Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

NF90 stabilizes cyclin E1 mRNA through phosphorylation of NF90-Ser382 by CDK2.

Nuclear factor 90 (NF90), an RNA-binding protein, has been implicated in regulating interleukin-2 (IL-2) and the immune response. It was recently reported that NF90 is upregulated in hepatocellular carcinoma (HCC) tissues and promotes HCC proliferation through upregulating cyclin E1 at the posttranscription level. However, the regulation of NF90 in HCC remains unclear. We demonstrate here that cyclin-dependent kinase (CDK) 2 interacts with NF90 and phosphorylated it at serine382. Mechanistically, phosphorylation of NF90-Ser382 determines the nuclear export of NF90 and stabilization of cyclin E1 mRNA. We also demonstrate that the phosphorylation deficient mutant NF90-S382A inhibits cell growth and induces cell cycle arrest at the G1 phase in HCC cells. Moreover, an NF90-S382A xenograft tumor had a decreased size and weight compared with the wildtype NF90. The NF90-S382A xenograft contained a significantly lower level of the proliferation marker Ki-67. Additionally, in HCC patients, NF90-Ser382 phosphorylation was stronger in tumor than in non-tumor tissues. Clinically, phosphorylation of NF90-Ser382 is significantly associated with larger tumor sizes, higher AFP levels, and shorter overall survival rates. These results suggest NF90-Ser382 phosphorylation serves as a potential diagnosis and prognostic marker and a promising pharmacological target for HCC.

NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma.

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP- ribosylation enzyme and plays important roles in a variety of cellular processes, including DNA damage response and tumor development. However, the post-transcriptional regulation of PARP1 remains largely unknown. In this study, we identified that the mRNA of PARP1 is associated with nuclear factor 90 (NF90) by RNA immunoprecipitation plus sequencing (RIP-seq) assay. The mRNA and protein levels of PARP1 are dramatically decreased in NF90-depleted cells, and NF90 stabilizes PARP1's mRNA through its 3'UTR. Moreover, the expression levels of PARP1 and NF90 are positively correlated in hepatocellular carcinoma (HCC). Finally, we demonstrated that NF90-depleted cells are sensitive to PARP inhibitor Olaparib (AZD2281) and DNA damage agents. Taken together, these results suggest that NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma cells, and NF90 is a potential target to inhibit PARP1 activity.

MiR-608 rs4919510 is associated with prognosis of hepatocellular carcinoma.

Single nucleotide polymorphisms (SNPs) within microRNAs (miRNAs) are considered potential markers for risk and prognosis of various cancers. In the current study, we aimed to determine whether miR-608 rs4919510 affected hepatocellular carcinoma (HCC) prognosis. We genotyped rs4919510 using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. Associations between rs4919510 and overall survival (OS) and demographic characteristics and clinical features were estimated using the Cox proportional hazards model. Results showed that HCC patients who carried the rs4919510 CC genotype had a significantly longer OS compared to those who carried the GG genotype (P = 0.013, hazard ratio [HR] = 0.600, 95 % confidence interval [CI] 0.402-0.897) and the CG + GG genotype (P = 0.033, HR = 0.681, 95 % CI 0.479-0.970) in univariate analysis. Similar results were obtained in multivariate analysis. Further stratification analysis indicated that rs4919510 was significantly associated with OS in patients who were satisfied with one of the following criteria: male gender, HbsAg-positive, α-fetoprotein (AFP)-positive, tumor size >5 cm, cirrhosis, solitary tumor, I + II pTNM stage, or no tumor capsule. Finally, a significantly higher frequency of rs4919510 CC genotype was observed in patients with cirrhosis (22.9 %, 55/240) than those without cirrhosis (14.0 %, 17/121) (P = 0.047). In conclusion, our results illustrated the potential role of miR-608 rs4919510 as a prognostic marker for HCC patients undergoing surgical resection of the tumor.

miR-492G>C polymorphism (rs2289030) is associated with overall survival of hepatocellular carcinoma patients.

Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) are considered potential markers of cancer risk and prognosis in various cancers. In the current study, the primary aim is to determine whether the miR-492G>C polymorphism (rs2289030) altered hepatocellular carcinoma (HCC) prognosis. The SNP rs2289030 of miR-492 was genotyped using DNA from blood samples of 362 HCC patients that had undergone surgical resection of a HCC tumor. The associations between overall survival and demographic characteristics, clinical features, and the SNP rs2289030 were estimated using the Cox proportional hazards model. Results showed that patients who carried the CG genotype (P = 0.015, hazard ratio [HR] = 0.704, 95 % confidence interval [CI] 0.530-0.934) and CG+GG genotype (P = 0.011, HR = 0.703, 95 % CI 0.536-0.924) had significantly decreased risk of death compared to those with the CC genotype. Similar results were found in the multivariate analysis adjusted by tumor size and venous invasion. Further stratification analysis indicated that the effect of rs2289030 had more prominence in patients ≤50 years old and that reported ever using alcohol, male gender, a family history of HCC, being HbsAg or alpha fetoprotein (AFP) positive, differentiation I + II, presence of venous invasion or cirrhosis, multiple tumors, and pTNM stage I + II. Results from this study illustrate the potential use of miR-492 rs2289030 as a prognostic marker for HCC patients that have undergone a surgical resection of the tumor.

Genetic polymorphisms in apoptosis-related genes and the prognosis of hepatocellular carcinoma.

The apoptotic pathway is important in the control of vital processes of hepatocellular carcinoma (HCC). In the current study, we aimed to determine whether apoptotic gene-related polymorphisms modified HCC prognosis. We genotyped 16 single nucleotide polymorphisms (SNPs) in 10 core genes (TP53, TP53INP1, TP53BP1, CDKN2A, CDKN1A, CDKN1B, MDM2, BAX, CCDN1 and BCL2) in the apoptotic pathway by using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. The associations between genotypes/haplotypes of the 10 genes and overall survival (OS) of HCC patients were assessed using the Cox proportional hazards model. We found one CDKN1B haplotype CCT/ACT (constructed by rs36228499 C>A, rs34330 C>T and rs2066827 T>G) significantly associated with decreased OS of HCC patients, compared to the common haplotype ACT/CTT both in univariate analysis (P=0.013, HR=1.198, 95% CI: 1.039-1.381) and multivariate analysis (P=0.006, HR=1.224, 95% CI: 1.059-1.413). We also find two SNPs (rs560191 G>C and rs2602141 T>G) in TP53BP1 shown to be marginally significantly associated with decreased OS of HCC patients. However, none of the other SNPs or haplotypes were significantly associated with HCC OS. Our results illustrated the potential use of CDKN1B haplotype as a prognostic marker for HCC patients with surgical resection of tumor.

Genetic variations in STAT4,C2,HLA-DRB1 and HLA-DQ associated with risk of hepatitis B virus-related liver cirrhosis.

Recent genome-wide associated studies (GWASs) have revealed several common loci associated with the risk of hepatitis B virus (HBV)- or hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). We selected 15 single nucleotide polymorphisms (SNPs) identified through GWASs on HBV- or HCV-related HCC, and genotyped them in two independent Chinese cohorts of chronic HBV carriers, including 712 LC cases and 2601 controls. The association of each SNP with the risk of HBV-related LC was assessed by meta-analysis of the two cohorts. Of the 12 SNPs reported in HBV-related HCC GWASs, five SNPs (rs7574865 in STAT4, rs9267673 near C2, rs2647073 and rs3997872 near HLA-DRB1 and rs9275319 near HLA-DQ), were found to be significantly associated with the risk of HBV-related LC (rs7574865: P = 1.79 × 10(-2), OR = 1.17, 95% CI = 1.03-1.34; rs9267673: P = 4.91 × 10(-4), OR = 1.37, 95% CI = 1.15-1.63; rs2647073: P = 3.53 × 10(-5), OR = 1.63, 95% CI = 1.29-2.06; rs3997872: P = 4.22 × 10(-4), OR = 1.86, 95% CI = 1.32-2.62; rs9275319: P = 1.30 × 10(-2), OR = 1.32, 95% CI = 1.06-1.64). However, among the three SNPs associated with the risk of HCV-related HCC in previous GWASs, none of them showed significant association with the risk of HBV-related LC. Our results suggested that genetic variants associated with HBV-related hepatocarcinogenesis may already play an important role in the progression from CHB to LC.

Meta-analysis of gene expression profiles indicates genes in spliceosome pathway are up-regulated in hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is among the commonest kind of malignant tumors, which accounts for more than 500,000 cases of newly diagnosed cancer annually. Many microarray studies for identifying differentially expressed genes (DEGs) in HCC have been conducted, but results have varied across different studies. Here, we performed a meta-analysis of publicly available microarray Gene Expression Omnibus datasets, which covers five independent studies, containing 753 HCC samples and 638 non-tumor liver samples. We identified 192 DEGs that were consistently up-regulated in HCC vs. normal liver tissue. For the 192 up-regulated genes, we performed Kyoto Encyclopedia of Genes and Genomes pathway analysis. To our surprise, besides several cell growth-related pathways, spliceosome pathway was also up-regulated in HCC. For further exploring the relationship between spliceosome pathway and HCC, we investigated the expression data of spliceosome pathway genes in 15 independent studies in Nextbio database ( https://www.nextbio.com/b/nextbioCorp.nb ). It was found that many genes of spliceosome pathway such as HSPA1A, SNRPE, SF3B2, SF3B4 and TRA2A genes which we identified to be up-regulated in our meta-analysis were generally overexpressed in HCC. At last, using real-time PCR, we also found that BUD31, SF3B2, SF3B4, SNRPE, SPINK1, TPA2A and HSPA1A genes are significantly up-regulated in clinical HCC samples when compared to the corresponding non-tumorous liver tissues. Our study for the first time indicates that many genes of spliceosome pathway are up-regulated in HCC. This finding might put new insights for people's understanding about the relationship of spliceosome pathway and HCC.

Genetic variants in five novel loci including CFB and CD40 predispose to chronic hepatitis B.

UNLABELLED: Hepatitis B virus affects more than 2 billion people worldwide, 350 million of which have developed chronic hepatitis B (CHB). The genetic factors that confer CHB risk are still largely unknown. We sought to identify genetic variants for CHB susceptibility in the Chinese population. We undertook a genome-wide association study (GWAS) in 2,514 CHB cases and 1,130 normal controls from eastern China. We replicated 33 of the most promising signals and eight previously reported CHB risk loci through a two-stage validation totaling 6,600 CHB cases and 8,127 controls in four independent populations, of which two populations were recruited from eastern China, one from northern China and one from southern China. The joint analyses of 9,114 CHB cases and 9,257 controls revealed significant association of CHB risk with five novel loci. Four loci are located in the human leukocyte antigen (HLA) region at 6p21.3, including two nonsynonymous variants (rs12614 [R32W] in complement factor B [CFB], Pmeta =1.28 × 10(-34) ; and rs422951 [T320A] in NOTCH4, Pmeta = 5.33 × 10(-16) ); one synonymous variant (rs378352 in HLA-DOA corresponding to HLA-DOA*010101, Pmeta = 1.04 × 10(-23) ); and one noncoding variant (rs2853953 near HLA-C, Pmeta = 5.06 × 10(-20) ). Another locus is located at 20q13.1 (rs1883832 in the Kozak sequence of CD40, Pmeta = 2.95 × 10(-15) ). Additionally, we validated seven of eight previously reported CHB susceptibility loci (rs3130542 at HLA-C, rs1419881 at TCF19, rs652888 at EHMT2, rs2856718 at HLA-DQB1, rs7453920 at HLA-DQB2, rs3077 at HLA-DPA1, and rs9277535 at HLA-DPA2, which are all located in the HLA region, 9.84 × 10(-71) ≤ Pmeta ≤ 9.92 × 10(-7) ). CONCLUSION: Our GWAS identified five novel susceptibility loci for CHB. These findings improve the understanding of CHB etiology and may provide new targets for prevention and treatment of this disease.

Epidermal growth factor receptor pathway polymorphisms and the prognosis of hepatocellular carcinoma.

The EGFR signaling pathway is important in the control of vital processes in the carcinogenesis of hepatocellular carcinoma (HCC), including cell survival, cell cycle progression, tumor invasion and angiogenesis. In the current study, we aim to assess if genetic variants in the genes of the EGFR signaling pathway are associated with the prognosis of HCC. We genotyped 36 single nucleotide polymorphisms (SNP) in four core genes (EGF, EGFR, VEGF, and VEGFR2) by using DNA from blood samples of 363 HCC patients with surgical resection. The associations between genotypes and overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) and 95% confident intervals (CIs) were estimated for the multivariate survival analyses by Cox proportional hazards regression models, adjusting for age, gender, family history, HBsAg and AFP. We found that five SNPs in the VEGFR2 gene were significantly associated with clinical outcomes of HCC patients. Among them, four SNPs (rs7692791, rs2305948, rs13109660, rs6838752) were associated with OS (p=0.035, 0.038, 0.029 and 0.028, respectively), and two SNPs (rs7692791 and rs2034965) were associated with DFS (p=0.039 and 0.017, respectively). Particularly, rs7692791 TT genotype was associated with both reduced OS (p=0.037) and DFS (p=0.043). However, only one SNP rs2034965 with the AA genotype was shown to be an independent effect on DFS (p=0.009) in the multivariate analysis. None of the other 31 polymorphisms or 9 haplotypes attained from the four genes was significantly associated with OS or DFS. Our results illustrated the potential use of VEGFR2 polymorphisms as prognostic markers for HCC patients.

Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma.

To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, P(meta) = 2.48 × 10(-10), odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, P(meta) = 2.72 × 10(-17), OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (P(trend) = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10(-14)).

An insertion/deletion polymorphism within RERT-lncRNA modulates hepatocellular carcinoma risk.

The Prolyl hydroxylase 1 (EGLN2) is known to affect tumorigenesis by regulating the degradation of hypoxia-inducible factor. Polymorphisms in EGLN2 may facilitate cancer cell survival under hypoxic conditions and directly associate with cancer susceptibility. Here, we examined the contribution of a 4-bp insertion/deletion polymorphism (rs10680577) within the distal promoter of EGLN2 to the risk of hepatocelluar carcinoma (HCC) in Chinese populations. The contribution of rs10680577 to HCC risk was investigated in 623 HCC cases and 1,242 controls and replicated in an independent case-control study consisting of 444 HCC cases and 450 controls. Logistic regression analysis showed that the deletion allele of rs10680577 was significantly associated with increased risk for HCC occurrence in both case-control studies [OR = 1.40; 95% confidence interval (CI) = 1.18-1.66, P < 0.0001; OR = 1.49; 95% CI = 1.18-1.88, P = 0.0007]. Such positive association was more pronounced in current smokers (OR = 3.49, 95% CI = 2.24-5.45) than nonsmokers (OR = 1.24, 95% CI = 1.03-1.50; heterogeneity P = 0.0002). Genotype-phenotype correlation studies showed that the deletion allele was significantly correlated with higher expression of both EGLN2 and RERT-lncRNA [a long noncoding RNA whose sequence overlaps with Ras-related GTP-binding protein 4b (RAB4B) and EGLN2)] in vivo and in vitro. Furthermore, RERT-lncRNA expression was also significantly correlated with EGLN2 expression in vivo, consistent with in vitro gain-of-function study that showed overexpressing RERT-lncRNA upregulated EGLN2. Finally, in silico prediction suggested that the insertion allele could disrupt the structure of RERT-lncRNA. Taken together, our findings provided strong evidence for the hypothesis that rs10680577 contributes to hepatocarcinogenesis, possibly by affecting RERT-lncRNA structure and subsequently EGLN2 expression, making it a promising biomarker for early diagnosis of HCC.

Microbial removal of NOX at high temperature by a novel aerobic strain Chelatococcus daeguensis TAD1 in a biotrickling filter.

The removal of NO(X) at high temperature by Chelatococcus daeguensis TAD1 in a biotrickling filter was studied. Media components of the recycling liquid were screened using Plackett-Burman design and then were optimized using response surface methodology, which enhanced the efficiency of nitrate removal by TAD1. The optimal medium was used to perform long-term experiments of NO(X) removal in a biotrickling filter under high concentrations of O(2) and NO in simulated flue gas. Results showed that the biotrickling filter was able to consistently remove 80.2-92.3% NO(X) when the inlet NO concentration was 600ppm under the conditions of oxygen concentration ranging between 2% and 20% and empty bed residence time (EBRT) being 112.5s. Analyses by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) indicated that TAD1 was always predominant in the biofilm under a flue gas environment. Overall, the present study demonstrated that utilizing a biotrickling filter inoculated with the aerobic denitrifier TAD1 to remove NO(X) at high temperature was practically feasible.

[Start-up and affecting factors of biofilter for treatment of NO(x) under aerobic conditions].

An optimized aerobic denitrifying bacteria was applied to a biofilter for the removal of NO(x). The removal process of NO(x) was investigated, and the relationship between environmental factors and NO(x) removal efficiency as well as the NO(x) transfermechanism under aerobic conditions are discussed. The results show that the biofilter finished start-up after 26 days and the presence of oxygen has no evident negative effect on the efficiency of NO(x) removal. Mainly happening at the middle and under part of the biofiltration system, the removal of NO(x) get a high efficiency of 93.6% at the EBRT of 59s and the inlet NO(x) concentration of 286.4 mg/m3 with 10% O2. A special biomembrane system was formulated by nitrobacteria, facultative denitrifying bacteria and aerobic denitrifying bacteria in the biofiltration system.