Objective To investigate the effects of down-regulation of polo-like kinase-1 (PLK1) gene by RNA interference (RNAi) on the invasion of a human malignant melanoma cell line A375 and their possible mechanisms.Methods Cultured A375 cells were classified into several groups:blank control group receiving no treatment,liposome group transfected with lipofectamine only,and three siRNA groups transfected with three concentrations of a small interference RNA (siRNA) targeting PLK1 respectively.After additional culture,real time quantitative PCR and Western blot analysis were performed to quantify the expressions of PLK1 mRNA and protein in A375 cells respectively,Transwell invasion assay to evaluate the invasive capacity of A375 cells,agarose gel electrophoresis and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling (TUNEL) to detect anoikis in A375 cells.The colony-forming capacity was also evaluated for A375 cells.Statistical analysis was carried out by one-factor analysis of variance.Results There was a significant decrease in PLK1 mRNA and protein expressions as well as in colony-forming units in the siRNA groups compared with the blank control group (all P ＜ 0.05).The invasive capacity of A375 cells was significantly inhibited in the siRNA groups with the number of migrating cells in Transwell assay being 39 ± 5,19 ± 5 and 9 ± 3 in A375 cells transfected with 3.125,6.250 and 12.500 nmol/L siRNAs respectively,compared to 56 ± 5 in the blank control group (all P ＜ 0.05).A characteristic DNA ladder was observed on agarose gel electrophoresis in the siRNA (6.250 nmol/L) group.Compared with the blank control group and liposome group,the three siRNA groups showed increased apoptotic index (3.86％ ± 0.35％ (3.125 nmol/L siRNA),7.35％ ± 0.36％ (6.250 nmol/L siRNA) and 17.56％ ± 0.38％ (12.500 nmol/L siRNA) vs.1.15％ ± 0.25％ (blank control group) and 1.18％ ± 0.22％ (liposome group),all P ＜ 0.05).Conclusions PLK1 siRNA can inhibit the invasion of malignant melanoma cells,likely by inducing anoikis in these cells.

Objective To study the effects of Forkhead box protein M1 (FoxM1) down regulation by small interfering RNA(siRNA) on chemosensitivity and mechanism of human pancreatic cancer cell and its mechanism.Methods Three FoxM1 siRNAs were designed and constructed.All cancer cells were divided into different groups,after transfected with FoxM1 siRNA for different time,the cultured cells were harvested to carry on the next tests.Expression of FoxM1 were determined by red-time PCR and Western blot,and prolifearion and chemosensitivity were evaluated by MTT assay,and the phosphorylation of Akt protein was examined by Western blot.Results FoxM1 siRNA could down-regulate the FoxM1 expression in a dose-and time-dependent manner.The MTF results showed that the inhibit rates was 17.78％,17.56％,35.39％,52.81％,70.98％ indifferentgroups [Con-A + Gemcitabine,Con-B + Gemcitabine,siRNA (3.125nM) + Gemcitabine,siRNA (6.25nM) + Gemcitabine and siRNA(12.5nM) + Gemcitabine,respectively.The phosphorylation of Akt protein was inhibited in a dose-dependent manner.Conclusions FoxM1 siRNA could sensitize human pancreaticr cancer cells chemotherapy sensitivity,it is the one of the important mechanisms through down-regulate Akt phosphorylated levels,but the molecular mechanism need to be explored further.

Objective To investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.Methods Small interfering RNA targeting at miR-10a (miR-10a-siRNA) was constructed,then it was transfected into pancreatic cancer AsPC-1 cells,and nonsense siRNA (Nc-siRNA) group and blank control group was established.Real time PCR assay was used to detect the expression of miR-10a in the 3 groups,and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells.The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.Results The miR-10a levels in control group,NC-siRNA group and miR-10a-siRNA group were 1.05 ±0.08,1.03 ±0.06,0.02 ±0.01 ; and the number of transmembrane cell were (150 ± 2.6),(145 ± 2.2),(62 ± 1.8),the levels of MMP-13 in the supernatant were (108.5 ± 2.8),(107.8 ± 2.5),(35.8 ± 1.5) pg/ml.The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P ＜ 0.01).The distance of cultured clone in miR-10a treated cancer cells (736± 18 μm) was significantly longer than those in the controls (385 ±5 μm) and NC-siRNA group (395± 13 μm,P＜0.01).Conclusions Down-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells,and the downregulated expression of MMP-13 may be one of the important mechanisms.

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Base Sequence ; China ; Cluster Analysis ; Gene Components ; Genetic Variation ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; genetics