5-氮杂-2’-脱氧胞苷通过反转原钙黏蛋白10表达抑制人乳腺癌细胞系MDA-MB-231的侵袭和迁移

目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-CdR)诱导抑癌基因原钙黏蛋白10(PCDH10)重新表达对人乳腺癌细胞MDA-MB-231体外侵袭迁移能力的影响并初步探讨其机制。方法体外培养人乳腺癌细胞MDA-MB-231,设置对照组和5-Aza-CdR药物处理组,分别采用反转录聚合酶链反应(RT-PCR)检测PCDH10 mRNA的表达水平;Transwell法和划痕实验检测细胞的侵袭迁移能力;Western blotting检测PCDH10、DNA甲基转移酶(DNMT) 3A、DNMT3B、核因子(NF)-κB p65和基质金属蛋白酶(MMP)-2、MMP-9蛋白表达的变化。结果 5-Aza-CdR能够反转PCDH10的mRNA和蛋白表达;PCDH10表达恢复后MDA-MB-231细胞的侵袭迁移能力受到抑制;Western blotting检测发现,MDA-MB-231细胞经5-Aza-CdR处理后DNMT3A、DNMT3B、NF-κB p65、MMP-2和MMP-9的表达下调。结论 5-Aza-CdR可抑制MDA-MB-231细胞DNMT3A和DNMT3B的表达,使抑癌基因PCDH10表达恢复,从而通过阻滞NF-κB p65的活化,下调MMP-2和MMP-9表达而抑制乳腺癌细胞的侵袭转移。

Side population cells isolated from KATO Ⅲ human gastric cancer cell line have cancer stem cell-like characteristics

AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells indifferent human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO Ⅲ human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO Ⅲ human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO Ⅲ, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem celllike characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer.

Expression and location of α-fetoprotein during rat colon development

AIM： To investigate the expression of α-fetoprotein （AFP）, a cancer-associated fetal glycoprotein, and its involvement during rat colon development. METHODS： Colons from Sprague-Dawley rat fetuses, young and adult （8 wk old） animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reversetranscriptase PCR （RT-PCR） and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. RESULTS： The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 （e18.5）. Compared to e18.5 d, the AFP expression was significantly decreased during rat development （85% for e20.5, P 〈 0.05, 58% for postnatal day 0.5 （P〈0.5）, P 〈 0.05, 37% for P7, P 〈 0.05, 24% for P14, P 〈 0.05, and 11% for P21, P 〈 0.05） and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested. CONCLUSION： This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

Expression and localization of Wolfram syndrome 1 gene in the developing rat pancreas

AIM： To investigate the expression and function of Wolfram syndrome 1 gene （WFS1） during the development of normal pancreas. METHODS： Pancreas from Sprague-Dawley rat fetuses, embryos, young and adult animals were used in this study. Expression levels of WFS1 in pancreas of different development stages were detected by reverse transcription-polymerase chain reation （RT-PCR） and Western blotting. To identify the cell location of WFS1 in the developing rat pancreas, double-immunofluorescent staining was performed using antibodies to specific cell markers and WFS1, respectively. RESULTS： Compared to E15.5, the highest level of WFSl mRNA was detected at E18.5, the level of WFSl mRNA in E15.5 and P0 was less, and at a lowest at adult （P 〈 0.05 vs P0 and adult）, respectively. Compare to E15.5, the highest level of WFS1 was at P14 and lowest at P21 （P 〈 0.05 vs P14 and P21）, respectively. The WFSl positive staining is expressed in the normal developing rat pancreas mainly in the islet beta-cells and mesenchyme at each stage tested. CONCLUSION： These results indicate that WFSl may be involved in some aspects of pancreatic development and further research on WFS1 may provide new evidences to prove the interactions between mesenchyma and epithelia at the same time.