Regulation Of β-catenin Protein Expression And Nuclear Localization By WNT5A

PartⅠExpression and significance ofβ-catenin protein in leukemic cell lines Objective:To study the expression ofβ-catenin protein in leukemic cell lines, and interpret the role of it in the occurrence of leukemia.Methods:The expression and localization ofβ-catenin protein in leukemic cell lines(K562,HL-60,Jurkat,Thp-1) and normal peripheral blood mononuclear cells were respectively assayed by means of Western Blot techniques and immunol cytochemistry techniques.Results:(1)The expression level ofβ-catenin protein was up-regulated in Jurkat and Thp-1 cell lines.(2)β-catenin protein express in 4 cell lines.β-catenin protein express in the cytoplasm and nucleus of Jurkat and Thp-1 cell line, and express in the cytoplasm of K562 and HL-60 cell lines without nucleus stain.By contrast, the expression ofβ-catenin protein was detected in the cytoplasm of the normal peripheral blood mononuclear cells.Conclusions:High expression and nuclear localization ofβ-catenin protein might be involved in leukemia development. PartⅡThe study on correlativity between WNT5A mRNA andβ-catenin protein over-expression in leukemic cell linesObjective:To establish a SYBR GreenⅠreal-time reverse transcription-polymerase chain reaction(RT-PCR) for quantitating humanβ-catenin mRNA and WNT5A mRNA and study the correlativity between WNT5A mRNA andβ-catenin over-expression in leukemic cell lines.Methods:Total RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA.SYBR GreenⅠreal-time PCR was used to quantitate mRNA expression ofβ-catenin , WNT5A in leukemic cell lines(K562,HL-60,Jurkat,Thp-1),and two normal peripheral blood mononuclear cells, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase )as a reference gene.The specific amplicons were measured by the characteristic of target genes'melting temperatures ?.Results:(1)β-catenin mRNA was detected in all the leukemic cell lines. K562 and HL-60 (with low expression level ofβ-catenin protein)showed a high expression level ofβ-catenin mRNA,and Jurkat and Thp-1(with high expression level ofβ-catenin protein) showed a low expression level ofβ-catenin mRNA.(2)WNT5A mRNA was detected in all the leukemic cell lines.Jurkat and Thp-1 cell lines showed a high expression level and HL-60 and K562 cell lines showed a low expression level.and There was no expression in 2 normal peripheral blood mononuclear cells.Conclusions:(1) SYBR GreenⅠreal-time quantitative RT-PCR is a rapid, sensitive and good specific method to detectβ-catenin and WNT5A mRNA. (2) The abnormal localization ofβ-catenin protein in Jurkat and Thp-1 cell lines is independent ofβ-catenin mRNA expression level, which is perhapes caused by high expression level of WNT5A mRNA. T5A mRNA; leukemic cell linesPartⅢConstruction of eukaryotic vector WNT5A shRNA and its regulation toβ-catenin protein expression and localization in Jurkat cellsObjective:To construct eukaryotic vector expressing short hairpin RNA(sh RNA) of WNT5A, to knock-down WNT5A mRNA expression in Jurkat cells with high expression level ofβ-catenin protein. And to detect the effect onβ-catenin mRNA expression level and localization ofβ-catenin protein and the effect on WNT signalling pathway downstream target genes mRNA expression.Methods:According to the relative documents, using the software of Invivogen company, computer aided designing a short hairpin(sh) RNA targeting the coding sequence of the WNT5A. a recombinant plasmid harboring 21nt-long small interfering(si) RNA (psiRNA-hH1-WNT5A)was constructed by inserting the designed shRNA to the eukaryotic expression vector psiRNA-hH1neo, and transfected into Jurkat cell with DMRIE-C. After transfected with psiRNA-WNT5A, Jurkat cells were selected by G418. The WNT5A,β-catenin mRNA expression levels of cells transfected with psiRNA-WNT5A were monitored by SYBR GreenⅠreal-time RT-PCR.Meanwhile, c-myc,c-jun mRNA expression level were quantitated using real-time RT-PCR.Western Blot analysis was used to dectectβ-catenin protein expression level.Results:The constructed eukaryotic vectors WNT5A shRNA was identified by enzyme digestion analysis.psiRNA-WNT5A was transfected into Jurkat cells as compared with untransfected Jurkat cells. The WNT5A mRNA expression level was significantly suppressed by 63.5%. However,β-catenin mRNA expression level had no statistic difference.Western Blot analysis showedβ-catenin protein expression level was decreased significantly. After transfected with psiRNA- WNT5A , and selected by G418,real time RT-PCR showed that c-myc and c-jun mRNA expression in psiRNA- WNT5A-Jurkat were decreased by 32.7% and 45.2% respectively.Conclusions:psiRNA-WNT5A can efficiently knock-down WNT5A mRNA expression in Jurkat cells. WNT5A under-expression in Jurkat cells has reducedβ-catenin protein expression and reduced the mRNA expression of target genes (c-myc and c-jun)of WNT signalling pathway.Therefore, WNT5A may be a regulation factors ofβ-catenin protein expression and nuclear localization in Jurkat cells.