Exploring The Roles Of Liver-intestine Cadherin (CDH17) In Gastric Cancer And Screening Of CDH17-associated Proteins

BackgroundGastric cancer (GC) has the highest incidence of any cancer in the Western Pacific countries, which includes China. It was the third leading cause of cancer-related mortality. The prognosis of GC is directly correlated with stage at presentation. Unfortunately a large majority of patients experience a relapse or present with unresectable locally advanced or metastatic disease. Systemic chemotherapy is the only effective treatment available in these situations, but it has extremely poor results and the median survival time is invariably shorter than one year. Gastrocarcinogenesis is a complex process in which alterations in proliferation and migration properties of cancer cells have an important role in cancer infiltrative growth and metastasis. Researches about the genes and their products involved in GC development may deepen our knowledge in the mechenism of cancer invasion, and further provide theoretical basis of early diagnosis markers and therapy target.Cadherins are a group of functionally related transmembrane glycoproteins that mediate calcium-dependent cell adhesion. They are divided into subclasses such as E-, P-, and N-cadherins, which are distinct in immunological specificity and tissue distribution. Researches have demonstrated that cadherins play a role in the construction of tissues and of the whole animal body and have strong implications in tumorigenesis. CDH17 (liver-intestine cadherin), also known as L1-cadherin, has been identified as a novel member of the cadherin superfamily, which is distinguished from classic cadherins by its unique structural and functional features. More and more studies have indicated the close correlation of CDH17 and tumor differentiation or tumor invasion. However, researches have identified the different even opposite role of CDH17 in colon carcinoma and hepatoma or GC, which suggestted CDH17 to be a multifounctional protein. AimsIn order to explore the specific role and possible mechanism of CDH17 in gastric carcinogenesis, the expression levels of CDH17 in gstric adenocarcinoma tissues were examined by quantum-dots based immunochemistry and CRI optical spectra imaging system, the correlation of CDH17 and clinicalpathological fators were alsso anylzed. Second, we constructed a stable transfected cell strain with CDH17 knockdown by the lentivirus-mediated RNA interference (RNAi) technology. Third, cell proliferation, opoptosis, invasive and matastasis ability aftrer CDH17 silencing was examined in vitro. The primary tumor growth, the local infiltration and systemic tumor dissemination were evaluated using orthotopic implantation technique. The therapeutic value of CDH17 inhibition was examined by intratumoral administration of CDH17-RNAi-Lentivirus in established GC tumor xenograft mice model. Furthmore, we used two dimensional polyacrylamide gel electrophoresis (2-DE) followed by tandem mass spectrometry (MS) to perform a proteome-wide profiling of GC cells stratified for the absence of CDH17 to identify proteins associated with CDH17 expression.Methods1. Quantum-dots immunohistochemistry technology was used to examine the expression of CDH17 in GC tissues. The expression levels of CDH17 in gstric adenocarcinoma tissues were examined by quantum-dots based immunochemistry and CRI optical spectra imaging system, the correlation of CDH17 and clinicalpathological fators were alsso anylzed.2. Four precursor microRNAs (pre-miRNA) sequences targeting to CDH17 (GenBank accession number NM004063) and a negative control were designed and inserted into pcDNA rM6.2-GW/EmGFP-miR expression vector (Invitrogen) to construct recombinant plasmid pcDNA-CDH17-miR-SR₁,-SR₂,-SR₃ and-SR₄, respectively. To verify the recombinants, the recombinant plasmids were extracted for sequence detection. Using the instructions for Lipofectamine 2000 (Invitrogen), we transient transfected the recombinant plasmids in cultured BGC823 cells. After 24 h, we observed the transfection effect under a fluorescent microscope. After 48 h, real time RT-PCR and western blot testing were used to identify the target site with the highest interfering efficiency. We packaged the recombinant lentiviral vector for CDH17 RNAi with the highest interfering efficiency using the BLOCK-iT Lentiviral Polâ…¡miR RNAi Expression System. The lentiviral vectors were transfected into BGC823 cells. To produce stable transfection cell line, the cells were cultured in a selection medium.3. CCK-8 and clon formation assay were used to assess the proliferation ability of BGC823 cells with CDH17 knockdown in vitro. Cell apoptotsis was assessed by Hoechst 33258 staining and Annexin V-APC/PI. Cell cycle distributions and related proteins cyclin-D1 and cyclin-E were analyzed by flow cytometry and western blot. Wound healing assays were performed to evaluate the motility and migration ability of cells. We also evaluated the primary tumor growth, assessed the local infiltration and systemic tumor dissemination using orthotopic implantation technique. Furthermore, the therapeutic value of CDH17 inhibition was examined by intratumoral administration of CDH17-RNAi-Lentivirus in established GC tumor xenograft mice model. Immunochesmistry was used to examin the expression level of CDH17 in transplantation tumor tissues and apoptosis of cancer cells were tested by TUNEL assay.4. Two-dimensional electrophoresis (2-DE) and mass spectrography (MS) technology were utilized to identify differentially expressed proteins in BGC823 and lenti-CDH17-miR-B cells with CDH17 knockdown.Results1. CDH17 was mainly located in cancer cell membrane and partly cytoplasm of gastric epithelium mucosa. The expression levels of CDH17 in GC tissues (5.3768±4.0292) were much higher than that of normal gastric mucosa (0.0874±0.0933) (P=0.000). The expression levels of CDH17 were gradually reduced in well, moderate and poor differentiated cancer tissues with 11.470±6.104,5.799±2.791 and 3.414â– 2.407 respectively (P=0.000). The expression levels of CDH17 were also viared between cancer tissues with or without lymphonode invasion (6.167±4.188 vs 2.669±1.699, P=0.041).2. Four recombinant plasmid expression vectors encoding pre-miRNA against CDH17 and a nagative cintrol were constructed correctly and viritified by enzyme digestion and sequencing. GFP was observed under the fluorescence microscope after the plasmids were transient transfected into the BGC823 cells 24 hours later. Then pcDNA-CDH17-miR-SR₄ was identified to have the highest interfering efficiency. We packaged the plenti6/V5 recombinant lentiviral expression vector by pDNOR221 and plenti6/V5-DEST for CDH17 RNA interference with pcDNA-CDH17-miR-SR₄ which had the highest interfering efficiency tested by real time RT-PCR and western blot. To construct the stable transfectants, the cells were cultured in a selection medium containing 6μg/ml of blasticidin for 2 weeks and the stable cell lines after selection were obtained with 3μg/ml of blasticidin. Of note, more than 90% knockdown of CDH17 expression in BGC823 cells was obtained by miRNA technique.3. Down-regulation of CDH17 inhibited proliferation and migration of the poorly differentiated GC cells BGC823 in vitro as well as induced cell cycle arrest at G0/G1 phase [the percentage(%) of cells at G0/G1 phase in BGC823, lenti-CDH17-miR-neg and lenti-CDH17-miR-B group were 57.4±2.32,60.7±2.40 and 72.5±1.65 respectively, P<0.05], and the decrease of cyclin-Dland increase of cyclin-E after CDH17 silencing were showed by western blot assay. The percentage(%) of apoptotic cells in BGC823, lenti-CDH17-miR-neg and lenti-miR-CDH17-B group was 0.720±0.085,0.767±0.110 and 1.947±0.391 respectively (P=0.001), which suggested that CDH17 knockdown could increase apoptosis. Using an in vivo tumor growth assay, we showed that metastases in the liver and lung both appeared earlier and more frequently in animals with tumors derived from BGC823 or lenti-CDH17-miR-neg cells than in tumors derived from lenti-CDH17-miR-B cells. Furthermore, the average tumor volume [(1.2600±0.21652) cm3] and weight [(7.0638±1.06035) g] in CDH17-RNAi- Lentivirus treated group were significantly lower than those in the sodium treated group[tumor volume:(1.6967±0.14706) cm3, tumor weight:(8.1353±1.14351) g] and GFP-Lentivirus treated group [tumor volume:(1.80830±0.25895) cm3, tumor weight: (4.7853±1.25603) g] (P<0.05). Immunochesmistry showed that CDH17 was greatly reduced in the CDH17-RNAi-Lentivirus treated group. Furthermore, apoptosis cells in the cancer tissue from CDH17-RNAi-Lentivirus treated group were increased examined by TUNEL assay, which suggested that CDH17-RNAi-Lentivirus may partial inhibited tumor growth in vivo through apoptosis in cancer cells.4. Fifteen different proteins, nine downregulated such as Nucleophosmin (NPM), Tropomyosin alpha-3 chain (TPM3), Glutathione S-transferase P (GSTP1) and Alpha-enolase (ENOA), six upregulated such as Clathrin light chain A (CLCA), Interleukin-1 alpha (ILIA) and Vinculin (VINC) were identified to mainly located in cytoplasm (30.3%), nucelus (12.12%) or peripheral membrane (9.09%) after CDH17 silencing in BGC823 cells. These proteins comprised a variety of cytoskeleton (25%) and chaperone (25%) proteins, as well as proteins involved in cell adhesion molecule (6.25%), signaling molecule (6.25%), nucleic acid binding (6.25%), lysase(6.25%), kinase (6.25%), oxidoreductase (6.25%), membrane traffic protein (6.25%), transferase (6.25%), etc.ConclusionTaken together, we showed that CDH17 maybe a positive regulator for proliferative and invasive behaviors of GC cells. Based on our investigations, we proposed that CDH17 may be an oncogene up-regulating invasive features of GC cells through interactions with many orther proteins.