Multiplex identification of sepsis-causing Gram-negative pathogens from the plasma of infected blood.

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram-negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram-negative pathogens and also validated whether our system can identify Gram-negative pathogens with the cell-free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram-negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX-M group 1 to identify the ESBL producing Gram-negative pathogens. All six target-specific peaks were clearly separated without any non-specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram-negative clinical isolates, all of them were successfully identified without any non-specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis-causing, drug-resistant Gram-negative pathogens and also the major ESBL producing Gram-negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram-negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.

A missense mutation in EBF2 was segregated with imperforate anus in a family across three generations.

The etiology of imperforate anus, a major phenotype of anorectal malformation (ARM), is still unknown and not a single gene has been reported to be associated with it. We studied a Korean family with six affected members with imperforate anus across three generations by whole exome sequencing and identified a missense mutation in the EBF2 gene (c.215C > T; p.Ala72Val). This mutation is completely segregated with the disease phenotype in the family and is evolutionarily highly conserved among diverse vertebrates. Also, this mutation was predicted to be functionally damaging. These results support that missense mutation in the EBF2 c.215C > T (p.Ala72Val) is very likely to contribute to the pathogenesis of ARM in this family.

Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system.

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood.

Association of common variants in the calcium-sensing receptor gene with serum calcium levels in East Asians.

Calcium is a universal intracellular messenger that has an important role in controlling various cellular processes. In this study, we explored genetic polymorphisms to identify novel loci influencing serum calcium levels in East Asians through a two-stage genome-wide association study with the sample of 8642 unrelated Koreans (4558 for discovery and 4093 for replication). Using single-nucleotide polymorphism (SNP) arrays, we discovered 963 associated SNPs in stage 1, and replicated 105 SNPs among them in stage 2. We examined them in a combined set of stage 1 and 2 samples and observed that 65 SNPs were significantly associated with serum calcium levels. Among them, rs13068893 in the CASR gene showed the strongest significance (P=3.85 × 10(-8)). Considering the high allele frequency and significance level of the rs13068893C>G in the CASR gene, this SNP may have a key role in regulating the serum calcium level. We also successfully replicated the four loci (CASR, CSTA, DGKD and GCKR) using our data set that have been previously reported to be significantly associated with calcium levels in Europeans and Indians. Further studies with more East Asian subjects or meta-analyses on them may enable validation of our results and identification of novel genetic loci associated with serum calcium levels.

Deletion variants of RABGAP1L, 10q21.3, and C4 are associated with the risk of systemic lupus erythematosus in Korean women.

OBJECTIVE: Several copy number variations (CNVs) have been found to be associated with systemic lupus erythematosus (SLE) through the target gene approach. However, genome-wide features of CNVs and their role in the risk of SLE remain unknown. The aim of this study was to identify SLE-associated CNVs in Korean women. METHODS: Genome-wide assessments of CNVs were performed in 382 SLE patients and 191 control subjects, using an Illumina HumanHap610 BeadChip genotyping platform. SLE-associated CNV regions that were identified by genome-wide association study (GWAS) were replicated in quantitative polymerase chain reaction (PCR) and deletion-typing PCR analyses in an independent sample set comprising 564 SLE patients and 511 control subjects. RESULTS: Of 144 common CNV regions, 3 deletion-type CNV regions in 1q25.1, 8q23.3, and 10q21.3 were found to be significantly associated with SLE by GWAS analysis. In the independent replication, the CNV regions in 1q25.1 (RABGAP1L) and 10q21.3 were successfully replicated (odds ratio [OR] 1.30, P=0.038 and OR 1.90, P=3.6×10(-5), respectively), and the associations were confirmed again by deletion-typing PCR. The CNV region in the C4 gene, which showed a potential association in the discovery stage, was included in the replication analysis and was found to be significantly associated with the risk of SLE (OR 1.88, P=0.01). Through deletion-typing PCR, the exact sizes and breakpoint sequences of the deletions were defined. Individuals with the deletions in all 3 loci (RABGAP1L, 10q21.3, and C4) had a much higher risk of SLE than did those without any deletions in the 3 loci (OR 5.52, P=3.9×10(-4)). CONCLUSION: These CNV regions can be useful to identify the pathogenic mechanisms of SLE, and might be used to more accurately predict the risk of SLE by taking into consideration their synergistic effects on disease susceptibility.

Polymorphisms in PDE4D are associated with a risk of COPD in non-emphysematous Koreans.

Despite extensive effort, only a few chronic obstructive pulmonary disease (COPD)-associated genes have been suggested, indicating that there must be additional risk-associated loci. Here we aimed to identify additional COPD-associated SNPs and to explore the potential relationship between COPD subgroups and the SNPs in the Korean population. We performed a genome-wide association study (GWAS) with 990 Korean individuals; 102 COPD cases and 544 controls for GWAS using Affymetrix SNP array 5.0, and 173 COPD cases and 171 controls for replication. After validating the candidate single nucleotide polymorphisms (SNP), we performed subgroup analysis by disease phenotype. Through GWAS, we identified a novel SNP in the phosphodiesterase-4D (PDE4D) gene [rs16878037 (C>T), p = 1.66 ◊ 10(-6)] that was significantly associated with COPD. This signal in PDE4D was successfully replicated in the independent set (p = 0.041). When we combined the discovery and replication data, the association signal became more significant (p = 5.69 ◊ 10(-7)). In the COPD subgroup analysis, the T allele of rs16878037 was significantly more frequent in COPD patients without severe diffusion capacity impairment (mild mixed and obstruction-dominant group) than in patients with severe impairment (severe mixed and emphysema-dominant groups). This result supports that PDE4D polymorphisms might be involved in the susceptibility to COPD especially in non-emphysematous individuals and that they could also affect the responsiveness of the PDE4 inhibitor treatment.

De novo copy number variations in cloned dogs from the same nuclear donor.

BACKGROUND: Somatic mosaicism of copy number variants (CNVs) in human body organs and de novo CNV event in monozygotic twins suggest that de novo CNVs can occur during mitotic recombination. These de novo CNV events are important for understanding genetic background of evolution and diverse phenotypes. In this study, we explored de novo CNV event in cloned dogs with identical genetic background. RESULTS: We analyzed CNVs in seven cloned dogs using the nuclear donor genome as reference by array-CGH, and identified five de novo CNVs in two of the seven clones. Genomic qPCR, dye-swap array-CGH analysis and B-allele profile analysis were used for their validation. Two larger de novo CNVs (5.2 Mb and 338 Kb) on chromosomes X and 19 in clone-3 were consistently validated by all three experiments. The other three smaller CNVs (sized from 36.1 to 76.4 Kb) on chromosomes 2, 15 and 32 in clone-3 and clone-6 were verified by at least one of the three validations. In addition to the de novo CNVs, we identified a 37 Mb-sized copy neutral de novo loss of heterozygosity event on chromosome 2 in clone-6. CONCLUSIONS: To our knowledge, this is the first report of de novo CNVs in the cloned dogs which were generated by somatic cell nuclear transfer technology. To study de novo genetic events in cloned animals can help understand formation mechanisms of genetic variants and their biological implications.

CNVRuler: a copy number variation-based case-control association analysis tool.

SUMMARY: The method for genome-wide association study (GWAS) based on copy number variation (CNV) is not as well established as that for single nucleotide polymorphism (SNP)-GWAS. Although there are several tools for CNV association studies, most of them do not provide appropriate definitions of CNV regions (CNVRs), which are essential for CNV-association studies. Here we present a user-friendly program called CNVRuler for CNV-association studies. Outputs from the 10 most common CNV defining algorithms can be directly used as input files for determining the three different definitions of CNVRs. Once CNVRs are defined, CNVRuler supports four kinds of statistical association tests and options for population stratification. CNVRuler is based on the open-source programs R and Java from Sun Microsystems. AVAILABILITY: CNVRuler software is available with an online manual at the website, www.ircgp.com/CNVRuler/index.html.

Genome-wide association study of lung cancer in Korean non-smoking women.

Lung cancer in never-smokers ranks as the seventh most common cause of cancer death worldwide, and the incidence of lung cancer in non-smoking Korean women appears to be steadily increasing. To identify the effect of genetic polymorphisms on lung cancer risk in non-smoking Korean women, we conducted a genome-wide association study of Korean female non-smokers with lung cancer. We analyzed 440,794 genotype data of 285 cases and 1,455 controls, and nineteen SNPs were associated with lung cancer development (P < 0.001). For external validation, nineteen SNPs were replicated in another sample set composed of 293 cases and 495 controls, and only rs10187911 on 2p16.3 was significantly associated with lung cancer development (dominant model, OR of TG or GG, 1.58, P = 0.025). We confirmed this SNP again in another replication set composed of 546 cases and 744 controls (recessive model, OR of GG, 1.32, P = 0.027). OR and P value in combined set were 1.37 and < 0.001 in additive model, 1.51 and < 0.001 in dominant model, and 1.54 and < 0.001 in recessive model. The effect of this SNP was found to be consistent only in adenocarcinoma patients (1.36 and < 0.001 in additive model, 1.49 and < 0.001 in dominant model, and 1.54 and < 0.001 in recessive model). Furthermore, after imputation with HapMap data, we found regional significance near rs10187911, and five SNPs showed P value less than that of rs10187911 (rs12478012, rs4377361, rs13005521, rs12475464, and rs7564130). Therefore, we concluded that a region on chromosome 2 is significantly associated with lung cancer risk in Korean non-smoking women.

Copy number variations in East-Asian population and their evolutionary and functional implications.

Recent discovery of the copy number variation (CNV) in normal individuals has widened our understanding of genomic variation. However, most of the reported CNVs have been identified in Caucasians, which may not be directly applicable to people of different ethnicities. To profile CNV in East-Asian population, we screened CNVs in 3578 healthy, unrelated Korean individuals, using the Affymetrix Genome-Wide Human SNP array 5.0. We identified 144,207 CNVs using a pooled data set of 100 randomly chosen Korean females as a reference. The average number of CNVs per genome was 40.3, which is higher than that of CNVs previously reported using lower resolution platforms. The median size of CNVs was 18.9 kb (range 0.2-5406 kb). Copy number losses were 4.7 times more frequent than copy number gains. CNV regions (CNVRs) were defined by merging overlapping CNVs identified in two or more samples. In total, 4003 CNVRs were defined encompassing 241.9 Mb accounting for approximately 8% of the human genome. A total of 2077 CNVRs (51.9%) were potentially novel. Known CNVRs were larger and more frequent than novel CNVRs. Sixteen percent of the CNVRs were observed in > or =1% of study subjects and 24% overlapped with the OMIM genes. A total of 476 (11.9%) CNVRs were associated with segmental duplications. CNVS/CNVRs identified in this study will be valuable resources for studying human genome diversity and its association with disease.

Stuffer-free multiplex ligation-dependent probe amplification based on conformation-sensitive capillary electrophoresis: a novel technology for robust multiplex determination of copy number variation.

Developing diagnostic tools based on the application of known disease/phenotype-associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user-friendly multiplex CNV detection method, termed stuffer-free MLPA-CE-SSCP, that combines a variation of multiplex ligation-dependent probe amplification (MLPA) with CE-SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar-sized stuffer-free MLPA products, we adopted CE-SSCP rather than length-dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X-chromosome copy number (1-5) revealed that copy number determinations using stuffer-free MLPA-CE-SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.

Simultaneous copy number gains of NUPR1 and ERBB2 predicting poor prognosis in early-stage breast cancer.

BACKGROUND: The full extent of chromosomal alterations and their biological implications in early breast carcinogenesis has not been well examined. In this study, we aimed to identify chromosomal alterations associated with poor prognosis in early-stage breast cancers (EBC). METHODS: A total of 145 EBCs (stage I and II) were examined in this study. We analyzed copy number alterations in a discovery set of 48 EBCs using oligoarray-comparative genomic hybridization. In addition, the recurrently altered regions (RARs) associated with poor prognosis were validated using an independent set of 97 EBCs. RESULTS: A total of 23 RARs were defined in the discovery set. Six were commonly detected in both stage I and II groups (> 50%), suggesting their connection with early breast tumorigenesis. There were gains on 1q21.2-q21.3, 8q24.13, 8q24.13-21, 8q24.3, and 8q24.3 and a loss on 8p23.1-p22. Among the 23 RARs, copy number gains on 16p11.2 (NUPR1) and 17q12 (ERBB2) showed a significant association with poor survival (P = 0.0186 and P = 0.0186, respectively). The patients simultaneously positive for both gains had a significantly worse prognosis (P = 0.0001). In the independent replication, the patients who were double-positive for NUPR1-ERBB2 gains also had a significantly poorer prognosis on multivariate analysis (HR = 7.31, 95% CI 2.65-20.15, P = 0.0001). CONCLUSIONS: The simultaneous gain of NUPR1 and ERBB2 can be a significant predictor of poor prognosis in EBC. Our study will help to elucidate the molecular mechanisms underlying early-stage breast cancer tumorigenesis. This study also highlights the potential for using combinations of copy number alterations as prognosis predictors for EBC.

Strategy for high-fidelity multiplex DNA copy number assay system using capillary electrophoresis devices.

Structural variation of human genome such as duplications and deletions, collectively termed copy number variation (CNV), is one of the major genetic variations. Reliable and efficient measurement of CNV will be essential to develop diagnostic tools for CNV-related diseases. We established a strategy based on multiplex PCR and capillary electrophoresis (CE) for reliable CNV assay. Multiplex-PCR was performed using five primer sets for target loci and a diploid control (DC). We designed primers satisfying three conditions: different size of each PCR product for CE separation, unified annealing temperature for multiplex PCR, and suitability for quantitative PCR (qPCR). We defined the accurate PCR cycles for quantification of copy numbers at which the amplifications for all targets were supposed to be exponential, named maximum doubling cycle. CE was carried out with PCR product and the ratio of the peak areas (target/diploid control) was calculated. Our multiplex PCR-CE analysis reliably determined copy numbers of X chromosome with variable copies ranging from 1 to 5 and showed higher reliability than qPCR (correlation coefficient 0.996 versus 0.898). When measuring the six randomly selected autosomal CNV targets using our multiplex PCR-CE, the results agreed with those from qPCR. In addition, our strategy was validated for the broad application to commonly used CE devices. Taken together, this assay will be useful for accurate analysis of multiple disease-associated CNVs in a clinical setting.

Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma.

BACKGROUND: Since hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms. METHODS: TPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays. RESULTS: Silencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells. CONCLUSIONS: Based on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway.

PathCluster: a framework for gene set-based hierarchical clustering.

MOTIVATION: Gene clustering and gene set-based functional analysis are widely used for the analysis of expression profiles. The development of a comprehensive method jointly combining the two methods would allow for greater biological insights. RESULTS: We developed a software package, PathCluster for gene set-based clustering via an agglomerative hierarchical clustering algorithm. The distances between predefined gene sets are illustrated in a dendrogram in which the relationships between gene sets can be visually assessed. Valuable biological insights can be obtained according to the type of gene sets, e.g. coordinated action of molecular functions (functional gene sets) and putative motif synergy (promoter gene set) in a biological process. The combined use of gene sets further enables the interrogation of different biological themes and their putative relationships, such as function-versus-regulatory motif or drug-versus-function. PathCluster can also be used for knowledge-based sample partitioning or class categorization for clinical purposes. With extended applicability, PathCluster will facilitate the gleaning of meaningful biological insights and testable hypotheses in the contexts of given expression profiles. AVAILABILITY: PathCluster executable files can be freely downloaded at http://www.systemsbiology.co.kr/PathCluster/.

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array.

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q.

To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor-related genes, using whole-genome array-CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high-grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1-q32.1 (64.5%), 1q32.1-q44 (59.2%), 8q11.21-q24.3 (48.7%) and a loss in 17p13.3-p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2-p21.1 and gain in 8q11.21-q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC.

Trends in cervical cancer mortality in Korea 1993-2002: corrected mortality using national death certification data and national cancer incidence data.

Cervical cancer is a major health problem for Korean women, accounting for 9.8% of new female cancer cases, even though incidence rates have been decreasing. The Korean cervical cancer mortality rate for 1993-2002 based on National Statistical Office data shows an increasing trend, but the actual rates are thought to have decreased by epidemiologists, clinicians and other cancer experts. To explain this gap and solve this problem, we corrected the number of cervical cancer deaths by comparing death certificate cases of unspecified uterine cancer data with the national cancer incidence databases of entire cancer registries in Korea. We used 2 different methods to make a correction. First, we considered "uterus, unspecified" deaths previously registered as "cervix, uterine" cases misclassified and added them to the cervical cancer deaths. Alternatively, we multiplied the total number of registered unspecified uterine cancer deaths by age-specific proportions of registered incident cervical cancer cases among all cancers and added the product to cervical cancer deaths. The overall corrected age-standardized cervical cancer mortality rates per 100,000 women decreased from 5.2 in 1993 to 3.9 in 2002 (estimated annual percentage change (EAPC): -4.05%, 95% CI: -4.88, -3.22). While cervical cancer mortality showed a decreasing tendency in women aged 30-69 years, it increased substantially in women aged > or =70 years (EAPC: 3.62%, 95% CI: 1.92-5.35). Results of this study will provide evidence-based foundation for the evaluation of the existing cervical cancer-screening programs.

Overexpression of TFF3 is involved in prostate carcinogenesis via blocking mitochondria-mediated apoptosis.

The overexpression of trefoil factor family 3 (TFF3) is observed in a variety of cancers, including prostate cancer (PCa), and its potential role in carcinogenesis, such as activating the PI3K/AKT pathway, is suggested. However, its role and its related mechanisms in prostate tumorigenesis remain unknown. To elucidate the role of TFF3 overexpression in PCa, we silenced TFF3 in two PCa cell lines that overexpressed TFF3 and explored the molecular mechanism behind its antiapoptotic role. We also examined TFF3 expression in 108 Korean PCa specimens and 106 normal prostate tissues by immunohistochemistry (IHC) analysis. The mean TFF3 IHC score in the tumor tissues was significantly higher than that in the normal tissues (4.702 vs. 0.311, P = 2.52 × 10-24). TFF3-silenced cells showed suppressed tumor cell growth and migration. TFF3 silencing decreased BCL2 and increased BAX expression. The translocation of BAX to the mitochondria was also confirmed. After TFF3 silencing, the expression of the mitochondrial proapoptotic proteins, cytochrome C and Smac/DIABLO, was elevated, and these proteins were released from the mitochondria to the cytosol. Downstream mediators of mitochondrial apoptosis, including cleaved caspase-3, caspase-9, and PARP, were also elevated. Accordingly, the proportion of apoptotic cells was significantly higher among TFF3-silenced cells. There was no difference in extrinsic apoptosis-related molecules after TFF3 silencing. All the results support that TFF3 silencing induces the downstream signaling pathway of mitochondria-mediated apoptosis. This study provides a better understanding of the mechanism of prostate tumorigenesis, suggesting TFF3 as a potential biomarker and therapeutic target of PCa.