Screening And Identification Of PRL-3 Interacting Protein And Its Preliminary Studies

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is a common malignant tumor leading to death of a lot of people. The incidence of CRC in our country is consistently increasing in recent decades. Metastasis is the main cause resulting in most death of patients with CRC. It is critical to elucidate molecular mechanisms of metastasis and find the key factors controlling tumor metastasis, it will also be helpful to develop the effective preventive and therapeutic strategies.It is well-known that PRL-3 (Phosphatase of regenerating liver-3, PRL-3) is a key gene related to progression and metastasis of CRC. The role of PRL-3 in the pathogenesis of liver metastasis of CRC was firstly identified by analyzing gene expression profiles of CRC by analysis of Serial Analysis of Gene Expression (SAGE) in metastatic CRC. Recent studies also found that PRL-3 could promote proliferation, migration, invasion and adhesion of cancer cells and play an important role in tumor angiogenesis. PRL-3 belongs to a subclass of PTP family (protein tyrosine phosphates), which has 3 members including PRL-1, PRL-2 and PRL-3. Homology of amino acid sequences among three members exceeds 75%, which suggest their functional similarity. In their catalytic domain, PRLs lack Serine-Threonine residues essential for catalysis of the phosphatases. PRLs have only one catalytic domain, characteristic of a CAAX sequence in C-terminus for prenylation. PRLs is located in the cytoplasma when prenylated and in the nucleus when non-prenylated, which suggests PRLs participate as signal molecules in signal pathway network.The role of PRL-3 in tumor metastasis of CRC has been identified. However, there are still many questions to be answered, such as its biological properties, the substrate catalyzed, the mechanisms promoting metastasis and invasion of tumor cells, the signal pathways PRL-3 participating in. In order to illuminate the questions, Screening and identification of PRL-3 interacting proteins will be the key step to find the clues of PRL-3 related signal pathway.Screenings of PRL-3 interacting proteins were firstly analyzed by bioinformatical methods and yeast two-hybrid screening systems, some of PRL-3 interacting candidate proteins would be identified by GST-pull down, Co-immunoprecipitation and Co-localization analysis, ant its role in CRC would also be studied.METHODS1. Expression of PRL-3 and its clinical significance in CRCProtein and mRNA expression of PRL-3 in normal mucosa, primary CRC and metastatic lymph nodes and its possible clinical significance in carcinogenesis, progression, invasion and metastasis were studied by immunohistochemistry and Quantitative RT-PCR. 2. Screening and identification of PRL-3-interacting proteins1) Bioinformatics prediction and analysis of PRL-3-interacting proteinsPRL-3-interacting proteins were predicted by bioinformatical methods, and gene expression patterns of the candidate proteins in human tissues or organs were analyzed by Digital northern blot in NCBI online database.2) Screening of PRL-3- interacting protein in yeast two-hybrid screening systemsComplement cDNA of PRL-3 was cloned and yeast expression bait vector of pGBKT7-PRL-3 used in yeast two-hybrid screening systems was constructed. And Homo Sapien fetal brain cDNA Library was used to screen PRL-3-interacting proteins.3. Identification and bioinformatical analysis of CDH22, a candidate PRL-3-interacting proteinInteraction between PRL-3 and CDH22, a candidate PRL-3- interacting protein was identified by GST-pull down assay, co-immunoprecipitation assay and co-localization analysis in vivo and in vitro.1) GST-pull downFirstly, complement cDNA of CDH22 was cloned and eukaryotic expression vector of CDH22 was constructed to obtain fusion proteins of CDH22 in vitro. Secondly, prokaryotic expression vector of pGEX-4T-1-PRL-3 was constructed, and PRL-3 fusion proteins with GST label were induced and purified. Thirdly, interaction between PRL-3 and CDH22 in vitro was identified by GST-pull down assay.2) Co-immunoprecipitation (CO-IP)Interaction between PRL-3 and CDH22 was examined respectively in exogenous systems and endogenous cell models.Two eukaryotic expressing vectors expressing PRL-3 with HA label and CDH22 with FLAG label were constructed respectively and co-transfected into HEK293A cells. Cells were lysed, and proteins were purified by Anti-HA Affinity Gel or Anti-FLAG M2 Affinity Gel respectively, affinitive proteins were analyzed by western blots with anti-FLAG or anti-HA monoclonal antibodies.Endogenous interaction between PRL-3 and CDH22 was studied in colorectal cancer cells. Cells were lysed and purified by anti-PRL-3 Affinity Gel or anti-CDH22 Affinity Gel respectively, protein pellets were analyzed by western blots with anti-CDH22 or anti-PRL-3 antibodies.3) Co-localization analysisEukaryotic expression vector of pEGFP-PRL-3 was constructed firstly, and eukaryotic expression vector expressing pEGFP-PRL-3 and HA-CDH22 were co-transfected into SW480 cells. Cells were stained with anti-HA antibodies followed by TRITC conjugated antibodies 24 hours after transfection and observed under confocal microscopy. Green fluorescence and red fluorescence of cells were merged to localize the contacting sites of both proteins.Meanwhile, pcDNA3-FLAG-PRL-3 and pcDNA3-HA-CDH22 were co-transfected into HEK293A. Cells were stained with anti-HA or anti-FLAG antibodies followed by TRITC or FITC conjugated specific antibodies 24 hours after transfection. Co-localization analysis of the two proteins was performed as what mentioned above.4) Bioinformatical analysis of CDH22 proteinFunctions, expression patterns and possible interaction networks of CDH22 protein were predicted by several kinds of bioinformatics software and online database.4. Expression of CDH22 in colorectal carcinomas and its clinical significanceProtein and mRNA expression of CDH22 in normal mucosa, CRC and metastatic lymph nodes and its possible clinical significance in carcinogenesis, progression, invasion and metastasis were studied by immunohistochemistry and Quantitative RT-PCR.5. Lentivirus-mediated silencing of CDH22 gene and the influence on human CRC cellsFour short hairpin RNAs were designed to silence CDH22 expression, and recombinant lentivirus vectors under the control of the U6 promoter with four short hairpin RNAs were constructed. 293FT cells were co-transfected with recombinant lentivirus vectors and adjunctive plasmid. Virus supernatants were harvested and stored in-80℃before used in the next experiments, virus titer was determined as routine.SW480/EGFP+, a kind of human CRC cells, were infected with virus supernatants obtaining CDH22 specific RNAi lentiviral vectors, SW480/EGFP+ infected with virus supernatants obtaining mocked RNAi lentiviral vectors were regarded as controls. Cells CDH22 stably silenced were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to examine the effectiveness of RNA interference. Effects of CDH22 silence on cell proliferation was assessed by MTT assay, plate colony formation assay and flow cytometry in vitro. Moreover, proliferation of tumor cells was also observed in ectopic-transplanted mice models in vivo by whole-body visualizing methods. RESULTS1. Expression of PRL-3 in CRC and its clinical significanceProtein expression of PRL-3 in primary colorectal carcinoma samples with lymph node metastasis was significantly higher than those without lymph node metastasis (Z=-2.083, P＜0.05) by immunohistochemistry, And PRL-3 expression in metastatic lymph nodes was significantly higher than that in its corresponding primary colorectal carcinoma (X~2=-4.591, P=0.032). Additionally, protein expression of PRL-3 correlated positively to degree of invasion (P＜0.05). There is no correlation between PRL-3 expression and differentiations or types of cancer (P＞0.05). Similar results were observed in mRNA level by Quantitive RT-PCR.We confirmed that PRL-3 expression correlated positively to invasion and metastasis of colorectal carcinoma. Ceils of colorectal carcinoma with high PRL-3 expression tended to be more invasive and easy to metastasize.2. Screening of PRL-3- interacting proteins1) Bioinformatics prediction and analysis of PRL-3-interacting proteinsIt was found that human PRL-3 gene was homologous to dPRL-1, PRLs member in drosophila by phylogenetic analysis of PRLs family in different species. And dPRL-1 was found as an important bridge between CG11678-1A protein and CG17819-PA protein in drosophila. Homolog of CG11678-1A protein in human is ARP6 (actin-related protein 6 homolog), and homolog of CG17819-PA protein in human is called ARL-3, a member of ADP-ribosylation factor-like protein family. Gene expression patterns of PRL-3 and its related proteins in human tissues or organs were found similar by use of digital northern blot in online NCBI database.2) Screening of PRL-3-interacting proteins in yeast two-hybrid screening systems Four positive clones in our yeast two-hybrid screening systems were found after Homo sapien fetal brain cDNA Library had been screened. Two of four selected clones were sequenced and confirmed as gene CDH22. CDH22 was regarded as a candidate PRL-3-interacting protein to be identified.3. Identification and bioinformatical analysis of CDH22, a candidate PRL-3-interacting protein1) GST-pull downGST-pull down assay showed that GST-PRL-3 or GST-CDH22 fusion proteins could pull down its exogenous counterparts. Our results confirmed the interaction between PRL-3 and CDH22 in vitro.2) Co-immunoprecipitationCo-immunoprecipitation indicated that both exogenous and endogenous PRL-3 interacted with CDH22.3) Co-localization analysisConsistent with our co-immunoprecipitation results, PRL-3 and CDH22 were found to be colocalized at cytoplasm.4) Bioinformatics analysis of CDH22CDH22 has 5 repetitive extracellular domains conserved in Cadherin family. The results of digital Northern blots showed that CDH22 was expressed in the majority of CRC cells. Wnt1 was predicted as one of CDH22-interacting proteins by bioinformatical methods, which suggested that PRL-3-CDH22 may be involved in regulation of WNT signaling pathway.4. Expression of CDH22 in colorectal carcinomas and its clinical significance CDH22 was mainly expressed in intracytoplasm by immunohistochemistry. Expression of CDH22 was significantly different among groups of the normal colorectal mucosa, adenoma, carcinomas and lymphatic metastasis of colorectal carcinoma (χ~2=32.672, P＜0.01). Protein expression of CDH22 correlated positively with invasion and metastasis of colorectal cancer. Similar results in mRNA level were also observed by Quantative RT-PCR.5. Effects of CDH22 silencing on biological behaviors of human SW480 cells1) Establishment of SW480 cells stably CDH22 silenced by RNA interferenceOne clone, in which 85.4％of cellular CDH22 proteins were blocked, was selected to be investigated in our next experiments.2) Effects of CDH22 silencing on the biological behaviors of human SW480 cellsA significantly time-dependent inhibitory proliferation was found in SW480/EGFP~+/CDH22~- cells as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells by in vitro MTT assay (F=5.633, P＜0.001). In addition, SW480/EGFP~+/CDH22~- cells had a significant impaired ability to form colonies in plates as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells(F=72.337, P=0.003). Interestingly, SW480/EGFP~+/CDH22~- cells showed a significantly decreased amount in S period by flow cytometry. The results indicated that silence of CDH22 partially blocked proliferation of SW480 cells.SW480/EGFP~+/CDH22~- cells had significantly reduced motility and migration as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells determined by in vitro Boyden chamber assay(F=51.083, P=0.000).The effects of CDH22 on in vivo tumor growth was assessed by subcutaneous growth of SW480/EGFP~+/CDH22 and SW480/EGFP~+/mock cells in ectopic-transplanted mice models for thirty days. Silence of CDH22 led to a pronounced decrease of tumor growth in SW480/EGFP+/CDH22~- cells group 15 days after subcutaneous injection.CONCLUSION1. PRL-3 gene plays an important role in invasion and metastasis of CRC.2. CDH22 protein can directly interact with PRL-3 protein in vivo and in vitro. CDH22 is a new PRL-3 interacting protein.3. CDH22 gene significantly correlates to proliferation, invasion and metastasis of CRC.4. CDH22 promotes proliferation and migration of colorectal cancer cells.5. PRL-3-CDH22 related signal pathway plays a role in invasion and metastasis of CRC.