Effects Of Lentiviral-mediated MiRNA Against Liver-intestine Cadherin On The Bioligical Behavior Of Gasteic Cancer

Background and objectives:Gastric cancer （GC） is one of the most prevalent and lethal malignancies worldwide owing to the difficulty of early detection and high postsurgical recurrence rate Almost two-thirds of the cases occur in developing countries and 42% in China alone. Patients afflicted with GC are often asymptomatic, and the lack of sensitive and reliable biomarkers for early detection of GC means that diagnosis normally occurs late, when surgical intervention is not an option. Today, there are no effective drugs for curing gastric cancer. Using integrative genomic and proteomic approaches, researchers have begun to identify novel oncogenes and tumor suppressors in GC.Previous studies using clinical cohorts identified a cell surface adhesion molecule, cadherin-17 （CDH17）, also known as liver-intestine cadherin, as a potential disease marker for GC. In contrast to the conserved cytoplasmic domain of classical cadherins as E-, P-, and N-cadherin with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. The independence of its adhesive function from cytoskeletal anchorage clearly distinguishes CDH17 from E-cadherin and other classical cadherinsIn human, CDH17 is expressed exclusively in the intestinal epithelial cells and a fraction of pancreatic ductal epithelial cells. It is not found in the healthy adult liver and stomach, while altered CDH17 has been reported in colorectal, liver and gastric cancer, which means that CDH17 may play an important role in tumor progression. In our earlier studies, we identified that the expression of CDH17 was significantly higher in chronic atrophic gastritis with intestinal metaplasia than in gastric cancer and was lower in poorly differentiated tumors than in well and moderately differentiated tumors. Furthermore, we demonstrated a significant inverse correlation between expression levels of CDH17 and Galectin-3 and indicated CDH17 as an independent factor associated with lymph node metastasis. However there were some inconsistencies in the previous researches, as some researchers found that CDH17 was positively correlated to T and N stage; while other researchers reported that CDH17 was negatively correlated to T and N stage. So whether CDH17 was a negative or a positive regulator for cancer progression is uncertain now. In order to explore the role of CDH17 in GC, we analyzed the effects of stable CDH17 silencing with recombinant lentivirus mediated miRNA both in vitro and in vivo.Methods:1、Construction and Transfection of Lentiviral Vectors with Specific miRNAs for CDH17Four precursor miRNA sequences （SRI-4） targeting to CDH17 （GenBank accession number NM₀04063） and negative control were designed using an Internet application system. These double-stranded oligonucleotides were inserted into pcDNATM6.2-GW/EmGFP-miR expression vector and then transient transfected into BGC823 cells according to the operating manual of Lipofectamine 2000. Real-time PCR and western blot showed that the SR4 yielded the best suppression efficiency, therefore, this construct was chosen to package the recombinant lentiviral vector for CDH17 RNAi with pDONRTM 221 vector and pLenti6/V5-DEST using Gateway Technology. The new miRNA expression vector （pLenti6/V5-GW/EmGFP-miR） was then transfected into 293FT to assemble the lentiviral stocks named lenti-CDH17 The lentivirus was used to transduce BGC823 cells and the stable transfectants named lenti-CDH17-miR-B were selected using blasticidin for 2 weeks. Flow cytometry was performed to detect transfection efficiency. Two controls were included; BGC823 cells received no treatment or an RNAi vector with mismatch sequence （Lenti-CDH 17-miR-neg, Mock）.2、Characterization of changes in biological properities of Detection of lenti-CDH17-miR-B cellsThe cellular proliferation of transfected cells was measured via MTT assay. Cell cycle was analyzed by flow cytometry using a FACStar PlusTM. The ability of cells to migraton and invade through a Matrigel-coated filter was measured in transwell chambers following the manufacturer’s instructions. Cell Adhesion Assay was detected by MTT method and comparing OD value.Tumor Growth and metastasis Assay In Vivo3、the change in cancergenesis and metastasis of lenti-CDH17-miR-B cells in vivo.cell suspension was inoculated into nude mice subcutaneously in the dorsal area.The animals were monitored every other day, and tumor volume was calculated. Tumors were also evaluated with H&E （hematoxylin and eosin） and the expression of CDH17 were examined by immunohistochemistry.in the orthotopic implantation,The animals’abdomens were opened and the stomach was gently exteriorized. One donor tumor fragment was placed into the gastric tissue and fixed with one drop of tissue adhesive. The stomach was relocated into the abdominal cavity, which was then closed with absorbable suture.Four and six weeks after transplantation, animals were sacrificed and an autopsy was done to examine the growth of the tumor. Local infiltration was determined at liver, lung, spleen, and diaphragmatic muscle.Results1、Lentiviral stably-transfected BGC823 cells and Efficiently Suppressed CDH17 ExpressionThe lentiviral vector expression cassette allowed for the permanent expression of GFP and CDH17 miRNA in transduced cells. GFP expression and the percentage of GFP expression cells were determined by flow cytometry analysis, the efficient transduction of lenti-CDH17 after 3 d transfection is 98.8%. To test the knockdown efficiency, we also examined the expression of CDH17 on both mRNA and protein levels. Data showed that after being transfected with a MOI of 10 for 72 hours, the mRNA and protein levels of CDH17 were downregulated by 95% and 78% respectively in lenti-CDH17-miR-B cells compared with the control cells （lenti-CDH17-miR-neg）. Next, the surface expression of CDH17 in the cell lines was further confirmed at the protein level by immunofluorescence staining. CDH17 was high in both BGC823 and lenti-CDH17-miR-neg cells, while significantly downregulated in lenti-CDH17-miR-B cells.2、Effect of Down-Regulation of CDH17 on In Vitro Proliferation and Invasion of BGC823 CellsTo assess the potential effects of RNAi-mediated CDH17 silencing on cell proliferation and survival, we investigated cell growth in vitro. RNAi with mismatch sequence had no effect on the proliferative ability of BGC823 cells, whereas RNAi specific to BGC823 causes a dramatic reduction in the proliferation of cells （P<0.01） In the matrigel assays, the migrated cell numbers of lenti-CDH17-miR-B cells （24.4±4.4/HP） were much lower than that of the mock cells lenti-CDH17-miR-neg （115±9.5/HP） and the parental cells （113.0±14.0/HP） （P<0.01）, which suggests that lenti-CDH17 could significantly suppress the invasion of BGC823 cells. No significant difference was found between BGC823 and the lenti-CDH17-miR-neg cells. To assess the role of CDH17 in cell adhesion to ECM substrates, cells were plated on plastic plates coated with fibronectin. The lenti-CDH17-miR-B cells showed a significant reduction in their ability to adhere to fibronectin compared with untreated BGC823 cells or the mock cells lenti-CDH17-miR-neg. Cell cycle analysis was executed to determine whether the effect of miR-CDH17 on cell proliferation of BGC823 was due to cell cycle alterations. The result showed that comparing to the parental cells and the mock cells lenti-CDH17-miR-neg, the percentage of cells at G1 phase was increased from 57.1% and 60.7% to 72.5%（P<0.01）. These results indicated that downregulation of CDH17 induced a G1 cell cycle arrest in lenti-CDH17-miR-B cells.3、Knockdown of CDH17 decreased the growth and metastasis of the tumor derived from BGC-823 cells in vivoThe tumorigenicity of BGC823 cells after transfection was compared in nude mice model. Each mouse was inoculated with 1×106 cells. At 28 days postinoculation, obvious tumors were formed in the two control groups of mice inoculated with BGC823 cells or the lenti-CDH 17-miR-neg cells, while tumor growth was nearly completely suppressed in mice inoculated with lenti-CDH 17-miR-B cells, we observed only small tumors in mice inoculated with lenti-CDH 17-miR-B cells. All animals we found that All animals haven’t developed any metastases. The immunohistochemistry study confirmed that CDH17 protein expression was significantly suppressed in the lenti-CDH17-miR-B group compared with the BGC823 and lenti-CDH17-miR-neg groups.the primary orthotopic tumor volume in lenti-CDH17-miR-B group was much lower than that of the BGC823 and lenti-CDH17-miR-neg groups.nude mice in lenti-CDH17-miR-neg or BGC823 groups developed liver and lung metastases after 4 weeks, while neither of which were found in lenti-CDH17-miR-B group at that time. Moreover,6 weeks after transplantation, we found that mice with transplantation derived from lenti-CDH17-miR-B cells developed less metastases （lung 1/6 and liver 1/6, respectively） than animals in BGC823 group （lung 2/6 and liver 3/6, respectively）, and animals in lenti-CDH17-miR-neg group （lung 2/6 and liver 3/6, respectively）ConclusionsTo determine the role and mechanism of CDH17 in the process of gastric cancer invasive growth, miRNA-based CDH17 targeting pre-miRNA polâ…¡lentivirus vectors were constructed in our study. We found that lentiviruses could efficiently deliver CDH17 miRNAs into BGC823 cells and the CDH17 protein and mRNA levels were significantly decreased through transfection of CDH17 miRNA.Our present data showed that infection with the recombinant lentivirus expressing CDH17-specific miRNA dramatically reduced the proliferation of the gastric cancer cells BGC823 in MTT assay. This powerful effect was associated with a strong decrease in the expression of CDH17 in cells treated with the miRNA. Our adhesion and invasion assays showed that knockdown of CDH17 expression contributed to the significant reduction of ECM adhesion and invasion potency through the matrigel of BGC823 cells. Furthermore, our tumor growth assay in vivo demonstrated that knockdown of CDH17 expression can obviously slow the growth of gastric cancer derived from BGC823 cells.In our research, we demonstrated that knockdown of CDH17 induced cell cycle arrest. CDH17 silencing cells showed G0/G1 phase arrest and S phase reduction, suggesting that the reducing growth of the cells and tumor may associate with the cell cycle arrest.These observations, together with the ability of RNAi targeting for CDH17 to inhibit invasion and growth of gastric cancer cells, suggested the main role of CDH17 in induction of invasiveness, and the knockdown of CDH17 might result in marked suppression of the proliferation and invasive potential of GC cells. All the experiments should be reexamined with other GC cells to investigate the biological significance of CDH17 in gastric cancer.Taken together, our results indicated that the silencing of CDH17 expression by RNAi suppressed the proliferation and invasion of gastric cancer cells both in vitro and in vivo. CDH17 could be taken as an important determinant of malignant cellular behavior and maybe a promising target for therapeutic intervention of GC.