The Expression And Functional Study CypB In Gastric Cancer And Selection Of CypB-specific Aptamer

【Background】Gastric cancer is one of the most common malignant tumors in China. Tumorbiomarkers can help physicians to diagnose gastric cancer earlier and make more effectivetreatment decisions to improve cure rates and reduce deaths. So far, there are still no suchspecific and sensitive biomarkers for early gastric cancer. Therefore it is urgent to developnovel biomarkers for gastric caner and discover new targets for treatment of gastric caner.Cyclophilin B (CypB) is a ubiquitously expressed protein which is known to be secretedinto body fluids such as milk and blood. Recently, researchers found that CypB isover-expressed in cancer including breast caner, hepatoma, colorectal cancer andpancreatic cancer. CypB may play an important role in cancer development. Howeverthere are still no reports about the role of CypB in gastric cancer.Aptamers are single-stranded nucleic acid ligands selected against a specific target molecule for desired functions. Aptamers are selected using an in vitro procedure termedsystematic evolution of ligands by exponential (SELEX) enrichment. Aptamers have beenstudied as a bio-material in numerous investigations concerning their use as a diagnosticand therapeutic tool, biosensing probe, and in the development of new drugs, drugdelivery systems. We hope to search for aptamers specific binding CypB, and propse theselected aptamer as potencial cancer imaging agent by targeting gastric canceroverexpressioning CypB.【Objectives】This study aimed:1. to investigate the expression of CypB in both serum and tissuesfrom gastric cancer patients;2. to determine the effects of CypB on growth andproliferation of gastric cancer cells;3. to isolate CypB-specific aptamer by SELEX.【Methods】1. Blood samples from gastric cancer patients and healthy volunteers were collected.CypB, CEA, CA19-9levels in the sera were measured by ELISA.2. The expression of CypB in serum was correlated to the clinical and pathologicalfeatures of patients with gastric cancer.3. The diagonosis value of CypB was compared to CEA and CA19-9.4. The expression patterns of CypB were observed in gastric cancer and adjacentnon-tumor tissue using immunohistochemistry.5. The correlation of CypB and clinical parameters of gastric cancer patients wasanalyzed.6. CypB siRNA lentivirus （LV-CypB-si） and control lentivirus（LV-si-con）wasproduced.7. CypB expression in gastric cancer cell lines was detected by Western blot.BGC823and SGC7901cells were chosen to be infected with LV-si-con andLV-CypB-si，and stable transfectants were isolated..8. MTT and colony formation assays were used to examine the effect of CypB on thecell growth and proliferation in vitro. Cell cycle was analyzed with flowcytometry. 9. Tumorigenicity of gastric cancer cells in nude mice was applied to explore theeffect of CypB on the cell growth and cell proliferation in vivo.10. DNA aptamers specific to CypB were islolated by SELEX method.11. Binding affinity (Kd) was determined in direct binding assay by flow cytometryanalysis. The Kd of the aptamer-CypB interaction was obtained by fitting thedependence of fluorescene intensity of specific binding on the concentration of theaptamer to the equation Y=B max X/(Kd+X).(Y: fluorescene intensity; X:concentration of aptamer; B max: the number of binding site)【Results】1. Serum levels of CypB were increased in the gastric cancer patients.CypB levels were greatly elevated in gastric caner patients serum comparing tothe healthy volunteer (median6.13ng/ml versus4.34ng/ml, p<0.05). The CypBlevels in gastric cancer patients were significantly correlated with tumor stage andlymph node metastasis (p<0.05), but not with patient’s age, gender and tumordifferentiation. The ROC curve analyses suggested the diagnosis value of CypBwas better than CEA and CA19-9.2. CypB expression was significantly increased in gastric cancer tissues.CypB was mainly located in cytoplasm and only occasionally in nuclei in gastriccancer cell. The positive rate of CypB was82.6%(109of132patients) in gastriccancer tissues, and58.3%(35of60) in adjacent non-tumor tissues. Furtheranalyses showed association of CypB expression with tumor stage and lymphnode metastasis, but not with patients’ age, gender or tumor differentiation.3. Knock-down of CypB can inhibit gastric cancer cell growth, proliferation,cell cycle progress and tumorigenesis.CypB expression level was obviously higher in SGC7901and BGC823thanMKN28and GES. These two cell lines were infected with LV-si-con andLV-CypB-si respectively. MTT and cloney formation assays showed asignificantly decreased rate of cell proliferation in cells transfected withLV-CypB-si. Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G1. These findings indicated that CypBcould promote the G1-S transition of gastric cancer cell.4. Two aptmers which can bind to CypB protein with high affinity andspecificity were isolated through SELEX.Through eight rounds of selection using CypB-his protein as target and ni-beadsas counter target, we have isolated two single-stranded DNA aptamers C10andC11that specificly bind to CypB with Kd in the nanomolar range. C10aptamerconjugated with FITC showed specific binding to CypB positive gastric cancercells BGC823and SGC7901.【Conclusions】Our data suggests that CypB is over-expressed in both serum and tissues of gastriccancer patients. The CypB expression level was correlated with tumor stage and lymphnode metastasis. Knock-down of CypB decreases gastric cancer cells proliferation and invivo tumorigenesis. These findings indiccate CypB could be a potential biomarker andtherapeutic target for gastric cancer.CypB-specific aptamers were succusfully isolated, which may be used in further studyand future application of CypB.