loci in its transcriptome were searched in 18 040 unigenes (>=1 kb) by using MISA. SSR loci information was analyzed and SSR primers were designed by Primer 3. Furthermore, 110 pairs of primers were randomly selected for the polymorphic analysis on 20 plants collected from different habitats. A total of 5 640 SSRs were found in the transcriptome of A. membranaceus var. mongholicus, distributed in 4 462 unigenes with the distribution frequency of 31.26%. SSR loci occurred every 6 514 bp. Mono-nucleotide repeat was the main type, accounted for as much as 36.72% of all SSRs, followed by tri-nucleotide(32.57%) and di-nucleotide(27.73%) repeat motif. Among all 75 repeat types, A/T(2 026) was the predominant one followed by AG/CT(1 179), AAG/CTT(477). For validating the availability of the SSR primers designed using Primer 3, 110 pairs of primers were randomly selected for PCR amplification. Among them, 97 pairs of primers (88.18%) produced clear and reproductive bands. Using 19 pairs of primers showed polymorphism, 20 plants were divded into two groups by UPGMA. There are numerous SSRs in A. membranaceus var. mongholicus transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity, molecular identification, and marker-assisted breeding study for this plant.

[Prognostic value of PSA kinetics in locally advanced prostate cancer treated by maximal androgen blockade combined with brachytherapy].

OBJECTIVE: To evaluate the effect of post-treatment PSA kinetics on the prognosis of prostate cancer (PCa). METHODS: We retrospectively reviewed the clinical data of 114 cases of locally advanced PCa treated by maximal androgen blockade (MAB) combined with brachytherapy, and analyzed the association of the changes in PSA kinetics with the prognosis of the patients. RESULTS: The median survival time of the patients was 81 (15 - 144) months, with 1-, 3- and 5-year survival rates of 91. 23%, 78.07% and 68.42% , respectively. Univariate analysis indicated that the baseline PSA level, PSA nadir, the time of PSA decreasing to nadir, PSA doubling time, and the extent of PSA declining were all predictive factors for the survival time of the PCa patients. Multivariate analysis demonstrated that PSA nadir, the time of PSA decreasing to nadir, and the extent of PSA declining were three independent prognostic factors, which prolonged the long-term survival of the patients by 1.7, 3.2 and 6.8 times, respectively. CONCLUSION: For locally advanced PCa treated by MAB combined with brachytherapy, PSA nadir <1 micro g/L, the time to nadir <3 months, and the extent of PSA declining >96% are independent prognostic factors.

[Long-term outcomes of prostate cancer patients treated with combined brachytherapy and maximal androgen blockade].

OBJECTIVE: To explore the prognostic factors of prostate cancer (PCa) patients and evaluate the effect of brachytherapy on survival time. METHODS: A total of 289 PCa were recruited to collect their clinical and survival data. And their possible prognostic factors were analyzed. A further comparison of 5-year cumulative survival rate was made between the patients treated by maximal androgen blockade (MAB) and those on MAB plus brachytherapy. RESULTS: Their median survival time was 73 (7-144) months. And the 1, 3 and 5-year survival rates were 93.1%, 81.0% and 60.2% respectively. Univariate analysis indicated that prostate volume, basal level of prostate-specific antigen (PSA), Gleason score, tumor stage, PSA nadir, time PSA decreasing to nadir and brachytherapy were all predictive factors for survival time. And multivariate analysis further demonstrated that Gleason score, tumor stage and PSA nadir were independent prognostic indicators. And the combination therapy based on brachytherapy could significantly increase the 5-year cumulative survival rate than MAB-based monotherapy. CONCLUSION: Gleason score, tumor stage and PSA nadir may predict the prognosis of PCa patients. And brachytherapy significantly improves patient survival.

[Over-expression of hypoxia-inducible factor-1alpha induces epithelial-mesenchymal transition in LNCaP cells in vitro and in vivo].

OBJECTIVE: To investigate the effects of the over-expression of hypoxia-inducible factor-1alpha (HIF-1alpha) on the epithelial-mesenchymal transition (EMT) and invasive potency of LNCaP cells in vitro and in vivo. METHODS: We cultured LNCaP cells stably expressing HIF-1alpha (LNCaP/HIF-1alpha) and LNCaP cells, identified the over-expression of HIF-1alpha, determined the proliferation of the two cell lines by MTT assay and the level of PSA in the supernatant of culture medium, and detected the anchorage independent growth by soft-agar colony formation assay. A subcutaneous tumor model was established in nude mice by injecting LNCaP/HIF-1alpha and LNCaP cells followed by observation of the tumor growth. Tumor specimens were obtained for immunohistochemistry. RESULTS: The over-expression of HIF-1alpha was confirmed in the LNCaP/HIF-1alpha cells by immunofluorescence staining and Western blotting. The level of PSA was obviously decreased in LNCaP/HIF-1alpha as compared with that in LNCaP cells. MTT assay identified the increased proliferation of LNCaP/HIF-1alpha cells. The cell colony forming ability of the LNCaP cells was significantly lower than that of the LNCaP/HIF-1alpha cells. The rate of tumorigenesis was increased and its time shortened in the LNCaP/HIF-1alpha group. Immunohistochemistry revealed an up-regulated expression of vimentin and a down-regulated expression of E-cadherin in the tumor specimens. CONCLUSION: The overexpression of HIF-1alpha can up-regulate the expression of vimentin and down-regulate the expression of E-cadherin, which may enhance the invasive potency of LNCaP cells by inducing EMT.

[Over-expression of hypoxia-inducible factor 1 alpha increases angiogenesis of LNCaP cells in vivo].

OBJECTIVE: To observe the influence of hypoxia-inducible factor 1 alpha (HIF-1alpha) on angiogenesis in prostate carcinoma in vivo and to investigate its molecular mechanism. METHODS: LNCaP/HIF-1alpha and LNCaP cells were cultured, the level of PSA in the supernatant of the culture medium detected by ELISA assay before and after the transfection, and the cellular cycle measured by flow cytometry. Nude mouse models of subcutaneous tumor were established with LNCaP/HIF-1alpha and LNCaP cells, the tumor growth observed, and tumor specimens collected for immunohistochemical staining. RESULTS: Compared with the LNCaP cells, LNCaP/HIF-1alpha cells showed an obviously decreased PSA level (t = 8.243, P < 0.05) and enhanced proliferous activity. The tumorigenesis rate increased and the tumorigenesis time advanced in the LNCaP/HIF-1alpha group of the nude mice. Immunohistochemistry displayed higher expressions of VEGF, iNOS and Ang-2 in the LNCaP/HIF-1alpha than in the LNCaP group. CONCLUSION: The over-expression of HIF-1alpha can up-regulate VEGF and iNOS involved in angiogenesis in vivo and contribute to the invasive potency of LNCaP cells. HIF-1alpha may have no influence on Ang-2 either in vitro or in vivo, while the expression of Ang-2 is regulated by other factors in vivo.

[Over-expression of hypoxia-inducible factor-1 alpha increases angiogenesis of LNCaP cells].

OBJECTIVE: To evaluate the effect of hypoxia-inducible factor-1 alpha (HIF-la) on angiogenesis in human prostate cancer cells. METHODS: Human prostate cancer cells of the line LNCaP were cultured and transfected by the recombinant plasmid pcDNA3. 1(-)/HIF-1alpha containing the gene HIF-1alpha with the Lipofectamine 2000 system. The positive clone cells were selected by G418 and confirmed by Western blotting and immunofluorescence (LNCaP/HIF-1alpha cells). The expressions of VEGF, iNOS and Ang-2 were detected by Western blotting. RESULTS: The expression of HIF-1alpha in the LNCaP/HIF-1alpha cells was distinctly higher than that in the LN-CaP cells. Compared with the LNCaP cells, the expressions of VEGF and iNOS were up-regulated, whereas Ang-2 remained unchanged in the LNCaP/HIF-1alpha cells. CONCLUSION: The over-expression of HIF-1alpha can induce an increase in angiogenesis proteins and improve the angiogenesis potency of prostate cancer.