The Effect Of HmSD Knockdown On Proliferation And Migration Of PC-3 Cell In Vitro

Prostate cancer is a leading cause of cancer-related deaths in the Western countries. in China, the incidence and mortality rates of prostate cancer have increased markedly, because of the chang of Life style and diet structure.because the effect of traditional chemotherapy, radiotherapy and surgical treatment are not very ideal , a new treatment of tumor gene therapy is thought highly of increasingly.Sialidase is a kind of glycosidase,which can hydrolyzing sialic acid from non-deoxidized end glycogen and glycolipid.Human sialidases are overexpressed in mang tumors.human membrane-associated membrane (Neu3) is one of sialidases .In some tumors ,With the adjacent non-tumor tissue compared to the expressional level of Neu3 mRNA and protein in the tumor tissue significantly increased. the neu3 expression is higher, the tumor malignancy is higher. Therefore,Targeted silencing of hmSDis a promising approach in cancer control.Small RNA (siRNAs) are short double-stranded RNA molecules that can target complementary mRNAs for degradation via a cellular process termed RNA interference (RNAi). Tumor is due to the proliferation and differentiation of cells control disorders and the surrounding tissue disorder, including the activation of oncogenes and tumor suppressor gene inactive- ation and apoptosis-related gene expression abnormalities in the process of longevity to promote the growth of tumor growth factor.Therefore, the use of RNAi technology without affecting the normal gene function under the premise inhibit gene mutation or overexpression of expression, so as to achieve the purpose of gene therapy.Objective:To investigate the efficacy of hmSD gene silencing in growth inhibition of PC-3 cell and to elucidate the underlying mechanisms. Methods:1. Plasmid constructionAccording to the design principles, three suitable target sites against hmSD were selected and the specificities were determined by BLAST. The complementary oligonucleotides were annealed and ligated into the lineared pGCsi-U6/Neo/GFP siRNA expression vector, to construct recombinant plasmids pGCsi-hmSD1 , pGCsi-hmSD2and pGCsi-hmSD3 . Construction of good will have three recombinant plasmids were transformed into JM109 competent bacteria, positive clones selected strains, extracted plasmid, identified by restriction enzyme digestion, and sequenced.2. In vitro study(1)The prostate cancer cell line PC-3 were transfected with plasmids pGCsi-hmSD1, pGCsi-hmSD2 and pGCsi-hmSD3 and the efficiency of transfection is Appraiseed. To determine the expression levels of hmSD, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performed. cell morphology observed by Inverted microscope cell morphology. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and detected for the inhibition of cell proliferation by MTT. Cell migration was assayed by Scratch test.besides, Cell migration was assayed using a modified Boydenchamber containing a polycarbonate Transwell membranefilter (Corning Costar, Cambridge, MA, USA) coated with collagen type I. Cells were deposited on the upper chamber in IMDM containing 1% fetal bovine serum (FBS). The lower chamber contained IMDM with 10% (control) FBS. Cells were incubated for 6 h at 37°Cin 5% CO2/95% air. After scraping non-migrated cells from the upper surface of the membrane with a cotton swab, the migrated cells remaining on the bottom surface were stained with 0.5% crystal violet. The stained insert waswashed thoroughly, dissolved with 1% Triton X-100, andabsorbance (O.D.) at 595 nm was measured. To determine the expression levels of MMP-2, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performedResults:1.The plasmids pGCsi-hmSD containing siRNA-hmSD were constr- ucted, which have been confirmed by restriction enzyme digest and sequence analysis.2. The results from semi-quantitative RT-PCR analysis for the PC-3 cell samples in pGCsi-hmSD1,pGCsi-hmSD2, pGCsi-hmSD3 and pGCsi-scramble groups demonstrated that hmSD siRNA could specifically reduce hmSD mRNA expression. the number of cells are reduced ,Using an inverted microscope observation. The results of MTT demonstrated that inhibition of cell proliferation were present in treated groups. Significant hypodiploidy peaks before G1 phase were present in both treated cell samples in FCM analysis. The results of Scratch test and Transwell migration assay demonstrated that inhibition of cell migration. The results from semi-quantitative RT-PCR analysis demonstrated that the level of MMP-2 mRNA expression reduce in transfect pGCsi-hmSD groups compare withpGCsi-scramble groupConclusions:In our studies, hmSD knockdown showed that the expression levels of hmSD is reduced and cell proliferation is inhibited.It maybe the underlying mechanisms that the expression levels of hmSD, Cell migration is inhibited It maybe the underlying mechanisms that the pathway of MMP-2.