Jinyu Wei

The identification of an ESCC susceptibility SNP rs920778 that regulates the expression of lncRNA HOTAIR via a novel intronic enhancer

Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), which could induce genome-wide retargeting of polycomb-repressive complex 2, trimethylates histone H3 lysine-27 (H3K27me3) and deregulation of multiple downstream genes, is involved in development and progression of esophageal squamous cell carcinoma (ESCC). We hypothesized that the functional single nucleotide polymorphisms (SNP) in HOTAIR may affect HOTAIR expression and/or its function and, thus, ESCC risk. Therefore, we examined the association between three haplotype-tagging SNPs (htSNP) across the whole HOTAIR locus and ESCC risk as well as the functional relevance of an ESCC susceptibility SNP rs920778. Genotypes were determined in three independent case-control sets consisted of 2098 ESCC patients and 2150 controls. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was investigated in vitro and in vivo. We found that the HOTAIR rs920778 TT carriers had a 1.37-fold, 1.78-fold and 2.08-fold increased ESCC risk in Jinan, Shijiazhuang and Huaian populations, respectively, compared with the CC carriers (P = 0.003, 7.7 × 10(-4) and 5.9 × 10(-4)). During inspecting functional relevance of the rs920778 SNP, we identified a novel intronic HOTAIR enhancer locating between +1719bp and +2353bp from the transcriptional start site through reporter assays. Moreover, there is an allelic regulation of rs920778 on HOTAIR expression via this enhancer in both ESCC cell lines and normal esophageal tissue specimens, with higher HOTAIR expression among T allele carriers. These results demonstrate that functional genetic variants influencing lncRNA regulation may explain a fraction of ESCC genetic basis.

A functional lncRNA HOTAIR genetic variant contributes to gastric cancer susceptibility.

Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) acts as an oncogene in gastric cancer development. HOTAIR could induce genome‐wide retargeting of polycomb‐repressive complex 2, trimethylates histone H3 lysine‐27 (H3K27me3) and deregulation of multiple downstream genes. Additionally, as the ceRNA of miR‐331–3p, HOTAIR may modulate HER2 deregulation in gastric cancer cells. We hypothesized that the functional single nucleotide polymorphisms (SNP) in HOTAIR may affect HOTAIR expression and/or its function and, thus, gastric cancer risk. We examined the association between three haplotype‐tagging SNPs (htSNP) across the whole HOTAIR locus and gastric cancer risk as well as the functional relevance of a gastric cancer susceptibility SNP rs920778. Genotypes were determined in two independent hospital‐based case‐control sets that consisted of 800 gastric cancer patients and 1600 controls. The allele‐specific regulation on HOTAIR expression by the rs920778 SNP was examined in vitro and in vivo. We found that the HOTAIR rs920778 TT carriers had a 1.66‐ and 1.87‐fold increased gastric cancer risk in Jinan and Huaian populations compared with the CC carriers (P = 4.2 × 10−4 and 6.5 × 10−5). During inspecting functional relevance of the rs920778 SNP, we observed an allelic regulation of rs920778 on HOTAIR expression in both gastric cancer cell lines and tissue samples, with higher HOTAIR expression among T allele carriers. These findings elucidate that functional genetic variants influencing lncRNA expression may explain a portion of gastric cancer genetic basis.

A functional BRCA1 coding sequence genetic variant contributes to risk of esophageal squamous cell carcinoma.

As a tumor suppressor, breast cancer susceptibility gene 1 (BRCA1) plays a pivotal role in maintaining genomic stability. A functional rs799917 T>C polymorphism located in the BRCA1 coding sequence could influence miR-638-mediated regulation of BRCA1 expression. Therefore, we examined the association between this polymorphism and esophageal squamous cell carcinoma (ESCC) risk as well as its biological function. Genotypes were determined in two independent case-control studies consisted of 1128 ESCC patients and 1150 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The allele-specific regulation on BRCA1 expression by the polymorphism was investigated in vitro and in vivo. We found that the BRCA1 rs799917 CC genotype was significantly associated with increased ESCC risk compared with the TT genotype in both studies (Jinan population: OR = 1.28, 95% CI = 1.04-1.58, P = 0.020; Huaian population: OR = 1.46, 95% CI = 1.17-1.83, P = 0.001). Stratified analyses with pooled data indicated that a multiplicative interaction between rs799917 and smoking or drinking in intensifying ESCC risk was evident (gene-smoking: P interactio n = 5.8 × 10(-5); gene-drinking: P interaction = 7.1 × 10(-7)). In vitro experiments indicate that miR-638 could negatively regulate BRCA1 expression and enhance proliferation of ESCC cells. In vivo BRCA1 messenger RNA expression analyses showed that the rs799917 C allele carriers had significantly decreased BRCA1 expression in both normal and cancerous esophagus tissues compared with T allele carriers, suggesting that lower BRCA1 expression may lead to higher risk for malignant transformation of esophagus cells. These results suggest that BRCA1 functional rs799917 polymorphism is involved in susceptibility to developing ESCC, alone and in a gene-environment interaction manner.

Whole exome sequencing identifies lncRNA GAS8-AS1 and LPAR4 as novel papillary thyroid carcinoma driver alternations

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. However, we know little of mutational spectrum in the Chinese population. Thus, here we report the identification of somatic mutations for Chinese PTC using 402 tumor-normal pairs (Discovery: 91 pairs via exome sequencing; validation: 311 pairs via Sanger sequencing). We observed three distinct mutational signatures, evidently different from the two mutational signatures among Caucasian PTCs. Ten significantly mutated genes were identified, most previously uncharacterized. Notably, we found that long non-coding RNA (lncRNA) GAS8-AS1 is the secondary most frequently altered gene and acts as a novel tumor suppressor in PTC. As a mutation hotspot, the c.713A>G/714T>C dinucleotide substitution was found among 89.1% patients with GAS8-AS1 mutations and associated with advanced PTC disease (P = 0.009). Interestingly, the wild-type lncRNA GAS8-AS1 (A713T714) showed consistently higher capability to inhibit cancer cell growth compared to the mutated lncRNA (G713C714). Further studies also elucidated the oncogene nature of the G protein-coupled receptor LPAR4 and its c.872T>G (p.Ile291Ser) mutation in PTC malignant transformation. The BRAF c.1799T>A (p.Val600Glu) substitution was present in 59.0% Chinese PTCs, more frequently observed in patients with lymph node metastasis (P = 1.6 × 10(-4)). Together our study defines a exome mutational spectrum of PTC in the Chinese population and highlights lncRNA GAS8-AS1 and LPAR4 as potential diagnostics and therapeutic targets.

Association of functional FEN1 genetic variants and haplotypes and breast cancer risk.

AIM As a tumor suppressor, FEN1 plays an essential role in preventing tumorigenesis. Two functional germline variants (-69G>A and 4150G>T) in the FEN1 gene have been associated with DNA damage levels in coke-oven workers and multiple cancer risk in general populations. However, it is still unknown how these genetic variants are involved in breast cancer susceptibility. METHODS We investigated the association between these polymorphisms and breast cancer risk in two independent case-control sets consisted of a total of 1100 breast cancer cases and 1400 controls. The influence of these variations on FEN1 expression was also examined using breast normal tissues. RESULTS It was found that the FEN1-69GG genotypes were significantly correlated to increased risk for developing breast cancer compared with the -69AA genotype in both sets [Jinan set: odds ratios (OR)=1.41, 95% confidence interval (CI)=1.20-1.65, P=1.9×10(-5); Huaian set: OR=1.51, 95% CI=1.22-1.86, P=1.7×10(-4)]. Similar results were observed for 4150G>T polymorphism. The genotype-phenotype correlation analyses demonstrated that the -69G or 4150G allele carriers had more than 2-fold decreased FEN1 expression in breast tissues compared with -69A or 4150T carriers, suggesting that lower FEN1 expression may lead to higher risk for malignant transformation of breast cells. CONCLUSION Our findings highlight FEN1 as an important gene in human breast carcinogenesis and genetic variants in FEN1 confer susceptibility to breast cancer.

Replication study of ESCC susceptibility genetic polymorphisms locating in the ADH1B-ADH1C-ADH7 cluster identified by GWAS.

China was one of the countries with highest esophageal squamous cell carcinoma (ESCC) incidence and mortality worldwide. Alcohol drinking has been identified as a major environmental risk-factor related to ESCC. The alcohol dehydrogenase (ADH) family are major enzymes involved in the alcohol-metabolizing pathways, including alcohol dehydrogenase 1B (ADH1B) and ADH1C. Interestingly, ADH1B and ADH1C genes locate tandemly with ADH7 in a genomic segment as a gene cluster, and are all polymorphic. Several ESCC susceptibility single nucleotide polymorphisms (SNPs) of the ADH1B-ADH1C-ADH7 cluster have been identified previously through a genome-wide association study (GWAS). In the study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC. Genotypes were determined in two independent case-control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Our data demonstrated that these ADH1B-ADH1C-ADH7 cluster SNPs confer susceptibility to ESCC in these two case-control sets, which were consistent to results of the previous GWAS.

Association of a functional RAD52 genetic variant locating in a miRNA binding site with risk of HBV‐related hepatocellular carcinoma

As an important member in homologous recombination repair, RAD52 plays a crucial part in maintaining genomic stability and prevent carcinogenesis. Several cancer susceptibility RAD52 single nucleotide polymorphisms (SNPs) have been identified previously. However, little or nothing has been known about the RAD52 SNPs and their functional significance in hepatitis B viruses (HBV)‐related hepatocellular carcinoma (HCC). Therefore, we investigated the association between five RAD52 SNPs (rs1051669, rs10774474, rs11571378, rs7963551, and rs6489769) and HBV‐related HCC risk as well as its biological function in vivo. Genotypes were determined in two independent case–control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The allele‐specific regulation on RAD52 expression by the functional genetic variant was examined with normal liver tissues. We found that only the RAD52 rs7963551 SNP was significantly associated with HCC risk, with the odds of having the rs7963551 CC genotype in patients was 0.59 (95% CI = 0.45–0.78, P = 1.5 × 10−4, HCC cases versus chronic HBV carriers) or 0.65 (95% CI = 0.52–0.81, P = 1.1 × 10−4, HCC cases versus healthy controls) compared with the AA genotype. In the genotype–phenotype correlation analyses of 44 human liver tissue samples, rs7963551 CC or AC was associated with a statistically significant increase of RAD52 mRNA expression, which are consistent to functional relevance of allelic regulation of RAD52 expression by rs7963551 SNP and miRNA let‐7 in cancer cells. Our data demonstrated that RAD52 functional rs7963551 SNP contributes to susceptibility to developing HCC.

The functional TP53 rs1042522 and MDM4 rs4245739 genetic variants contribute to Non-Hodgkin lymphoma risk.

As a heterogeneous kind of malignances, Non-Hodgkin lymphoma (NHL) is the most common hematologic cancer worldwide with the significantly increased morbidity in China. Accumulated evidences demonstrated that oncoprotein MDM4 plays a crucial role in the TP53 tumor suppressor signaling pathway. An rs4245739 A>C polymorphism locating in the MDM4 3′-untranslated region creates a miR-191 target site and results in allele-specific MDM4 expression. In this study, we examined the association between this polymorphism as well as the TP53 Arg72Pro (rs1042522 G>C) genetic variant and Non-Hodgkin Lymphoma (NHL) risk in a Chinese Han population. Genotypes were determined in 200 NHL cases and 400 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. We found significantly increased NHL risk among carriers of the TP53 72Pro allele compared with those with the 72Arg allele (P = 0.002 for the Pro/Pro genotype). We also observed a significantly decreased NHL risks among carriers of the MDM4 rs4245739 C allele compared with those with the A allele in Chinese (P = 0.014 for the AC genotype). Stratified analyses revealed the associations between these SNPs and NHL risk are especially noteworthy in young or male individuals. Additionally, the associations are much pronounced in NHL patients with B-cell lymphomas or grade 3 or 4 disease. Our results indicate that the TP53 Arg72Pro and the MDM4 rs4245739 polymorphisms contribute to NHL susceptibility and support the hypothesis that genetic variants in the TP53 pathway genes can act as important modifiers of NHL risk.

The identification of two regulatory ESCC susceptibility genetic variants in the TERT-CLPTM1L loci

The chromosome 5p15.33 TERT-CLPTM1L region has been identified by genome-wide association studies as a susceptibility locus of multiple malignancies. However, the involvement of this locus in esophageal squamous cell carcinoma (ESCC) development is still largely unclear. We fine-mapped the TERT-CLPTM1L region through genotyping 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) using a two stage case-control strategy. After analyzing 2098 ESCC patients and frequency-matched 2150 unaffected controls, we found that rs2853691, rs2736100 and rs451360 genetic polymorphisms are significantly associated with ESCC risk in Chinese (all P<0.05). Reporter gene assays indicated that the ESCC susceptibility SNP rs2736100 locating in a potential TERT intronic promoter has a genotype-specific effect on TERT expression. Similarly, the CLPTM1L rs451360 SNP also showed allelic impacts on gene expression. After measuring TERT and CLPTM1L expression in sixty-six pairs of esophageal cancer and normal tissues, we observed that the rs2736100 G risk allele carriers showed elevated oncogene TERT expression. Also, subjects with the rs451360 protective T allele had much lower oncogene CLPTM1L expression than those with G allele in tissue specimens. Results of these analyses underline the complexity of genetic regulation of telomere biology and further support the important role of telomerase in carcinogenesis. Our data also support the involvement of CLPTM1L in ESCC susceptibility.

Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma

B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57–0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29–0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32–0.62, P = 0.002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations.