Cap43 As A Tumor Marker In Nickel Induced Lung Cancer

ObjectiveTo detect Cap43 expression in tissue and serum of lung cancer patients, we initially studied the feasibility of Cap43 as a novel tumor marker of nickel induced lung cancer.Materials and Methods1. Object of studySurgically resected 79 lung cancer tissue specimens confirmed pathologically and their surrounding normal controls were obtained from the First Affiliated Hospital of China Medical University for immunohistochemistry detection. 28 fresh lung cancer tissue specimens were collected from 79 specimens in immunohistochemistry detection from the First Affiliated Hospital of China Medical University for western blot detection. 28 fresh lung cancer tissue specimens were collected from 79 specimens in immunohistochemistry detection from the First Affiliated Hospital of China Medical University for realtime RT-PCR. 91 serum specimens of lung cancer patients were collected from the First Affiliated Hospital of China Medical University. 79 of the total specimens come from the same patients as immunohistochemistry detection. 5 lung cancer patients, 20 grinder's disease patients and 200 common people serum specimens were collected from Jilin Nickel Industry Co.Ltd. All the serum specimens were collected for double antibody sandwich ELISA detection.2. Materials(1) ReagentGoat anti-human ndrg1 polyclonal antibody(PcAb)(Santa Cruz Corp, US); Rabbit anti-human Cap43 PcAb(Invitrogen corp, US); HRP conjugated rabbit-anti-goat-IgG (Jackson ImmunoResearch, US); Standard of NDRG-1(US Biological corp,US); S-P kit for immunohistochemistry detection(Zsbio Corp; Beijing), Western bloting ECL kit(Santa Cruz Corp, US); PVDF membrane(Millipore Corp, US); Rabbit anti-humanβ-actin PcAb(Santa Cruz Corp, US); Protein marker and tissue lysate(Beyotime Biotechnology; Haimen, China); TRIZOL(Santa Cruz Corp, US); DEPC(Promega Corp, US); RT enzyme (MMLV Kit); dNTP(Huamei Corp, China); Oligo(dT)₁₈(Sangon Biological Engineering Cor, Shanghai,China); Sybergreen (Molecular Probes, US).(2) Laboratory apparatusELIASA(BIO-RAD Corp, US); Electrophoresis Gel System(Tanon Cor, Shanghai,China); FeroTec Gradien PCR gene amplification apparatus(Ferrotec Corporation, Japan); Rotor-Gene 3000 Realtime PCR apparatus(Corbett Research, Australia).3. Experimental methods(1) Immunohistochemistry detectionTumors tissues were processed and embedded in paraffin wax. Sections were made.SP detection methods were adopted. The goat anti-human NDRG-1 polyclonal antibody was incubated for 4℃overnight. DAB coloration , dehydration, clearing and mounting were developed as routine. The detection digital picture results were semiquantitatively analyzed by Image analysis system.(2) Western blot detection200mg tissues were obtained and homogenized by lysate liquid After quantitation by BCA Kit, protein samples were electrophoresed and electroblotted. After being blocked with rabbit serum overnight at 4℃, the membranes were incubated with goat anti-human ndrg1 PcAb for 2 h at room temperature. Then membranes were incubated in HRP-rabbit anti-goat IgG antibody for 1 h at room temperature. Blots were detected by using chemiluminescent detection kits. Then, the films were scanned with a digital imaging system. Protein expression levels were determined by densitometric analysis using imaging analysis system.(3) Realtime RT-PCR detection100mg tissues were obtained , dissolved in TRIZOL and then homogenized, two-dimensional separated. RNA precipitations were collected. Then ultraviolet absorption assay and RNA electrophoresis were developed for concentration and purity testing. Reverse transcription were used by MMLV methods for cDNA synthesis. Gradient deliquation DNA templates were prepared for developing the standard curve. After allocation Realtime PCR reaction system, all the reagents were reacted in realtime PCR appearance. According to the results of the realtime PCR appearance and the standard curve, the concentration of target gene andβ-actin gene expression were determined.(4) Double antibody sandwich ELISA detectionThe assay was optimized by checkerboard titration. Polystyrene 96-well microtiter plates were coated with goat anti-human ndrg1 PcAb at 4℃overnight. Then these plates were blocked for 60 min at room temperature. Ater washed, the plates were then coated with serum samples and NDRG-1 standards for 60 min at room temperature. The wells, after washed , were coated with rabbit anti-human Cap43 PcAb for 60 min at room temperature. Then after wash, the wells were coated with HRP-goat anti-rabbit IgG for 60 min at room temperature. TMB substrate solution was then added after washing at room temperature for 15 min. 2 N sulfuric acid was added. Optical density was determined photometrically at 450 nm by an ELISA reader.4. Positivity judgment criteria(1) Judgment criterion for immunohistochemistry assayDistributions of positive buffy particles are in cytoplasm and cellular nucleus; percentage of stained cells in total cells in highpower field is more than 20% under the common light microscope.(2) Judgment criterion for western blot assaySpecific protein bands indicated by marker are present and IDVof the protein bands(rectified by reference) are significantly higher than that of the control groups.(3) Judgment criterion for realtime RT-PCRThe mRNA expression concentration of cases (rectified by reference) are significantly higher than that of the negative control.(4) Judgment criterion for double antibody sandwich ELISA assay P/N(ratio of OD of case and negative control )>2.1.5. Statistical analysisAll analyses were performed using the SPSS Version 13.0 statistical package. All the statistics were expressed as 'mean±S'.. Differences were taken as statistically significant when P<0.05. Independent Sample t test, Fisher's exact test ,SNK-q test, x² test, Pearson correlation test were used for statistical analyses. Size of test(a) is 0.05. Results1. Cap43 expression in tumor tissues of non-nickel induced lung cancer patients.The results of immunohistochemistry, western blot and realtime RT-PCR assays incicated: Cap43 gene and protein expression were expressed high in NSCLC, its expression level was significantly higher than surrounding normal controls(P<0.05); its gene and protein expression in lung adenocarcinoma was obviously higher than squamous cell carcinoma(P<0.05); Results of western blot aslo showed the poorer the lung cancer differentiated, the higher the Cap43 expression degree was(P<0.05). All the results above indicated that Cap43 expression in tissues is closely correlated with the tumor histological pattern in Lung cancer.2. Cap43 expression in serum of non-nickel induced lung cancer patients.A double antibody sandwich ELISA was developed for detection of Cap43 protein in serum. Results of the assay showed the standard protein curve regression equation was : (y|^)= 0.53566+ 0.00046x;its linear detection was 10 to 3000 ng/ml (R² =0.9939,P <0. 0001). By evaluation of pathological diagnosis, this detection's sensivity, coincidence rate are respective 84.51%,73.63%; The intial quantitatibve detection showed the level of Cap43 protein expression in serum in lung cancer group were higher than healthy adults group(P <0.05); its expression in lung adenocarcinoma was obviously higher than squamous cell carcinoma(P<0.05).3. The correlation between the intensity of Cap43 expression in serum and tissues of non-nickel induced lung cancer patients.By comparison with results of immunohistochemistry assay(histological detection) and double antibody sandwich ELISA (serological detection) of 79 non-nickel induced lung cancer patients, it was found that Cap43 expression in serum was closely correlated with it in tissues(r=0.915,P=0.029<0.05).4. Cap43 expression in serum of nickel induced lung cancer patientsThe Cap43 was expressed higher in serum of nickel induced people(lung cancer, grinder's disease patients and health examination common people) than non-nickel induced nomal people; its expression in nickel induced lung cancer and grinder's disease patients were both obviouly higher than health examination common people of nickel mine(P<0.05).Conclusions(1) Cap43 expression is expressed high in tissues of NSCLC patients and its expression is closely correlated with the tumor histological pattern in lung cancer. Cap43 expression is also expressed high in serum and its expression is closely correlated with it in tissues of NSCLC patients(2) Cap43 probably become a important serological auxiliary marker for precaution of high risk (lung cancer and grinder's disease) for nickel miner.