Objective To investigate the role of mutated BRAFV599E gene in the growth of malignant melanoma cells. Methods In the previous study, plasmids containing small hairpin RNAs (shRNAs), braf1 and braf2 specific for mutated BRAFV599E gene, were designed and used to transfect A375 cells to inhibit the expression of BRAF gene in these cells. In this study, four kinds of A375 cells, including Abraf1 (transfect ed with braf1), Abraf2 (transfected with braf2), Aneg (transfected with negative plasmid) and A375 (untransfected) cells, were chosen and cultured in 96-well plate. MTT assay, plate clone forming assay, flow cytometry were applied to test the growth, clone formation, cell cycle and apoptosis of these cells respectively. Results Compared with A375 and Aneg cells, inhibited proliferation (F=25.48, P<0.001) and clone-forming rate (F=90.06, P<0.001) were observed in Abraf1 and Abraf2 cells; furthermore, flow cytometry showed a decrease in S-phase population(F=147.87, P<0.001) but an increase in G1-phase population (F=9.14, P<0.05)in Abraf1 and Abraf2 cells. However, neither Abrafl nor Abraf2 cells exhibited a significant increase in apoptosis ratio (F=2.27, P>0.05). Conclusions Mutated BRAFV599E gene could induce the switch from G1 phase to S phase in melanoma cells, subsequently accelerate the growth of melanoma cells, but it has no obvious influence on the apoptosis of these cells.

Plasma endothelin-1 (ET-1),von Willebrand factor ( vWF) and fibrin D-dimer (D-D) were determined in 14 children with schnlein-henoch purpuric nephritis (SHN),17 children with schnlein-hen och purp ura (SHP, no abnormal finding in the examination of the urinary system) and 12 children as normal controls, respectively. The results showed that plasma ET-1( 88.48±22.96ng/L),vWF(1.59±0.38U/ml) and D-D(1.45±0.39)mg/L in the SH N group were all significantly higher than those in both of the control group (4 3.73±17.89)ng/L, (0.99±0.3)U/ml and (0.28±0.23)mg/L and the SHP group (57. 54±20.92)ng/L, (1.5±0.31)U/ml and (0.64±0.34)mg/L although no significa ntly difference in the levels of vWF was observed between SHN and SHP groups. It was noticed that the levels of three parameters decreased significantly in the SHN g roup after treatment (all P＜0.01). There was a positive correlati on between the levels of plasma ET-1 and D-D with serum creatinine (all P＜0.01). It is suggested that excessive ET-1 induced by endothelial dam age of renal vessels, intravascular coagulation and secondary fibrinolysis are p robably involved in the process of renal damage.

Objective To select microRNAs (miRNA) associated with human cutaneous malignant melanoma (MM). Methods Total RNA was extracted from 6 tissue samples of MM and 9 human control samples of pigmented nevi, and small RNAs of less than 200 bp were enriched, miRNA microarray was used to select differentially expressed miRNAs between tissue samples of MM and pigmented nevi from 468 candi-dates. The expression of differentially expressed miRNAs was confirmed by fluorescence based real-time quan-titative PCR (qPCR) in all of these samples. Those miRNAs that were identified as differentially expressed with both miRNA microarray and qPCR were considered as significant miRNAs. Results Between the tissue samples of MM and pigmented nevi, 12.18% to 86.33% of miRNAs differentially expressed by more than 2 folds, 1.28% to 19.02% by more than 5 folds, and 0.43% to 5.34% by more than 10 folds. The expression of miRNA-21 was obviously up-regulated, while that of miRNA-320 and miRNA-494 was down-regulated in the MM samples. Conclusion There is an increase in the expression of homo sapiens miRNA-21 but a decrease in that of miRNA-320 and miRNA-494 in MM tissues.

Objective To construct the short hairpin RNA (shRNA)-expressing plasmid vectors specific for BRAF gene, and to test their effects in BRAF knockdown in human melanoma cell lines. Methods Two pairs of specific BRAF shRNA oligoes and a pair of randomly synthesized non-specific shRNA oligo were synthesized and inserted into plasmid pGenesil-1. Their fidelity was confirmed by double endonuclease digestion and sequencing. The constructed plasmids were transfected into human melanoma cell lines A375 and M14. The expression of BRAF mRNA and BRAF protein were detected by RT-PCR and Western blotting, respectively. Results The designed shRNA oligoes were precisely cloned into the plasmid pGenesil-1. The expression of BRAF mRNA and protein were down-regulated by specific plasmid braf 1 and braf 2, except to non-specific plasmid neg. The plasmid braf 1 was more effective, reducing BRAF gene expression by 90 per cent. Conclusions Plasmid mediated shRNA could successfully knockdown BRAF expression in human melanoma cells, and the suppression of the gene expression could maintain for 1 month at least.

Objective To investigate the expression of microRNA-21(miRNA-21)in two human malignant melanoma cell lines,A375 and M14,and its effect on the viability of these cells.Methods The expression of human miRNA-21 was assessed by quantitative fluorescent PCR in A375 and M14 cells.Then,three concentrations(90,180,270 nmol/L)of miRNA-21 inhibitor and were transfected both cells and a negative control were transfected a mixture of LipofectamineTM 2000 reagent,respectively.After another 3-day culture,the proliferation of cells was detected by cell counting kit-8,and R value was calculated to denote the relative activity of cells.Statistical analysis was carried out by SPSS13.0.Results The expression of miRNA-21 was higher on A375 cells than that on M14 cells with the average value of 2-deltaCT being 1.2928±0.1509 vs 0.1894±0.1803.With miRNA-21 inhibitor at the concentration of 90,180,270 nmol/L,the activity of A375 cells was significantly lowered in comparison with that in the control group,with the R value being 0.7362±0.1662.0.7248±0.3204 and 0.6767±0.2998 respectively(all P＜0.01).However,in the case of M14 cells,cell activity was only suppressed by miRNA-21 inhibitor at 90 nmol/L with the R value being 0.7295±0.1478.and no significant inhibition was observed with the inhibitor at 180 or 270 nmol/L (both P＞0.05).Conclusions miRNA-21 is expressed on human melanoma cell lines,A375 and M14,at different levels,with a promoting effect on the proliferation of both cells.Moreover,miRNA-21 may act as an oncogene-like gene via down-regulating the expression of some tumor-inhibiting factors.

Objective To investigate the effect of BRAFV600E mutation on the invasion capacity of a human melanoma cell line, A375. Methods Plasmids containing short hairpin RNAs (shRNA) specific for BRAF gene were prepared in previous study, and used to transfect A375 cells. Those cells transfected with negative plasmid and untransfected cells served as the controls. Transwell chambers were used to examine the invasion ability of melanoma cells in vitro. RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF), respectively, before and after the transfection. The activity of MMP-2 was also studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Compared with the negative control, the specific shRNA decreased the mRNA and protein expressions of MMP-2 by 35% and 85%, respectively, and those of VEGF by 45% and 14%, respectively. Additionally, the number of cells invading through Matrigel chambers reduced by 69% in those cells transfected with the positive plasmid. Conclusions The mutant BRAFV600E has the potential to enhance the invasion capacity of melanoma cells, whereas specific shRNA could suppress the increase in metastasis capacity likely by inhibiting the production of VEGF and MMP.

0.05). Conclusions Solcoderm is a simple, safe, and efficient method for the treatment of verruca vulgaris.

Objective To study LI gene sequence of HPV cp6108 from 5 cases of condyloma acuminata. Methods T-A cloning and direct sequencing of PCR product were used. Results The LI gene sequences of HPV cp6108 from 5 specimens were presented with the homology of 99% to reference sequence in GenBank. A total of 3 gene mutations were found, including a nonsense mutation of G70A, a missense mutation of D77N, and a missense mutation of Tl16P. Conclusions In comparison with the sequence in GenBank, at least 3 gene mutations of HPV CP6108, i.e. one nonsense mutation of G70A and missense mutations of D77N and Tl 16P, are found in the present study.

Objective To evaluate the in vitro effect on tumor cell uptake,tumor imaging and in vivo biodistribution of 99Tcm-epidermal growth factor receptor (EGFR) mRNA antisense PNA probe mediated by cationic liposome.Methods The oligonucleotide with sequence complementary to part of the EGFR mRNA antisense PNA was hybridized in an anti-parallel orientation targeted PNA.PNA hybridization complexes were labeled with 99Tcm by ligand exchange.The assembly of lipofectamine and 99Tcm-labeled heteroduplex was achieved by electrostatic interactions,and the radiolabeled purity was determined by reversedphase HPLC (RP-HPLC).The disparities of cell uptake in SKOV3 cells and the differences of biodistribution and molecular imaging in BALB/c nude mice bearing SKOV3 xenografts between lipofectanine-mediated 99Tcm-EGFR mRNA antisense PNA (group 1) and 99Tcm-EGFR mRNA antisense PNA (group 2) were analyzed.Two-sample t (or t') test and Wilcoxon rank sum test were used for statistical analysis.Results The labeling rates of both group 1 and group 2 were more than 95％ within 6 h.The cell uptake at 1,2,4,6,12,24 h after injection was (28.90±1.12)％,(32.76±1.20)％,(38.20±3.11)％,(41.23±1.60)％,(46.63±1.55)％ and (46.78±2.14)％ in group 1,and was (3.51±0.39)％,(3.90±0.40)％,(4.69±0.18)％,(5.91±0.26)％,(5.30±0.22)％ and (5.39±0.17)％ in group 2 respectively (t'=47.11-58.67,Z=2.80,all P＜0.05).The retention ratios showed significant difference between the two groups (t'=7.25-11.55,Z=2.80,all P＜0.05).The SKOV3 tumor could be visualized in both groups at 1 h post injection but much better visualized in group 1.The T/NT ratios were higher in group 1 at all time points (t =3.96,t'=12.65-14.69,Z=2.83-5.29,all P＜0.05).The T/NT ratios at uptake peak were 5.02 and 3.95,respectively.The probe accumulated mainly in tumor,kidneys and liver.Tumor uptake increased with time ((1.49±0.09) ％ID/g and (2.15±0.21) ％ID/g at 1 h,(3.90±0.65) ％ID/g and (5.00±0.10) ％ID/g at 6 h) after lipofectamine treatment.The ratios of tumor to contralateral muscle were also higher in group 1 (t =11.24,t' =3.96-11.94,all P＜0.05).Conclusions Lipofectamine-mediation can significantly improve the intracellular delivery of radionuclide molecular probe.Lipofectamine-mediated 99Tcm-EGFR mRNA antisense PNA can greatly improve imaging contrast and visualization of EGFR-over-expressing tumors.

Objective To systematically investigate the molecular epidemiological profiles of human papillomavirus (HPV) in patients with condyloma acuminata(CA). Methods Two hundred and one samples of HPV DNA isolated from CA were PCR amplified by the PGMY09/11 primer system. The PCR products were simultaneously hybridized to 37 specific HPV probes immobilized on a nylon strip and then genotyped. All DNA templates were further PCR amplified using HPV 6 and 11 type specific primers for verification. Results All samples were HPV DNA positive consisting of totally 31 genotypes, the types of which were type 11(53.7%, 108/201), 6(43.8%, 88/201), 16(6.5%, 13/201), 52(6.0%, 12/201), 33(5.5%, 11/201), cp6108 (5.5%, 11/201) and 42 (5.0%, 10/201). The samples infected with a single and mixed types of HPV accounted for 60.2% (121/201) and 39.8% (80/201) respectively. Consistent results were found with the detection of HPV6 and 11 between hybridization assay and type-specific PCR. Conclusions At least 31 HPV genotypes are associated with CA. HPV 11 predominates while 68, 40, 54, 67, 73, 82, 35, 64 and 83 are rare in CA. Type cp6108 is detected in CA for the first time with a high prevalence. HPV26, 69, 70, 71,72 and IS39 might be not associated with CA. CA infected with a single and mixed HPV types accounts for 60.2% and 39.8%, respectively.