Effects And Mechanisms Of Suppressing Ndst1 And Hs2st With RNA Interference On Biological Behaviors Of Prostate Cancer Cell Line Myc-CaP

Objects1. To construct RNA interference vectors targeting Ndstl and Hs2st2. To observed effects of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaP3. To investigate preliminarily the influence mechanism of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPMethods1. Construction, screening and identification of RNA interference vectors targeting Ndst1 and Hs2stUsing OligoEngine Workstation 2.0 to analysis the mRNA sequences of Ndst1 and Hs2st, designed and synthesized three target sequences for each gene to recombined with the siRNA eukaryotic expression vector pSUPER.retro.neo+GFP, and obtain pSUPER-Ndst1 and pSUPER-Hs2st. After recombination interference vector was transfected into prostate cancer, Myc-Cap cells, we select the interference vector with the most significant inhibitory effect for follow-up experiments by detecting the mRNA and protein expression of Ndst1 and Hs2st in the transfected group cells2. In vitro effects of Ndstl and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPUsing drawing of the cell growth curve with CCK-8 method and cell counting method, soft colony forming assay and cell cycle detection by flow cytometry to detect cell proliferation; using Transwell chamber, cell scratch healing assay and Matrigel Invasion Chamber to detect migration and invasion capacity of Myc-CaP cells in the control group and experimental group; Using Annexin V-FITC/PI double staining method combined with flow cytometry to detect Myc-CaP cells apoptosis.3. In vivo effects of Ndst1 and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPInjected the Myc-CaP cells with Ndst1 and Hs2st suppression into nude mice subcutaneously, and observe the formation and growth of tumor. Detect tumor cell proliferation and apoptosis of transplanted tumor tissue by BrdU labeling and TUNEL method respectively. Injected the Myc-CaP cells stained with fluorochrome into nude mice through tail vein, and separate lung tissue for frozen section and observe of lung metastases under microscope4. Preliminary study on the influence mechanism of Ndstl and Hs2st suppression on the biological behaviors of prostate cancer cell line Myc-CaPAdded biotin-FGF-2 into Ndstl and Hs2st knockdown Myc-CaP cells, Then used theR-phycoerythrin-streptavidin-biotin to combined with biotin, detected the binding capacity of FGF-2 with Ndstl and Hs2st knockdown Myc-CaP cells by flow cytometry. In addition, we examined effect of FGF-2 on Ndstl and Hs2st knockdown Myc-CaP cells in the growth, invasion and apoptosis.Results1. Constructed successfully RNA interference vectors, pSUPER-Ndstl and pSUPER-Hs2st, targeting Ndstl and Hs2st, inhibition rates were85.6% and 80.2% separately.2. In vitro studies:Ndstl suppression Myc-CaP cells growth slow down; colony-forming ability decreased by 46.2%; migration and invasive ability decreased by 30.0% and 17.1%; apoptosis rate increased 1.2 times.3. In vitro study:Hs2st suppression Myc-CaP cells growth slow down; colony-forming ability decreased by 81.2%; migration and invasive ability decreased by 69.6% and 69.5%; apoptosis rate increased 2.9 times.4. In nude mice:Ndstl suppression Myc-CaP cells growth slow down; proliferation rate reduced by 39.3%; capacity of short-term lung metastases by 40.8%; apoptosis rate increased 1.6 times.5. In nude mice:Hs2st suppression Myc-CaP cells growth slow down; proliferation rate reduced by50.2%; capacity of short-term lung metastases by 65.6%; apoptosis rate increased 5.9 times.6. Binding capacity of Ndstl and Hs2st suppression Myc-CaP cells with FGF-2 decreased by 79.5%和38.8% separately, compared with the control group.7. Adding the FGF-2 (20ng/ml), the growth of Ndstl and Hs2st suppression Myc-CaP cells accelerated, but obviously slower than the control group; the control group, Ndstl group and Hs2st group Myc-CaP cells migration capacity increased by 5.8,3.6 and 2.1 times; apoptosis rate decreased by 81.9%,51.4% and 22.0% respectively.Conclusions1. Suppression of Ndstl or Hs2st can inhibit Myc-CaP cells proliferation, migration and invasion and promote apoptosis, in which suppression of Hs2st has more pronounced effect.2. Changes in the biological behaviors of Ndstl or Hs2st suppression Myc-CaP cells are related to the binding capacity decline of FGF-2 with Myc-CaP cells.