A3G-induced mutations show a low prevalence and exhibit plus-strand regional distribution in hepatitis B virus DNA from patients with non-hepatocellular carcinoma (HCC) and HCC.

APOBEC3G (A3G) cytidine deaminase is an innate immune restriction factor that can edit and inhibit hepatitis B virus (HBV) replication. The preferred target of A3G is deamination of the third cytosine of 5'CCC to form a mutant marker 5'CC C → K. However, the distribution of A3G-induced mutations on HBV DNA during infection is not well characterized. To provide clarity, we obtained the HBV DNA sequences from HBV infected individuals with and without hepatocellular carcinoma (HCC and non-HCC, respectively), from the NCBI database, and calculated the r values of A3G-induced 5'CC C → K mutation prevalence in HBV DNA. A3G-induced mutations were weakly prevalent and mainly distributed in the plus strand of HBV DNA (r = 1.407). The mutations on the minus strand were weaker (r = .8189). There were A3G-induced mutation regions in the 1200 to 2000 nt region of the plus strand and the 1600 to 1500 nt region of the minus strand. There was no significant difference in the r values of A3G-induced mutations in HBV DNA between the HCC and non-HCC groups. However, the rvalue of the plus strand 2400 to 2800 nt regions of HCC derived HBV DNA (r = 4.2) was significantly higher than that of the same regions of non-HCC derived HBV DNA (r = 1.21). These findings clarify the weak prevalence and preferred plus-strand distribution of A3G-induced mutations on HBV DNA from HCC and non-HCC. These findings may provide valuable clues regarding the interaction mechanism between A3G and HBV DNA and inform HCC screening.

A long-acting isomer of Ac-SDKP attenuates pulmonary fibrosis through SRPK1-mediated PI3K/AKT and Smad2 pathway inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease with a poor prognosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development. However, it's extremely short half-life greatly limits its applications. Previously, we reported an Ac-SDKP analog peptide in which Asp and Lys residues were replaced with D-amino acids (Ac-SDD KD P). Ac-SDD KD P exhibits better resistance to angiotensin-1-converting enzyme (ACE)-mediated degradation and a longer half-life than Ac-SDKP in rat and human sera. The objective of this study was to explore the potential application of Ac-SDD KD P for the treatment of IPF and to clarify the underlying mechanisms. We found that Ac-SDD KD P exerted similar antifibrotic effects as Ac-SDKP on human fetal lung fibroblast-1 (HFL-1) proliferation, α-smooth muscle actin (α-SMA), collagen I and collagen III expression, and Smad-2 phosphorylation in vitro. In vivo, Ac-SDD KD P exhibited significantly greater protective effects against bleomycin-induced pulmonary fibrosis than Ac-SDKP in mice. α-SMA, CD45, collagen I and collagen III expression, and Smad-2 phosphorylation were significantly decreased in the lungs of Ac-SDD KD P-treated but not Ac-SDKP-treated mice. Furthermore, a pull-down experiment was used to screen for molecules that interact with Ac-SDKP. Co-immunoprecipitation (Co-IP) and computer-based molecular docking experiments demonstrated an interaction between Ac-SDKP or Ac-SDD KD P (Ac-SDKP/Ac-SDD KD P) and serine/arginine-rich protein-specific kinase 1 (SRPK1) that caused inhibition SRPK1-mediated phosphatidylinositol-3 kinase/ serine/threonine kinase (PIK3/AKT) signaling pathway activation and Smad2 phosphorylation and thereby attenuated lung fibrosis. Our data suggest that long-acting Ac-SDD KD P may potentially be an effective drug for the treatment of pulmonary fibrosis. The interacting molecule and antifibrotic mechanism of Ac-SDKP/Ac-SDD KD P were also identified, providing an experimental and theoretical foundation for the clinical application of the drug.

Distribution and difference of APOBEC-induced mutations in the TpCpW context of HBV DNA between HCC and non-HCC.

Hepatitis B virus (HBV) DNA is vulnerable to editing by human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. However, the distribution of APOBEC-induced mutations on HBV DNA is not well characterized. To this end, we obtained the HBV DNA sequence of HBV-infected individuals with and without hepatocellular carcinoma (HCC and non-HCC groups, respectively) from NCBI database and calculated the rapo values of APOBEC-induced TpCpW→TpKpW mutation prevalence in HBV DNA. The results showed that the APOBEC-induced mutations were mainly distributed in the minus strand of non-HCC-derived HBV DNA (rapo = 2.04), while the mutation on the plus-strand was weaker (rapo = 0.99). There were high APOBEC-induced mutation regions in the minus strand of HBV DNA 1 to 1000 nucleotides (nts) region and in the plus-strand of HBV DNA 1000 to 1500 nts region; the mutations in the 1 to 1000 nts region were mainly TpCpW→TpTpW mutation types (total T/G: 111/18) and a number of these were missense mutations (missense/synonymous: 35/94 in P gene, 17/15 in S gene, and 5/10 in X gene). The difference between minus to plus-strand rapo of HCC-derived HBV DNA (1.96) was greater than that of the non-HCC group (1.05). The minus-strand rapo of HCC-derived HBV DNA regions 1000 to1500nts and 1500 to 2000 nts (rapo = 4.2 and 4.2) was also higher than that of the same regions of non-HCC-derived HBV DNA (rapo = 1.2 and 1.1). Finally, the ratio of minus to plus-strand rapo was used to distinguish HCC-derived HBV DNA from non-HCC-derived HBV DNA. This study unraveled the distribution characteristics of APOBEC-induced mutations on double strands of HBV DNA from HCC and non-HCC samples. Our findings would help understand the mechanism of APOBECs on HBV DNA and may provide important insights for the screening of HCC.

Dynamic Halbach array magnet integrated microfluidic system for the continuous-flow separation of rare tumor cells.

Circulating tumor cells (CTCs), the most representative rare cells in peripheral blood, have received great attention due to their clinical utility in liquid biopsy. The downstream analysis of intact CTCs isolated from peripheral blood provides important clinical information for personalized medicine. However, current CTC isolation and detection methods have been challenged by their extreme rarity and heterogeneity. In this study, we developed a novel microfluidic system with a continuously moving Halbach array magnet (dHAMI microfluidic system) for negative isolation CTCs from whole blood, which aimed to capture non-target white blood cells (WBCs) and elute target CTCs. The dynamic and continuous movement of the Halbach array magnet generated a continuous magnetic force acting on the magnetic bead-labelled WBCs in the continuous-flow fluid to negatively exclude the WBCs from the CTCs. Furthermore, the continuously moving magnetic field effectively eliminated the effect of magnetic bead aggregation on the fluid flow to realize the continuous-flow separation of the CTCs without a sample loading volume limitation. The experimental procedure for CTC negative isolation using the dHAMI microfluidic system could be completed within 40 min. Under the optimized experimental conditions of the dHAMI microfluidic system, including the flow rate and concentration of the immunomagnetic bead, the average CTC capture rate over a range of spiked cell numbers (50-1000 cancer cells per mL) was up to 91.6% at a flow rate of 100 µL min-1. Finally, the CTCs were successfully detected in 10 of 10 (100%) blood samples from patients with cancer. Therefore, the dHAMI microfluidic system could effectively isolate intact and heterogeneous CTCs for downstream cellular and molecular analyses, and this robust microfluidic platform with an excellent magnetic manipulation performance also has great application potential for the separation of other rare cells.

[Expression and activity identification of APOBEC3A-HBc fusion protein].

Objective To construct the expression vector of the fusion protein of apolipoprotain B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) and hepatitis B virus core proteins (HBc) with different sequences, and identify its expression, intracellular localization and cytosine deamination activity. Methods The APOBEC3A gene and the coding sequence of HBc and four kinds of truncated HBc containing C-termimal domain were amplified by PCR. The APOBEC3A with full-length HBc antigen or four kinds of truncated HBc were cloned into a eukaryotic expression vector pcDNA3.0 by In-Fusion method, and confirmed by DNA sequencing. The recombinants were then transfected into HEK293T cells. The expression and localization of the fusion proteins were detected by Western blotting and immunofluorescence cytochemistry. The cytosine deamination activity was analyzed by electrophoresis on a urea denaturing polyacrylamide gel. Results Five kinds of fusion protein were expressed in HEK293T cells successfully, and the expression of the vectors containing the truncated HBc were higher than that of the APOBEC3A-HBc vector containing the full-length HBc. The fusion protein of APOBEC3A-HBc was mainly expressed in the cytoplasm of HEK293T cells, and the fusion proteins of APOBEC3A-HBc144S, APOBEC3A-HBc144E, APOBEC3A-HBc144AAA, APOBEC3A-HBc144A were expressed mainly in the nuclei of HEK293T cells. The cytosine deamination activity of the fusion proteins containing the truncated HBc was higher than that of the fusion protein APOBEC3A-HBc. Conclusion The fusion protein expression vectors of APOBEC3A and HBc with different lengths and sequences have been constructed successfully. The expressing ability, intracellular localization and cytosine deamination activity are obviously different between the fusion protein APOBEC3A-HBc and the fusion proteins containing the truncated HBc.

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H2O2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H2O2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H2O2. Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

[Cloning,expression and activity identification of human innate immune protein apolipoprotein B mRNA editing enzyme catalytic subunit 3A(APOBEC3A)].

Objective: To construct the expression vector of apolipoprotein B mRNA editing enzyme catalytic subunit 3A( APOBEC3A),express APOBEC3 A in eukaryotic cells and identify its cytosine deaminase activity. Methods: The APOBEC3 A gene was obtained by PCR and inserted into the eukaryotic expression vector pc DNA3. 0( +). The recombinant vector pc DNA3. 0-APOBEC3 A was then transfected into HEK293 T and Hep G2 cells after confirmed by DNA sequencing. The recombinant protein was purified by Ni-NTA His Bind affinity column. Western blot analysis was used to detect the expression of APOBEC3 A protein. The localization of APOBEC3 A protein in HEK293 T and Hep G2 cel s was identified by immunofluorescence cytochemistry. The deaminase activity of APOBEC3 A protein was characterized by fluorescence polarization. Results: DNA sequencing confirmed that APOBEC3 A gene( 600 bp) was inserted into pc DNA3. 0-APOBEC3 A,which was expressed in HEK293 T and Hep G2 cells successfully. APOBEC3 A protein was mainly expressed in cytoplasm of HEK293 T cells and cytoplasm and nuclei of Hep G2 cells. APOBEC3 A protein showed cytosine deaminase activity on the TTCA sequence in single-stranded DNA. Conclusion: The study constructed successfully APOBEC3 A eukaryotic expression vector,identified the differential expression of APOBEC3 A protein in HEK293 T and Hep G2 cells,and confirmed that the APOBEC3 A protein had cytosine deaminase activity.

The hepatitis B virus (HBV) core protein enhances the transcription activation of CRE via the CRE/CREB/CBP pathway.

We previously reported that hepatitis B virus core protein (HBc) can bind to the Enhancer I (Enh I) domain and can accumulate with transcription coactivator cAMP response element (CRE). This raises the possibility that HBc may interact with CRE/CREB and regulate CRE transcription activation. In this study, we investigated the function and mechanisms of HBc in regulating CRE transcriptional activation using the HepG2 cell line. Our results showed the following: (1) HBc expression significantly increases HBV CRE transcriptional activation; (2) phosphorylation of the serine residues in the arginine-rich domain (ARD) of HBc protein impacts the function of transcriptional activation by the CRE; (3) HBc protein significantly increases HBV CRE transcriptional activation following forskolin treatment; (4) HBc nonspecifically binds to CRE and enhances the binding of the cAMP response element-binding protein (CREB) to CRE; and (5) HBc increases the concurrent accumulation of CREB and CBP at the CRE region. HBc activates Enh I through its binding to CRE, increasing the concurrent accumulation of CREB/CBP on CRE, and thus increases CRE transcriptional activation.

A novel IgM-H-ficolin complement pathway to attack allogenic cancer cells in vitro.

The pentameric serum IgMs are critical to immune defense and surveillance through cytotoxicity against microbes and nascent cancer cells. Ficolins, a group of oligomeric lectins with an overall structure similar to C1q and mannose-binding lectin (MBL) participate in microbe infection and apoptotic cell clearance by activating the complement lectin pathway or a primitive opsonophagocytosis. It remains unknown whether serum IgMs interplay with ficolins in cancer immunosurveillance. Here we report a natural cancer killing of different types of cancer cells by sera from a healthy human population mediated by a novel IgM-H-ficolin complement activation pathway. We demonstrate for the first time that H-ficolin bound to a subset of IgMs in positive human sera and IgM-H-ficolin deposited on cancer cells to activate complement attack in cancer cells. Our data suggest that the IgM-H-ficolin -mediated complement activation pathway may be another defensive strategy for human cancer immunosurveillance.

An aptamer-based immunoassay in microchannels of a portable analyzer for detection of microcystin-leucine-arginine.

The rapid detection of microcystin-leucine-arginine (MC-LR), the most highly toxic among MCs, is significantly important to environmental and human health protection and prevention of MC-LR from being used as a bioweapon. Although aptamers offer higher affinity, specificity, and stability with MC-LR than antibodies in the immunodetection of MC-LR due to steric hindrance between two antibodies and limited epitopes of MC-LR for use in a sandwich immunoassay, no sandwich immunoassay using an aptmer has been developed for MC-LR detection. This study is aimed at developing an aptamer-antibody immunoassay (AAIA) to detect MC-LR using a portable analyzer. The aptamers were immobilized onto the glass surface of a microchamber to capture MC-LR. MC-LR and horseradish peroxidase (HRP)-labeled antibody were pulled into the microchamber to react with the immobilized aptamer. The chemiluminescence (CL) catalyzed by HRP was tested by a photodiode-based portable analyzer. MC-LR at 0.5-4.0 µg/L was detected quantitatively by the AAIA, with a CL signal sensitivity of 0.3 µg/L. The assay took less than 35 min for a single sample and demonstrated a high specificity, detecting only MC-LR, but not MC-LA, MC-YR, or nodularin-R. The recovery of two spiked real environmental samples calculated as 94.5-112.7%. Therefore, this AAIA was proved to be a rapid and simple method to detect MC-LR in the field by a single analyst.

miR-99b-targeted mTOR induction contributes to irradiation resistance in pancreatic cancer.

BACKGROUND: Radiation exerts direct antitumor effects and is widely used in clinics, but the efficacy is severely compromised by tumor resistance. Therefore uncovering the mechanism of radioresistance might promote the development of new strategies to overcome radioresistance by manipulating activity of the key molecules. METHODS: Immunohistochemistry were used to find whether mTOR were over-activated in radioresistant patients' biopsies. Then Western blot, real-time PCR and transfection were used to find whether radiotherapy regulates the expression and activity of mTOR by modulating its targeting microRNA in human pancreatic cancer cell lines PANC-1, Capan-2 and BxPC-3. Finally efficacy of radiation combined with mTOR dual inhibitor AZD8055 was assessed in vitro and in vivo. RESULTS: Ionizing radiation promoted mTOR expression and activation in pancreatic cancer cells through reducing miR-99b expression, which negatively regulated mTOR. Novel mTOR inhibitor, AZD8055 (10 nM, 100 nM, 500 nM) synergistically promoted radiation (0-10 Gy) induced cell growth inhibition and apoptosis. In human pancreatic cancer xenografts, fractionated radiation combined with AZD8055 treatment further increased the anti-tumor effect, the tumor volume was shrinked to 278 mm3 after combination treatment for 3 weeks compared with single radiation (678 mm3) or AZD8055 (708 mm3) treatment (P < 0.01). CONCLUSIONS: Our data provide a rationale for overcoming radio-resistance by combined with mTOR inhibitor AZD8055 in pancreatic cancer therapy.

Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions.

BACKGROUND: The core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes' expression. RESULTS: Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS). CONCLUSION: HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state.

HBV covalently closed circular DNA (cccDNA) is the template for the transcription of HBV. HBV core protein (HBc) is a main component of the HBV cccDNA minichromosome. However, the function of HBc in cccDNA is not fully understood. In light of recent findings that HBV cccDNA may be regulated epigenetically, we analyzed the binding of HBc to cccDNA and the impact of HBc on cccDNA epigenetic profile in the liver biopsy samples of 22 patients with chronic Hepatitis B (CHB). We found that HBc binding to HBV cccDNA occurred preferentially at CpG island 2, an important region for the regulation of HBV transcription. Furthermore, the relative abundances of HBc binding to CpG island 2 were positively correlated with the ratios of relaxed circular DNA to cccDNA and the levels of serum HBV DNA in those patients. Interestingly, the relative abundances of HBc binding to CpG island 2 were associated with the binding of CREB binding protein (CBP) and with hypomethylation in CpG island 2 of HBV cccDNA minichromosomes. However, relatively higher amounts of HBc binding to CpG island 2 of cccDNA were accompanied by lower amounts of HDAC1 binding. Multivariate analysis revealed that the abundances of HBc binding to CpG island 2 of cccDNA and positive HBeAg were independent factors associated with the replication of HBV (p = 0.001 for both). Apparently, HBc is a positive regulator of HBV transcription and replication, maintaining the permissive epigenetic state in the critical region of the HBV cccDNA minichromosomes.

Soluble MHC I and soluble MIC molecules: potential therapeutic targets for cancer.

It has become clear that soluble MHC I (sMHC I) and soluble MIC (sMIC), which are highly elevated in sera of cancer patients, can be viewed to be tolerogenic, and that metalloproteinases are involved in their generation process. In this review, an overview is provided of the recent progress made in the sMHC I and sMIC fields, with emphasis on their structure, formation, and function, and the key-questions that still await answers are addressed. Understanding better their formation mechanism, it will become more feasible to modulate the immune responses in cancer patients by targeting molecules involved in their generation process.

O6-Methylguanine-DNA methyltransferase gene promoter methylation detection in glioma tumors by a novel fluorescence polarization assay.

O6-Methylguanine-DNA methyltransferase methylation status has a very good predictive value for benefit from alkylating agent therapy. The stratified therapy assignment of patients according to the O6-Methylguanine-DNA methyltransferase methylation status requires a standardized diagnostic test. A novel method detecting the promoter methylation status of O6-Methylguanine-DNA methyltransferase in tissue samples by a fluorescence polarization assay was developed. A pair of primers was used to amplify a 266 bp fragment in the promoter region of the O6-Methylguanine-DNA methyltransferase gene. Two probes specific for either methylated or unmethylated DNA labeled with different fluorophores hybridized with their target amplicons, and the hybridization increased the fluorescence polarization values. The methylation status was determined by the increased fluorescence polarization values. Ninety-seven glioma tumor samples were analyzed in parallel with the new assay and the nested gel-based methylation-specific polymerase chain reaction assay. The results of the methylation status of the fluorescence polarization assay were in good concordance with the results obtained with the nested gel-based methylation-specific polymerase chain reaction assay. The sensitivity and stability of the fluorescence polarization assay have been measured. The coefficient of variation of the reproducibility for the fluorescence polarization assay was <10%. The minimum detection level established with the fluorescence polarization assay was 20 copies/µL. The fluorescence polarization assay allowed the discrimination of the O6-Methylguanine-DNA methyltransferase methylation status at individual CpG sites directly in the solution without the 2-step approach with nested primers.

A novel fluorescence polarization based assay for 14 human papillomavirus genotypes in clinical samples.

A specific and practical method was developed for high throughput 14 human papillomavirus (HPV) genotypes assay in clinical samples by a single PCR. GP5+/6+ polymerase chain reaction (PCR) system was used to amplify HPV DNA in 1127 samples. The PCR product was assayed by AcycloPrime reaction with fluorescence polarization (FP). Fourteen HPV genotypes specific sequence primers designed within GP5+/GP6+ amplification polymorphism regions of L1 genes for corresponding HPV genotypes were annealed with the type specific PCR products and special fluorescent terminator was added to the end of the primer under direction of the PCR products. AcycloPrime-FP analysis showed specific anneal and incorporation without any cross-reaction. The types detected with FP showed an excellent overall agreement with sequence when the individual monotype results were taken into account. The proposed method could detect more than one type of HPV infection, but the sequence method was limited. AcycloPrime-FP could reach the detection level: 100 ag for representative phylogenetically distant HPV genotypes: HPV6, 18, 31, 39, 42, 51 and 58. The results of AcycloPrime-FP showed excellent reproducibility. The proposed method allowed an economical detection of HPV genotypes without any use of labeled probe. It is expected to be an extremely useful tool for HPV genotypes screening.

A microarray-based gastric carcinoma prewarning system.

AIM: To develop a microarray-based prewarning system consisting of gastric cancer chip, prewarning data and analysis software for early detection of gastric cancer and pre-cancerous lesions. METHODS: Two high-density chips with 8,464 human cDNA sites were used to primarily identify potential genes specific for normal gastric mucosa, pre-cancerous lesion and gastric cancer. The low-density chips, composed of selected genes associated with normal gastric mucosa, precancerous lesion and gastric cancer, were fabricated and used to screen 150 specimens including 60 specimens of gastric cancer, 60 of pre-cancerous tissues and 30 of normal gastric mucosa. CAD software was used to screen out the relevant genes and their critical threshold values of expression levels distinguishing normal mucosa from pre-cancerous lesion and cancer. All data were stored in a computer database to establish a prewarning data library for gastric cancer. Two potential markers brcaa1 and ndr1 were identified by Western blot and immunohistochemistry. RESULTS: A total of 412 genes associated with three stages of gastric cancer development were identified. There were 216 genes displaying higher expression in gastric cancer, 85 genes displaying higher expression in pre-cancerous lesion and 88 genes displaying higher expression in normal gastric mucosa. Also 15 genes associated with metastasis of gastric cancer and 8 genes associated with risk factors were screened out for target genes of diagnosis chip of early gastric cancer. The threshold values of 412 selected genes to distinguish gastric cancer, pre-cancerous lesion from normal gastric mucosa were defined as 6.01+/-2.40, 4.86+/-1.94 and 5.42+/-2.17, respectively. These selected 412 genes and critical threshold values were compiled into an analysis software, which can automatically provide reports by analyzing the results of 412 genes obtained by examining gastric tissues. All data were compiled into a prewarning database for gastric cancer by CGO software. Northern blot and immunohistochemistry analysis confirmed that gene and protein of brcaa1 displayed lower expression in normal gastric mucosa and higher expression in gastric cancer tissues, conversely, ndr1 displayed lower expression in gastric cancer and higher expression in normal gastric mucosa. CONCLUSION: The microarray-based prewarning system for gastric cancer was developed. This system consisted of gastric cancer-associated gene chip, prewarning data and analysis software, which has a high potential for applications in the early detection of gastric cancer. The two potential markers brcaa1 and ndr1 identified may be used to distinguish cancer status fand non-cancer status.