Regulation Of CD44 EXpression By Tumor Necrosis Factor-α And Its Potential Role In Breast Cancer Cell Migration

Objective1. To detect CD44s, CD44v3 and CD44v6 mRNA and protein expression in ER(+)breast cancer cell lines MCF-7 and ER(-) cell lines MDA-MB-231 treated withTNF-αand the effect on cell migration.2. To investigated the possible pathways via which TNF-αinduces CD44 expressionin MCF-7 and MDA-MB-231 cells as well.Methods1. MTT method was used to assay the cytotoxicity of TNF-α(0,2,20,200ng/mL)on the two different breast cancer cell lines MCF-7 and MDA-MB-231.2. Expression of CD44s, CD44v3 and CD44v6 mRNA treat with TNF-α(0,2,20,200 ng/mL)was detected by RT-PCR assay.3. Expression of CD44s, CD44v3 and CD44v6 protein treat with TNF-α(0,2,20,200 ng/mL)was detected by western blot assay.4. Transwell was used for detecting cells migration in vitro treat with TNF-α(0,2,20,200 ng/mL).5. A specific CD44s siRNA oligonucleotide and a pcDNA3.1(+)/CD44s plasmidwere constructed. The CD44s siRNA was transient transfected into MDA-MB 231 cells that have high baseline expression of CD44s,and the pcDNA3.1(+)/CD44s plasmid was transient transfested into MCF-7 cells that have low baselineexpression of CD44s. MCF-7 and MDA-MB-231 transfectants were analyzed forCD44s expression by Western blotting. Transwell was carried out for detectingcells migration after transfection.6. MCF-7 and MDA- MB-231 cells were treated with TNF-α(20 ng/ml), andwestern blot was used for detecting the phosphorylation of MAPK pathwayproteins (JNK, p38, ERK).7. CD44 expression and cells migration in vitro were detected after pre-treatmentwith the specific inhibitors of activated phosphorylation protein.Results1. TNF-αremarkably decreased growth of both MCF-7 and MDA-MB-231 cells at ahigh concentration (200 ng/mL), and had no significant effect at a lowconcentration (≤20 ng/mL).2. TNF-αtreatment reduced CD44s mRNA, and increased CD44v3 and CD44v6mRNAs in MCF-7 cells.And TNF-αtreatment increased CD44s, CD44v3 andCD44v6 mRNAs in MDA-MB-231 cells.3. TNF-αtreatment reduced CD44s protein, and increased CD44v3 and CD44v6proteins in MCF-7 cells. TNF-αtreatment increased CD44s, CD44v3 and CD44v6proteins in MDA-MB-231 cells.4. MCF-7 and MDA-MB-231 cells migration in vitro increased by 21%-48% and22%- 90%, respectively.5. Down-regulation of CD44s expression at the protein level was obtained usingsiRNA transfection, and in vitro migration decreased when CD44s was knockeddown. Up-regulation of CD44s expression at the protein level was obtained usingpcDNA3.1(+)/CD44s plasmid transfection., and in vitro migration increased. 6. When treated with TNF-α, JNK phosphorylation of MCF-7 cells enhanced, andp38 phosphorylation of MDA-MB-231 cells enhanced.7. Both of CD44 expression and cell migration in vitro induced by TNF-αwerereduced after pretreatment with paired specific inhibitors.ConclusionTNF-αregulates the CD44s, CD44v3 and CD44v6 expression via JNK pathway inMCF-7 cells, and via p38 pathway in MDA- MB-231 cells. All CD44s, CD44v3 andCD44v6 premote breast cancer cells migration.