CD14, a novel surface marker of esophageal cancer stem cells.

Studies on targeting cancer stem cells (CSCs) have not yielded satisfactory results regarding solid tumor treatments; one of the reasons for this is the difficulty associated with the identification of a relatively specific antigen in solid tumors. CD14, which is mainly expressed in certain immune cells, is associated with tumor recurrence, growth, metastasis and resistance to treatment, which is in conformity with the characteristics of CSCs. It was thus hypothesized that esophageal CSCs (ECSCs) express CD14. In the present study, paraffin­embedded sections of human esophageal carcinoma were used to determine the co­expression of CD14 and the ECSC marker aldehyde dehydrogenase­1 (ALDH1) using immunofluorescence. CD14+ cells were then isolated using immunomagnetic separation for stemness detection, including proliferation, migration, invasion and tumorigenicity. Cell Counting Kit­8 (CCK­8), EdU and colony­formation assays were utilized to investigate the proliferative ability, the metastatic capacity was examined using Transwell and wound­healing assays and a xenograft assay was performed to investigate the tumorigenic ability. It was indicated that the ALDH1­labeled ECSCs expressed CD14 and primary CD14+ cells possessed the characteristics of CSCs. On the whole, the results of the present study suggest the potential utility of CD14 as a novel surface marker for ECSCs.

Risk-associated single nucleotide polymorphisms of mitochondrial D-loop mediate imbalance of cytokines and redox in rheumatoid arthritis.

BACKGROUND: We have identified rheumatoid arthritis (RA) risk-associated single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) including the major alleles of nucleotides 195T/C, 16260C/T, and 16519C/T as well as the minor alleles of nucleotides 146T/C and 150C/T previously. OBJECTIVE: We evaluated the potential relationships of these SNPs with status for oxidative stress and inflammation cytokines. METHODS: The DNA was extracted from blood samples of RA patients, and the SNPs of DNA D-loop were verified by polymerase chain reaction amplification and sequence analysis. Serum levels of inflammatory cytokines including interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-10, and tumor necrosis factor-α (TNF-α) were determined by cytometric bead array. Plasma reactive oxygen species (ROS) levels were measured by fluorescent probe technology. RESULTS: The RA risk-related allele 16519C was significantly associated with high IFN-γ levels (100.576 ± 11.769 vs 64.268 ± 8.199, 95% confidence interval [CI] -66.317 to -6.299, P = 0.018). This allele also associated with ROS at borderline statistics level (619.295 ± 36.687 vs 526.979 ± 25.896, 95% CI -186.145 to -1.513, P = 0.054). The subsequent analysis also showed that the ROS levels were positively correlated with IFN-γ levels (R = 0.291, P = 0.002). Further analysis showed that RA patients with high C-reactive protein levels displayed a higher ROS level (P = 0.001). CONCLUSION: Our results imply that the 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA, thereby modifying RA development. REMARK: The potential relationships of RA-associated SNPs in the mitochondrial D-loop with status for oxidative stress and inflammation were evaluated. The 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA to modify RA development.

Dicer Enhances Bevacizumab-Related Inhibition of Hepatocellular Carcinoma via Blocking the Vascular Endothelial Growth Factor Pathway.

PURPOSE: Vascular endothelial growth factor (VEGF) family members contribute greatly to the development and angiogenesis of hypervascular hepatocellular carcinoma (HCC). We have previously shown that Dicer inhibited HCC growth. In this study, we aimed to determine the relationship between Dicer and VEGF in HCC. METHODS: Gain-of-function studies were performed to determine the effect of different treatments on the proliferation, migration, and invasion of HCC cells. Expression of VEGF-A in xenograft tumor tissues was analysed using Western blotting, and that of CD31 using immunohistochemical analysis. RESULTS: We found that Dicer inhibited proliferation, migration and invasion of HCC cells by suppressing VEGF-A expression. Interestingly, VEGF-A165, which is the most prominent VEGF-A isoform, counteracted Dicer-induced inhibition of HCC cells. In addition, a monoclonal anti-VEGF antibody (bevacizumab) enhanced Dicer-induced inhibition of HCC in vitro and in vivo. Further, immunohistochemical analysis of CD31 indicated bevacizumab and Dicer synergized to reduce tumor microvessel density. CONCLUSION: Our data demonstrated that Dicer enhanced bevacizumab-related inhibition of HCC cell via the VEGF pathway; therefore, Dicer in coordination with bevacizumab may provide another potential approach for HCC therapy.

Association of dietary diversity with uterine fibroids among urban premenopausal women in Shijiazhuang, China: A cross-sectional study.

BACKGROUND AND OBJECTIVES: Uterine fibroids (UFs) are the most common neoplasm affecting reproductive-age women. The purpose of the present study is to explore the association between dietary diversity and risk of UFs in a cross-sectional study of urban premenopausal women. METHODS AND STUDY DESIGN: A total of 248 urban premenopausal women with age of 20-45 were recruited in 3 randomly chosen hospitals in Shijiazhuang, China. Dietary diversity was assessed from food frequency intake data using dietary diversity score (DDS), Prime Diet Quality Score (PDQS) and food variety score (FVS). UFs were diagnosed by the methods of ultrasound, pelvic exam, or surgery. Binary logistic regression was used to estimate the relationship between dietary diversity and risk of UFs. RESULTS: 37 of the study subjects (14.9%) had UFs. Participants with a low education level and single marital status participants had a lower DDS and PDQS, respectively. After adjustment for confounding factors, a higher DDS 24 was associated with decreased UF risk (OR=0.22, 95% CI=0.05-1.01). Similar trends were observed for the plantbased FVS (ptrend=0.025). Carrot (OR=0.04, 95% CI=0.00-0.48) and kiwi fruit (OR=0.03, 95% CI=0.00-0.47) were also inversely associated with risk of UFs after adjustment for confounding factors. CONCLUSIONS: Multifarious food groups and the increase of variety of plant-based food, especially carrot and kiwi fruit, may be associated with the lower risk of UFs; they may play an important role in inhibiting the formation of UFs.

Association of an miR-502-binding site polymorphism in the 3'-untranslated region of SET8 with colorectal cancer.

The histone methyltransferase SET8 is regulated by microRNA-502 through the binding site in its 3'-untranslated region, and the rs16917496 polymorphism at the miR-502-binding site in the SET8 gene has been implicated in a number of cancer types. The rs16917496 polymorphism including CC, CT and TT genotypes was analyzed in patients with colorectal cancer; the CC genotype was identified to be independently associated with longer post-operative survival times using multivariate analysis (relative risk, 2.406; 95% confidence interval, 1.017-5.691; P=0.046). In addition, decreased SET8 expression was associated with the SET8 CC genotype and longer survival times for patients with colorectal cancer. The results of the present study indicated that miR-502 mediates SET8 expression at least partly by altering the binding affinity between miR-502 and SET8 so as to modify the colorectal cancer outcome. The results indicate that SET8 may be a novel target for colorectal cancer therapy.

Genetic polymorphisms of microRNA machinery genes predict overall survival of esophageal squamous carcinoma.

BACKGROUND: MicroRNA (miRNA)-related single nucleotide polymorphisms (miR-SNPs) in miRNA processing machinery genes are implicated in carcinogenesis, as they change the expression profiles of miRNA. Six miR-SNPs in miRNA processing machinery genes, including Dicer (rs3742330), RAN (rs14035), XPO5 (rs11077), TNRC6B (rs9623117), GEMIN3 (rs197412), and GEMIN4 (rs2740348), were evaluated for their association with esophageal squamous cell carcinoma (ESCC). METHODS: The miR-SNP of the miRNA processing genes were genotyped using the polymerase chain reaction-ligase detection reaction (PCR-LDR) assay, while the XPO5 expression levels in ESCC tissues were measured by immunochemistry methods. RESULTS: Patients carrying the rs11077 AA allele exhibited a significantly increased lifespan than AC+CC carriers, as determined by univariate and multivariate analyses (relative risk: 2.490; 95% confidence interval [CI]: 1.225-5.058; P=.012). Furthermore, the rs11077 AA genotype displayed a trend for high XPO5 expression in ESCC tissues by immunochemistry analysis, and these high XPO5 expression levels were also associated with high survival rates among ESCC patients. CONCLUSION: Our results suggested that the miRNA machinery gene expression-associated miR-SNPs would modify cancer outcomes; in this light, XPO5 may be an important new target for ESCC therapy.

A polymorphism at the microRNA binding site in the 3'-untranslated region of C14orf101 is associated with the risk of gastric cancer development.

MicroRNAs (miRNAs) bind to the 3'-untranslated regions (3'-UTRs) of mRNAs, affecting translation and regulating cell differentiation, tumorigenesis and apoptosis. Genetic polymorphisms in these regions in target genes are able to affect the binding affinity between miRNA and target genes, ultimately affecting the expression of individual miRNAs. In the present case-control study, genotyping of 5 microRNA single nucleotide polymorphisms (SNPs) located at the binding site of the 3'-UTR of RYR3 (rs1044129), C14orf101 (rs4901706), KIAA0423 (rs1053667), GOLGA7 (rs11337) and KRT81 (rs3660) genes was assessed in order to investigate its role in gastric cancer (GC). The results indicated that the rs4901706 SNP, which is located in the 3'-UTR of C14orf101, was associated with GC development risk, as determined by χ2 analysis (relative risk, 1.630; 95% confidence interval, 1.070-2.483; P=0.022). A Renilla/luciferase reporter assay also indicated the different binding affinity between the SNP of rs4901706 and microRNA. In conclusion, rs4901706 SNP of C14orf101 gene in the microRNA binding site may be used as a valuable biomarker when predicting GC risk.

miR-502 medaited histone methyltransferase SET8 expression is associated with outcome of esophageal squamous cell carcinoma.

The histone methyltransferase SET8, whose expression is regulated by miR-502 though the binding site in the 3' UTR of SET8, implicated in cancer development. Single nucleotide polymorphism (SNP) of rs16917496 located in the miR-502 and SET8 binding site was analyzed in esophageal squamous cell carcinoma (ESCC) patients, the SET8 C/C genotype was independently associated with longer post-operative survival by multivariate analysis (relative risk, 2.250; 95% CI, 1.041-4.857; p = 0.039). Moreover, the reduced SET8 expression mediated by SET8 C/C genotype was associated with longer ESCC survival. Functional assay indicated that the SET8 knock down could inhibit proliferation and promote apoptosis of ESCC cells. The subsequent assay also showed the markedly inhibition of ESCC cell migration and invasion by SET8 knock down. Our data suggested that the altering SET8 expression, which is mediated at least partly by miR-502 through changing the binding affinity between miR-502 and SET8 3' UTR, could modify the ESCC outcome by inhibiting the proliferation and invasion as well as promoting the apoptosis of ECSS cell. Our data indicated that SET8 was a new target for ESCC therapy.

Prognostic significance of Dicer expression in hepatocellular carcinoma.

Dicer is a RNaseIII endonuclease of the microRNA processing pathway, which is implicated in carcinogenesis of various types of human cancer. The present study assessed the expression level of Dicer in hepatocellular carcinoma (HCC) tissue to evaluate its association with HCC tumorigenesis. A low expression of Dicer was significantly associated with a shorter postoperative survival time of patients with HCC, which was assessed using the log-rank test with Kaplan-Meier survival analysis. Multivariate analysis identified that Dicer expression was an independent predictor for HCC outcome (relative risk, 0.660; 95% confidence interval, 0.506-0.861; P=0.002). A functional assay demonstrated that Dicer overexpression inhibited the proliferation and promoted the apoptosis of HCC cells. In addition, a Transwell assay revealed that Dicer markedly inhibited the migration and invasion of HCC cells. The present findings indicate that Dicer expression modified the outcomes of HCC patients by inhibiting proliferation, promoting apoptosis and inhibiting metastasis of HCC cells.

Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with colorectal cancer outcome.

Single nucleotide polymorphisms (SNPs) in the displacement loop (D-Loop) of mitochondrial DNA (mtDNA) has been identified for their association with the risk and outcome in many cancers. We have identified risk associated D-loop SNPs for colorectal cancer previously, in the present study, we evaluate their prognostic value for postoperative survival of colorectal cancer (CRC). The minor haplotype of nucleotides 16290T and frequent haplotype of nucleotide 16298T in the hypervariable segment 1 (HV1) region of the D-loop were identified for their association with high survival rate of CRC. After adjusted with COX proportional hazard model, the nucleotide site of 16290 was identified as independent predictor for CRC (RR, 0.379; 95% CI, 0.171-0.839; p = 0.017). In conclusion, SNPs in the mtDNA D-Loop were found to be valuable markers for colorectal cancer outcome evaluation.

8-Hydroxy-2'-deoxyguanosine expression predicts outcome of esophageal cancer.

Esophageal cancer is characterized by increased oxidative stress and the production of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is one of the main mutagenic modifications of DNA. We analyzed the predictive value of 8-OHdG expression on postoperative survival of patients with esophageal cancer with univariate and multivariate analysis. The high levels of 8-OHdG are associated with significantly shorter survival time by log-rank test using Kaplan-Meier methods. Moreover, the level of 8-OHdG expression was identified as an independent predictor for esophageal cancer outcome using Cox proportional hazards model analysis (relative risk, 0.294; 95% confidence interval, 0.178-0.487; P = .000). These results suggest that oxidative damage marker of 8-OHdG is a useful prognostic marker in esophageal cancer. The analysis of 8-OHdG levels can help in the identification of patient subgroups that are at high risk for poor disease outcomes.

Mitochondrial DNA haplogroup N is associated good outcome of gastric cancer.

Accumulation of mutations and single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been identified for their association with cancer risk and disease outcome in a variety of cancers. We have identified cancer risk-associated D-loop SNPs in gastric cancer patients. In this study, we evaluated the predictive value of these SNPs for cancer outcome. Two SNP sites of nucleotides 489C/T and 523-524AC/del were identified for statistically significant prediction of postoperative survival in gastric cancer by univariate analysis with log-rank test. In addition, the mitochondrial DNA haplogroup N (489T) contributed to the good survival of gastric cancer patients compared with the mitochondrial DNA haplogroup M (489C) genotype (relative risk, 1.753; 95 %CI, 1.005-3.060; p = 0.048) by multivariate analysis with COX hazards model. In conclusion, analysis of genetic polymorphisms in the mitochondrial D-loop can help identify subgroups of patients who are at a high risk of a poor disease outcome.

A polymorphism at the microRNA binding site in the 3' untranslated region of C14orf101 is associated with non-Hodgkin lymphoma overall survival.

MicroRNAs (miRNAs) can bind to the 3' untranslated regions (UTRs) of messenger RNAs, where they interfere with translation and thereby regulate cell differentiation, apoptosis, and tumorigenesis. Genetic polymorphisms in the 3' UTRs targeted by miRNAs alter the strength of miRNA binding in a manner that affects the behavior of individual miRNAs. In this study, four miRNA binding-site single nucleotide polymorphisms (SNPs) located in the 3' UTR of RYR3 (rs1044129), C14orf101 (rs4901706), KIAA0423 (rs1053667), and GOLGA7 (rs11337) were genotyped in non-Hodgkin lymphoma (NHL) patients to assess their relationships with cancer risk and overall survival. rs4901706, located in the 3' UTR of C14orf101, was shown to be independently related to overall survival in NHL patients by multivariate analysis (relative risk, 1.770; 95% CI, 1.046-2.996; P = 0.033). The prognostic value of this SNP on tumor overall survival was supported in diffuse large B-cell lymphoma patients with a P value of 0.095 and validated in T-cell lymphoma patients with a P value of 0.037.

Single nucleotide polymorphisms in the mitochondrial displacement loop and age at onset of non-Hodgkin lymphoma.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) accumulated frequently in the mitochondrial displacement loop (D-loop) in many cancers. We had identified cancer risk-associated SNPs in the D-loop of non-Hodgkin lymphoma (NHL) patients previously, in this study, we investigated the association of age at onset and D-loop SNPs in NHL patients. MATERIALS AND METHODS: The D-loop region of mtDNA was sequenced for 133 NHL patients recorded at the Fourth Hospital of Hebei Medical University. The Kaplan-Meier method was used to identify age at onset-associated SNPs in the D-loop of NHL patients. The Cox proportional hazards model was used to identify independent risk factors for age at onset. RESULTS: The SNP sites of nucleotides 146C/T, 151T/C, 194T/C, 315C/C insert, 523Del/A, and 525Del/C were identified for their association with age at onset, by the logrank test. In an overall multivariate analysis, allele 146 (relative risk, 0.403; 95% confidence interval [CI]: 0.182-0.895) (P = 0.026), allele 151 (relative risk, 0.378; 95% CI: 0.165-0.868) (P = 0.022), and allele 315 (relative risk, 3.554; 95% CI: 1.344-9.400) (P = 0.011) were identified as independent predictors for age at onset in NHL patients. CONCLUSION: SNPs in the D-loop can predict age at onset in NHL patients. Analysis of the D-loop SNPs can help identify NHL patient subgroups at high risk of early onset.

Sirtuin-3 (SIRT3) expression is associated with overall survival in esophageal cancer.

Sirtuin-3 (SIRT3) was immunostained in 94 samples of esophageal cancer tissues and semiquantified using the HSCORE method to evaluate the predictive value of SIRT3 expression levels on esophageal cancer outcome. The relationship between SIRT3 expression and the 5-year survival rate of postoperational esophageal cancer patients was assessed with the Kaplan-Meier method. High expression of SIRT3 is associated with a shorter survival time in esophageal cancer patients, as shown by the log-rank test (P = .007), and the level of SIRT3 expression was identified as an independent predictor for esophageal cancer outcome using Cox proportional hazards model analysis (relative risk, 2.061; 95% confidence interval, 1.050-4.046; P = .036). SIRT3 expression was associated with esophageal cancer outcome. The analysis of SIRT3 levels can help in the identification of patient subgroups that are at high risk for poor disease outcomes.

Mitochondrial DNA haplogroup M is associated with late onset of hepatocellular carcinoma.

The accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been associated with various types of cancer. The association of SNPs with cancer risk and disease outcome has been exhaustively studied. In this study, we investigated the association of age-at-onset and SNPs in the mitochondrial D-loop using a population-based series of hepatocellular carcinoma (HCC) patients. Haplogroup M (489C) and allele 235G were identified for their association with the late onset of HCC by the log-rank test. In an overall multivariate analysis, haplogroup M (489C) was identified as an independent predictive factor for the age-at-onset of HCC at borderline significant levels [relative risk, 1.736; 95% confidence interval (CI), 0.967-3.115; p=0.065]. Genetic polymorphisms in the D-loop are predictive markers for age-at-onset in HCC patients. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop may help to identify HCC patient subgroups at high risk of early onset of the disease.

A polymorphism at the miR-502 binding site in the 3' untranslated region of the SET8 gene is associated with the risk of epithelial ovarian cancer.

MicroRNAs (miRNAs) bind to the 3' untranslated regions (UTRs) of messenger RNAs, where they interfere with the translation of genes that regulate cell differentiation, apoptosis, and tumorigenesis. SET8 reportedly methylates TP53 and regulates genomic stability. We analyzed a single nucleotide polymorphism (rs16917496) within the miR-502 mRNA seed region of the 3' UTR of SET8 in Chinese epithelial ovarian cancer (EOC) patients. The SET8 CC genotype was associated with a decreased risk of EOC in this case-control study. The analysis of genetic polymorphisms in miRNA binding sites may help identify subgroups of populations that are at high risk for EOC.

Molecular and organismal changes in offspring of male mice treated with chemical stressors.

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

8-Hydroxy-2'-deoxyguanosine expression predicts hepatocellular carcinoma outcome.

Hepatocellular carcinoma (HCC) is characterized by increased oxidative stress and the production of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is one of the main mutagenic modifications of DNA by oxidative stress. We analyzed the association of 8-OHdG with post-operative survival and revealed that low levels of 8-OHdG are associated with significantly shorter survival time. Moreover, levels of 8-OHdG were associated with HCC characteristics, including tumor size, tumor quantity, clinical staging, Child classification, portal vein thrombosis and ascites. These results suggest that oxidative damage is a useful prognostic marker in HCC when other clinical characteristics are present with 8-OHdG.

Single nucleotide polymorphisms in the mitochondrial displacement loop and age-at-onset of esophageal squamous cell carcinoma.

The accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for various types of cancer, and their association with cancer risk and disease outcome has been extensively identified. In the present study, we investigated the association between age-at-onset and SNPs in the mitochondrial D-loop using a population-based series of esophageal squamous cell carcinoma (ESCC) patients. The SNP sites of nucleotides 16266 C/T were identified for their association with age-at-onset using the log-rank test. The age-at-onset of patients with the minor allele T genotype was significantly lower than that of patients with the C genotype at the 16266 site (p=0.036). Genetic polymorphisms in the D-loop are predictive markers for age-at-onset in ESCC patients. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop may help identify ESCC patient subgroups at high risk of early onset.