Regulatory Effects Of PRL-3 On Cadherin Related Signaling Pathways In Colorectal Cancer

BACKGROUND & OBJECTIVEColorectal cancer(CRC) is a common malignant tumor leading to death in the world.The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of cancer patients.It is urgent to elucidate molecular mechanisms of metastasis and find out the preventive and therapeutic strategies.PRL-3(Phosphatase of Regenerating Liver-3) belongs to a subclass of protein tyrosine phosphatases(PTPs) family.Saha et al found that PRL-3 expression was dramatically up-regulated in metastatic colorectal carcinomas(CRCs).This key study raised the possibility that PRL-3 might be an excellent therapeutic target for metastatic CRC and also stimulated further studies into the prevalence of the association between levels of PRL-3 expression and cancer metastases.In our previous studies,CDH22 was found as a candidate PRL-3-interacting protein by yeast two-hybrid screening systems.Interaction between PRL-3 and CDH22 was identified by GST-pull down assay,co-immunoprecipitation assay and co-localization analysis.CDH22(PB-cadherin) and E-cadherin belong to the classical subgroups of cadherin family,which plays an important role in regulation of cadherin related signaling pathways of colorectal cancer.Our study aims to clarify the role of PRL-3 in regulation of cadherin related signaling pathways of colorectal cancer.SW480 cells with stable over-expression and knock-down of PRL-3 gene were established.Effects of PRL-3 on the biological behaviors of SW480 cells were studied.We try to find the the functional relationship between PRL-3 and cadherin.Molecular role of the PRL-3 in regulation of cadherin related signaling pathways of colorectal cancer were studied.METHODS1.Eukaryotic fluorescent expression vector of PRL-3,pEGFP-N1-PRL-3,was constructed and cell model of human colon carcinoma with PRL-3 gene stable over-expression was establishedFull length cDNA of PRL-3 was amplified by RT-PCR with total RNA extracted from the human colon carcinoma cells SW480 as template,and cloned into pGEM-T Easy vector or eukaryotic expression vector pEGFP-N1.The recombinant plasmid of pEGFP-N1-PRL-3 was identified by restriction endonuclease analysis and DNA sequencing.The pEGFP-N1-PRL-3 plasmid was transfected into SW480 cells.PRL-3 stable over-expression SW480 cells were screened by G418 selection.PRL-3 expression in transfectant was determined by real-time reverse transcription polymerase chain reaction(PCR) analysis and western blot.2.A lentiviral vector of RNA interference(RNAi) for PRL-3 gene was constructed and cell models of human colon carcinoma with PRL-3 gene stable knock-down were establishedPlasmids expressing two different short hairpin RNAs were constructed to target against PRL-3 under the control of the U6 promoter by lentiviral vector.An entry clone was generated in the pENTRTM/U6 Vector.After identification for sequencing, a recombinant lentiviral vector was obtained using the pENTRTM/U6 construct and pLenti6/BLOCK-iT TM-DEST vector.Recombinant lentivirus was harvested from 293FT cells cotransfected with the optimized ViraPower TM Packaging Mix and the pLenti6/BLOCK-iT TM -DEST expression construct.SW480 cells were infected with recombinant lentivirus.SW480 cells were infected with recombinant lentivirus and PRL-3 stable knock-down SW480 cells were screened by blasticidin selection.PRL-3 expression was determined by real-time reverse transcription polymerase chain reaction(PCR) analysis and western blotting.3.Effects of PRL-3 gene on the biological behaviors of human CRC. Effects of PRL-3 overexpression and knock-down on cell proliferation was assessed by MTT assay,plate colony formation assay and flow cytometry in vitro. Effects of PRL-3 overexpression and knock-down on cell motility and migration were assessed by in vitro Transwell chamber and adhesion assay.Morphological changes of colorectal cancer cells after PRL-3 overexpression or knock-down were studied by scanning electron microscope and Transmission electron microscope.4.Expression of cadherin related signaling molecules in SW480 cells with stable over-expression or knock-down of PRL-3 geneCadherin related signaling molecules including E-cadherin,CDH22,a-catenin,β-catenin,γ-catenin,Snail,β-actin,β-tubulin,GSK-3β,Phosphorylated GSK-3β, Ezrin,Paxilin were investigated by immunoblot and indirect immunofluorescent methods.RESULTSThe main results are as follows:1.Construction of pEGFP-N1-PRL-3 vector and its stable expression in SW480 cells Recombinant pEGFP-N1-PRL-3 plasmid with entirely coding elements of human PRL-3 gene was constructed and identified by restriction endonuclease analysis and DNA sequencing.PRL-3 expression in PRL-3 stable over-expression SW480 cells was 2.78 times of that in control group.Establishment of PRL-3 gene over-expression cell model of human colon carcinoma will provide a basic tool to study the role of PRL-3 gene in the metastasis of human colorectal carcinoma.2.Construction of lentiviral vector of RNA interference(RNAi) of PRL-3 gene and its stable expression in SW480 cellsA recombinant lentiviral vector expressing short hairpin RNAs to target against PRL-3 gene was successfully established and confirmed by DNA sequencing. Recombinant lentivirus was harvested from 293FT cells.The titer of virus was 6×10~5 pfu/L.Twelve clones of SW480 cells infected with recombinant lentivirus were selected.Clone1(Designated SW480-PRL-3-KD1) exhibited a dramatic knock-down of PRL-3 protein expression(72.9%).We also found that PRL-3 protein expression in clone 2(Designated SW480-PRL-3-KD2) was dramaticly knocked down. 3.Effect of PRL-3 silencing on the biological behaviors of human CRCSW480-PRL-3-KD1 cells showed a significantly reduced proliferation compared with SW480/EGFP/mock and SW480/EGFP cells as determined by in vitro MTT assay.In addition,SW480-PRL-3-KD1 cells had a significant reduction in their ability to form colonies in plate as compared with SW480/EGFP/mock and SW480/EGFP cells.The result of in vitro migration assay showed that SW480-PRL-3-KD1 cells had significantly reduced invasiveness as compared with SW480/EGFP/mock and SW480/EGFPcells.4.The role of PRL-3 in regulation of cadherin related signaling pathways of colorectal cancerPRL-3 induces epithelial mesenchymal transition by down-regulation of E-cadherin,CDH22 andγ-catenin and up-relation ofβ-Catenin andα-Catenin. Expression of E-cadherin was recovered by knock-down of PRL-3.Phosphorylated GSK-3βis involved in cadherin related signal pathway and regulation of PRL-3. PRL-3 reduces expression of paxillin and ezrin.CONCLUSION1.PRL-3 gene plays an important role in proliferation,adhesion and migration of CRC.2.PRL-3 induces epithelial mesenchymal transition by down-regulation of E-cadherin,CDH22 andγ-catenin and up- regulation ofβ-Catenin andα-Catenin.3.Phosphorylated GSK-3βis involved in cadherin related signaling pathway and regulation of PRL-3.