Rongrong Wei

Clinical Significance and Prognostic Value of microRNA Expression Signatures in Hepatocellular Carcinoma

Purpose: MicroRNAs (miRNAs) play important roles in the development and progression of cancer. The aim of this study is to identify miRNA expression signatures in hepatocellular carcinoma and delineate their clinical significance for hepatocellular carcinoma. Experimental Design: Patients with hepatocellular carcinoma, undergoing hepatectomy were randomly divided into training set (60 patients) and test set (50 patients). Other 56 patients were used as an independent cohort. The miRNA expression levels were detected by microarray and verified by quantitative real-time reverse transcription-PCR (qRT-PCR). Results: A 30-miRNA signature consisting of 10 downregulated and 20 upregulated miRNAs was established for distinguishing hepatocellular carcinoma from noncancerous liver tissues in the training set with 99.2% accuracy. The classification accuracies of this signature were 97% and 90% in the test set and independent cohort, respectively. The expression level of four miRNAs in the 30-miRNA signature was verified by qRT-PCR in the training set. Twenty miRNAs were then selected to construct prognostic signature in the training set. Of the 20 miRNAs, six were risk factors and 14 were protective factors. A formula based on the 20 miRNAs was built to compute prognostic index. Kaplan–Meier analysis showed that patients with a higher prognostic index had a significantly lower survival than those with a low index. This was verified in the test and independent sets. Multivariate analysis indicated that the 20-miRNA signature was an independent prognostic predictor. Conclusions: The 30- and 20-miRNA signatures identified in this study should provide new molecular approaches for diagnosis and prognosis of patients with hepatocellular carcinoma and clues for elucidating molecular mechanism of hepatocarcinogenesis. Clin Cancer Res; 19(17); 4780–91. ©2013 AACR.

A genome-wide integrative study of microRNAs in human liver

BackgroundRecent studies have illuminated the diversity of roles for microRNAs in cellular, developmental, and pathophysiological processes. The study of microRNAs in human liver tissue promises to clarify the therapeutic and diagnostic value of this important regulatory mechanism of gene expression.ResultsWe conducted genome-wide profiling of microRNA expression in liver and performed an integrative analysis with previously collected genotype and transcriptome data. We report here that the Very Important Pharmacogenes (VIP Genes), comprising of genes of particular relevance for pharmacogenomics, are under substantial microRNA regulatory effect in the liver. We set out to elucidate the genetic basis of microRNA expression variation in liver and mapped microRNA expression to genomic loci as microRNA expression quantitative trait loci (miR-eQTLs). We identified common variants that attain genome-wide significant association (p < 10-10) with microRNA expression. We also found that the miR-eQTLs are significantly more likely to predict mRNA levels at a range of p-value thresholds than a random set of allele frequency matched SNPs, showing the functional effect of these loci on the transcriptome. Finally, we show that a large number of miR-eQTLs overlap with SNPs reproducibly associated with complex traits from the NHGRI repository of published genome-wide association studies as well as variants from a comprehensive catalog of manually curated pharmacogenetic associations.ConclusionOur study provides important insights into the genomic architecture of gene regulation in a vital human organ, with important implications for our understanding of disease pathogenesis, therapeutic outcome, and other complex human phenotypes.

Impact of the Interaction between 3'-UTR SNPs and microRNA on the Expression of Human Xenobiotic Metabolism Enzyme and Transporter Genes.

Genetic variation in the expression of human xenobiotic metabolism enzymes and transporters (XMETs) leads to inter-individual variability in metabolism of therapeutic agents as well as differed susceptibility to various diseases. Recent expression quantitative traits loci (eQTL) mapping in a few human cells/tissues have identified a number of single nucleotide polymorphisms (SNPs) significantly associated with mRNA expression of many XMET genes. These eQTLs are therefore important candidate markers for pharmacogenetic studies. However, questions remain about whether these SNPs are causative and in what mechanism these SNPs may function. Given the important role of microRNAs (miRs) in gene transcription regulation, we hypothesize that those eQTLs or their proxies in strong linkage disequilibrium (LD) altering miR targeting are likely causative SNPs affecting gene expression. The aim of this study is to identify eQTLs potentially regulating major XMETs via interference with miR targeting. To this end, we performed a genome-wide screening for eQTLs for 409 genes encoding major drug metabolism enzymes, transporters and transcription factors, in publically available eQTL datasets generated from the HapMap lymphoblastoid cell lines and human liver and brain tissue. As a result, 308 eQTLs significantly (p < 10−5) associated with mRNA expression of 101 genes were identified. We further identified 7,869 SNPs in strong LD (r2 ≥ 0.8) with these eQTLs using the 1,000 Genome SNP data. Among these 8,177 SNPs, 27 are located in the 3′-UTR of 14 genes. Using two algorithms predicting miR-SNP interaction, we found that almost all these SNPs (26 out of 27) were predicted to create, abolish, or change the target site for miRs in both algorithms. Many of these miRs were also expressed in the same tissue that the eQTL were identified. Our study provides a strong rationale for continued investigation for the functions of these eQTLs in pharmacogenetic settings.

Relationship between Differential Hepatic microRNA Expression and Decreased Hepatic Cytochrome P450 3A Activity in Cirrhosis

Background and Aim Liver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis. Methods Hepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity. Results Hepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08). Conclusion The strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.

Genetic Polymorphism of Cytochrome P450 4F2, Vitamin E Level and Histological Response in Adults and Children with Nonalcoholic Fatty Liver Disease Who Participated in PIVENS and TONIC Clinical Trials

Vitamin E improved liver histology in children and adults with NAFLD who participated in TONIC and PIVENS clinical trials, but with significant inter-individual variability in its efficacy. Cytochrome P450 4F2 (CYP4F2) is the major enzyme metabolizing Vit E, with two common genetic variants (V433M, rs2108622 and W12G, rs3093105) found to alter its activity. We investigated the relationship between CYP4F2 genotypes, α-tocopherol levels and histological improvement in these two trials. V433M and W12G variants were genotyped in TONIC (n = 155) and PIVENS (n = 213) DNA samples. The relationships between CYP4F2 genotypes, plasma α-tocopherol levels at baseline and weeks 48 (w48) and 96 (w96) and histological end points (overall improvement in liver histology and resolution of NASH) were investigated. As a result, the V433M genotype was significantly associated with baseline plasma α-tocopherol in the TONIC trial (p = 0.004), but not in PIVENS. Among those receiving Vit E treatment, CYP4F2 V433M genotype was associated with significantly decreased plasma α-tocopherol levels at w48 (p = 0.003 for PIVENS and p = 0.026 for TONIC) but not at w96. The w96 α-tocopherol level was significantly associated with resolution of NASH (p = 0.006) and overall histology improvement (p = 0.021)in the PIVENS, but not in the TONIC trial. There was no significant association between CYP4F2 genotypes and histological end points in either trial. Our study suggested the a moderate role of CYP4F2 polymorphisms in affecting the pharmacokinetics of Vit E as a therapeutic agent. In addition, there may be age-dependent relationship between CYP4F2 genetic variability and Vit E pharmacokinetics in NAFLD.

TERT Polymorphism rs2736100-C Is Associated with EGFR Mutation–Positive Non–Small Cell Lung Cancer

Purpose: EGF receptor (EGFR) mutation–positive (EGFRmut+) non–small cell lung cancer (NSCLC) may be a unique orphan disease. Previous studies suggested that the telomerase reverse transcriptase (TERT) gene polymorphism is associated with demographic and clinical features strongly associated with EGFR mutations, for example, adenocarcinoma histology, never-smoking history, and female gender. We aim to test the association between TERT polymorphism and EGFRmut+ NSCLC. Experimental Design: We conducted a genetic association study in Chinese patients with NSCLC (n = 714) and healthy controls (n = 2,520), between the rs2736100 polymorphism and EGFRmut+ NSCLC. We further tested the association between the EGFR mutation status and mean leukocyte telomere length (LTL). The potential function of rs2736100 in lung epithelial cells was also explored. Results: The rs2736100-C allele was significantly associated with EGFRmut+ NSCLC [OR, 1.52; 95% confidence interval (CI), 1.28–1.80; P = 1.6 × 10−6] but not EGFRmut− NSCLC (OR = 1.07, 95% CI, 0.92–1.24, P = 0.4). While patients with NSCLC as a whole have significantly longer LTL than healthy controls (P ≤ 10−13), the EGFRmut+ patients have even longer LTL than EGFRmut− patients (P = 0.008). Meanwhile, rs2736100 was significantly associated with TERT mRNA expression in both normal and tumor lung tissues. All results remained significant after controlling for age, gender, smoking status, and histology (P < 0.05 for all tests). Moreover, the rs2736100 DNA sequence has an allele-specific affinity to nuclear proteins extracted from lung epithelial cells, which led to an altered enhancer activity of the sequence in vitro. Conclusions: Our study suggests that telomerase and telomere function may be essential for carcinogenesis of EGFRmut+ NSCLC. Further investigation for the underlying mechanism is warranted. Clin Cancer Res; 21(22); 5173–80. ©2015 AACR.

Epidermal Growth Factor Receptor (EGFR) Pathway Genes and Interstitial Lung Disease: An Association Study

The etiology and pathogenesis of idiopathic interstitial lung disease (ILD) remain incompletely understood. Genetic susceptibility to ILD has been demonstrated in previous studies. It is well known that EGFR inhibitors can induce ILD in human lung cancer patient with ethnic differences, which prompted us to hypothesize that genetic variation in EGFR pathway genes confer susceptibility to ILD. We aimed in this study to investigate whether functional polymorphisms of EGFR and its ligands genes (EGF and TGFA) were associated with ILD. Three EGFR [−216G/T (rs712830), −191A/C (rs712829), 497R > K(A/G) (rs2227983)], one EGF [61A/G, (rs4444903)] and one TGFA (rs3821262C/T) polymorphisms previously demonstrated to alter gene functions were genotyped in 229 sporadic idiopathic ILD patients and 693 normal healthy individuals. Allelic and genotypic association tests between these polymorphisms and ILD were performed. The EGF 61A/G polymorphism was significantly associated with elevated risk of ILD, with the frequency of G allele significantly increased in the ILD patient population (OR = 1.33, 95%CI = 1.07–1.66, P = 0.0099). None of the other polymorphisms were associated with risk of ILD. Our study suggested that the EGF 61A/G polymorphism may be associated with sporadic ILD. While a false positive finding cannot be excluded, independent studies are warranted to further validate this result.

Association between hTERT rs2736100 polymorphism and sensitivity to anti-cancer agents

Background: The rs2736100 single nucleotide polymorphism (SNP) is located in the intron 2 of human telomerase reverse transcriptase (hTERT) gene. Recent genome-wide association studies (GWAS) have consistently supported the strong association between this SNP and risk for multiple cancers. Given the important role of the hTERT gene and this SNP in cancer biology, we hypothesize that rs2736100 may also confer susceptibility to anti-cancer drug sensitivity. In this study we aim to investigate the correlation between the rs2736100 genotype and the responsiveness to anti-cancer agents in the NCI-60 cancer cell panel. Methods and Materials: The hTERT rs2736100 was genotyped in the NCI-60 cancer cell lines. The relative telomere length (RTL) of each cell line was quantified using real-time PCR. The genotype was then correlated with publically available drug sensitivity data of two agents with telomerase-inhibition activity: Geldanamycin (HSP90 inhibitor) and RHPS4/BRACO19 (G-quadruplex stabilizer) as well as additional 110 commonly used agents with established mechanism of action. The association between rs2736100 and mutation status of TP53 gene was also tested. Results: The C allele of the SNP was significantly correlated with increased sensitivity to RHPS4/BRACO19 with an additive effect (r = −0.35, p = 0.009) but not with Geldanamycin. The same allele was also significantly associated with sensitivity to antimitotic agents compared to other agents (p = 0.003). The highest correlation was observed between the SNP and paclitaxel (r = −0.36, p = 0.005). The telomere length was neither associated with rs2736100 nor with sensitivity to anti-cancer agents. The C allele of rs2736100 was significantly associated with increased mutation rate in TP53 gene (p = 0.004). Conclusion: Our data suggested that the cancer risk allele of hTERT rs2736100 polymorphism may also affect the cancer cell response to both TERT inhibitor and anti-mitotic agents, which might be attributed to the elevated telomerase-independent activity of hTERT, as well as the increased risk for TP53 gene mutagenesis conferred by the polymorphism. Detailed mechanisms need to be further investigated.