MAWBP and MAWD inhibit proliferation and invasion in gastric cancer

AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptasepolymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo . MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25±8.43, 12.75±4.49, 30±6.41 vs 336.75±22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25±16.54, 88.75±11.12, 341.75±22.23 vs 30.25±8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84±16.57, 98.33±9.8, 29±16.39 vs 298±11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67±14.57, 72.66±8.51, 330.67±20.55 vs 27±11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.

UO-126对HeLa细胞增殖及对FBW7表达的影响

目的:观察UO-126对人类宫颈癌细胞株HeLa细胞增殖及对F-盒和含蛋白7的WD重复结构域FBW7表达的影响。方法:采用MTT法观察不同浓度的UO-126对HeLa细胞增殖的影响;免疫荧光法检测FBW7在HeLa细胞中的定位和表达;RT-PCR、Western blot检测FBW7 mRNA及蛋白在UO-126阻断丝裂原活化蛋白激酶(mitogen-activated protein ki-nases,MAPK)信号通路前后的表达情况。结果:MTT结果显示各浓度UO-126具有抑制HeLa细胞增殖的作用,且具有剂量依赖性和时间依赖性(P<0.05)。免疫荧光检测显示UO-126处理HeLa细胞后FBW7阳性表达增强。RT-PCR及West-ern blot结果显示,UO-126作用后HeLa细胞FBW7 mRNA及蛋白表达水平较未处理HeLa细胞明显增高(P<0.05)。结论:MAPK信号通路阻断剂UO-126抑制HeLa细胞增殖,FBW7位于MAPK信号通路的下游。

Fbxw7上调对乳腺癌MDA-MB-231细胞侵袭、迁移及MAPK/ERK通路的影响

目的探讨F框/WD-40域蛋白7(Fbxw7)对乳腺癌MDA-MB-231细胞侵袭、迁移的影响,初步探究可能的作用机制。方法体外培养乳腺癌MDA-MB-231细胞,将Fbxw7过表达载体pEZ-M02-Fbxw7(Fbxw7过表达组)、空载体(pEZ-M02)转染至MDA-MB-231细胞,另外以未进行转染的MDA-MB-231细胞作为空白组。Western blot检测Fbxw7、p-ERK、p-p38MAPK、cadherin、vimentin、N-cadherin蛋白表达情况,CCK-8法检测各组细胞增殖能力,Transwell小室系统检测细胞迁移及侵袭能力。结果与空白组和空载组相比,Fbxw7过表达组MDA-MB-231细胞中Fbxw7蛋白表达、增殖抑制率明显升高,细胞侵袭、迁移数量明显降低,p-ERK1/2、p-p38MAPK、vimentin、N-cadherin蛋白表达明显降低,E-cadherin蛋白表达明显升高,差异有统计学意义(P<0.05)。结论过表达Fbxw7后能抑制MDA-MB-231细胞增殖、侵袭、迁移,其可能是通过抑制MAPK/ERK通路发挥对乳腺癌细胞的抑制作用。