Integrated analysis of key microRNAs /TFs /mRNAs/ in HPV-positive cervical cancer based on microRNA sequencing and bioinformatics analysis.

BACKGROUND: Cervical squamous cell carcinoma (CESC) is one of the most common malignancies associated with mortality in females. Its onset and prognosis are primarily concerned with persistent infection with high-risk types of human papillomavirus (HPV). However, the molecular mechanisms of HPV-positive CESC remain unclear. METHODS: In this study, we conducted a high-throughput sequencing to identify differentially expressed miRNAs (DEMs). Besides, three series were selected from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Then the miRNA-TF-gene regulatory network was constructed using bioinformatic methods. Genes in the network were performed functional enrichment analysis and protein-protein interaction (PPI) network analysis. Ultimately, the expression levels of six key miRNAs, TFs, and mRNAs were validated by 20 HPV-positive CESC tissues and 15 normal cervical samples. RESULTS: A total of 52 DEMs and 300 DEGs differed between the HPV-positive CESC and normal cervical samples. Then the miRNA-TF-gene regulatory network was constructed consisting of 22 miRNAs, 6 TFs, and 76 corresponding genes, among which miR-149-5p, miRNA-1248 and E2F4 acted as key regulators. PPI network analysis showed that ten genes including TOP2A, AURKA, CHEK1, KIF11, MCM4, MKI67, DTL, FOXM1, SMC4, and FBXO5 were recognized as hub genes with the highest connectivity degrees. Besides, five key molecules miRNA-149-5p, E2F4, KIF11, DTL, and SMC4 were suggested to play crucial roles in the development of HPV-positive CESC. CONCLUSION: These results present a unique insight into the pathological mechanisms of HPV-positive CESC and possibly provides potential therapeutic targets.

Development of a general logistic model for disease risk prediction using multiple SNPs.

Human diseases are usually linked to multiloci genetic alterations, including single-nucleotide polymorphisms (SNPs). Methods to use these SNPs for disease risk prediction (DRP) are of clinical interest. DRP algorithms explored by commercial companies to date have tended to be complex and led to controversial prediction results. Here, we present a general approach for establishing a logistic model-based DRP algorithm, in which multiple SNP risk factors from different publications are directly used. In particular, the coefficient ß of each SNP is set as the natural logarithm of the reported odds ratio, and the constant coefficient ß0 is comprehensively determined by the coefficient and frequency of each SNP and the average disease risk in populations. Furthermore, homozygous SNP is considered a dummy variable, and the SNPs are updated (addition, deletion and modification) if necessary. Importantly, we validated this algorithm as a proof of concept: two patients with lung cancer were identified as the maximum risk cases from 57 Chinese individuals. Our logistic model-based DRP algorithm is apparently more intuitive and self-evident than the algorithms explored by commercial companies, and it may facilitate DRP commercialization in the era of personalized medicine.

Positive Association between ANKRD55 Polymorphism 7731626 and Dermatomyositis/Polymyositis with Interstitial Lung Disease in Chinese Han Population.

Single nucleotide polymorphisms (SNPs) in TNFSF4 and ANKRD55 genes have been shown to be associated with several autoimmune diseases, although whether these genes are susceptibility genes for dermatomyositis/polymyositis (DM/PM) has, to date, not been reported. This study aimed to investigate the potential associations of these SNPs with DM/PM in a Chinese Han population. Five SNPs in TNFSF4 (rs2205960, rs844644, and rs844648) and ANKRD55 (rs6859219, rs7731626) genes were genotyped using the SequenomMassArray system in 2297 Chinese individuals. In total, 1017 DM/PM patients and 1280 gender-matched healthy controls were genotyped. No significant associations were observed in DM/PM patients for the five SNPs analyzed. The association between SNPs and interstitial lung disease (ILD) was also investigated. Both DM-ILD (Pc = 0.030, OR = 0.65, 95% CI: 0.47-0.88) and DM/PM-ILD (Pc = 0.015, OR = 0.67, 95% CI: 0.51-0.87) exhibited a significant association with the rs7731626-A allele. Rs7731626-A was less frequently found in DM-ILD and DM/PM-ILD patients compared with healthy controls. This is the first study to demonstrate a positive association between ANKRD55 polymorphism and DM-ILD and DM/PM-ILD. A decreased frequency of rs7731626-A in DM-ILD and DM/PM-ILD patients suggests that the A variant may be protective against DM/PM-ILD.

Single-nucleotide polymorphisms in the PLA2R1 gene are associated with systemic lupus erythematosus and lupus nephritis in a Chinese Han population.

Several novel susceptibility genes for systemic lupus erythematosus (SLE) and other nephropathy have been identified in recent genome-wide association studies. Membranous nephropathy is the most common diagnosis in adults with the nephrotic syndrome, and both idiopathic membranous nephropathy (IMN) and lupus nephritis (LN) are autoimmune diseases of the kidney; they may share common disease mechanisms that overlap with genetic susceptibility. Therefore, we sought to identify genetic variants associated with IMN in SLE/LN. The PLA2R1 single-nucleotide polymorphisms rs4664308, rs3828323, rs3792189, and rs3792192 were genotyped in a cohort of 1247 SLE patients and 1174 healthy controls, using the Sequenom MassArray system method. PLA2R1 displayed a nominally significantly genetic association with SLE [for rs4664308, P = 0.02, odds ratio (OR) 1.16, 95 % confidence interval (CI) 1.02-1.31; for rs3792192, P = 7.9 × 10(-3), OR 1.18, 95 % CI 1.05-1.34] and LN (for rs4664308, P = 0.04). The frequencies of genotypes of rs3792189 and rs3828323 were significantly different between the SLE patients and controls (all P < 0.05), and the haplotype (AA) formed by rs3792189 and rs3792192 was associated with SLE (P = 0.019). This was the first study to reveal that PLA2R1 polymorphisms were associated with SLE/LN patients, indicating that PLA2R1 might be a susceptibility gene for SLE/LN in a Chinese Han population.

An Integrative Analysis of the Putative Gefitinib-resistance Related Genes in a Lung Cancer Cell Line Model System.

The epidermal growth factor receptor-tyrosine kinase inhibitors (EGFRTKI), such as gefitinib and erlotinib have improved the survival of patients with nonsmall cell lung cancer (NSCLC). Unfortunately, acquired resistance will eventually develop in most patients who initially respond to the therapy. Currently known molecular mechanisms for such an acquired resistance may interpret only about 70% of clinical cases. In this study, using NSCLC cell model H1650, we constructed a gefitinib resistant cell line H1650GR through long term drug exposure with increased doses. RNA sequencing and whole genome SNP array were applied to investigate the transcriptome and genome alterations possibly involved in gefitinib resistance. By comparing the expression profiles between H1650GR and H1650 cells, we identified a large set of differentially expressed genes (DEGs), including FOXM1. In the PI3K/AKT pathway, AKT activity was predicted to be inhibited. However, genes that play important roles in gefitinib-induced apoptosis, including TP53, FOXO3 and BAD, were not up-regulated. Ingenuity Pathway Analysis (IPA) canonical pathway analysis showed that p53 signaling was inhibited in H1650GR cells, with down-regulation of pro-apoptosis genes FAS, PUMA, NOXA, and upregulation of anti-apoptosis genes BIRC5/Survivin. Besides, a large number of immune response-related genes were differently expressed, the role of which in gefitinib resistance requires further investigation. Whole genome copy number alterations (CNAs) were also analyzed and NOXA was located in the H1650GR unique copy number loss region, 18q21. Our results suggested that the much higher EGFR-TKI resistance in H1650GR may be produced by the integration of multi-aspect factors.

Systematic analysis of alternative first exons in plant genomes.

BACKGROUND: Alternative splicing (AS) contributes significantly to protein diversity, by selectively using different combinations of exons of the same gene under certain circumstances. One particular type of AS is the use of alternative first exons (AFEs), which can have consequences far beyond the fine-tuning of protein functions. For example, AFEs may change the N-termini of proteins and thereby direct them to different cellular compartments. When alternative first exons are distant, they are usually associated with alternative promoters, thereby conferring an extra level of gene expression regulation. However, only few studies have examined the patterns of AFEs, and these analyses were mainly focused on mammalian genomes. Recent studies have shown that AFEs exist in the rice genome, and are regulated in a tissue-specific manner. Our current understanding of AFEs in plants is still limited, including important issues such as their regulation, contribution to protein diversity, and evolutionary conservation. RESULTS: We systematically identified 1,378 and 645 AFE-containing clusters in rice and Arabidopsis, respectively. From our data sets, we identified two types of AFEs according to their genomic organisation. In genes with type I AFEs, the first exons are mutually exclusive, while most of the downstream exons are shared among alternative transcripts. Conversely, in genes with type II AFEs, the first exon of one gene structure is an internal exon of an alternative gene structure. The functionality analysis indicated about half and approximately 19% of the AFEs in Arabidopsis and rice could alter N-terminal protein sequences, and approximately 5% of the functional alteration in type II AFEs involved protein domain addition/deletion in both genomes. Expression analysis indicated that 20-66% of rice AFE clusters were tissue- and/or development- specifically transcribed, which is consistent with previous observations; however, a much smaller percentage of Arabidopsis AFEs was regulated in this manner, which suggests different regulation mechanisms of AFEs between rice and Arabidopsis. Statistical analysis of some features of AFE clusters, such as splice-site strength and secondary structure formation further revealed differences between these two species. Orthologous search of AFE-containing gene pairs detected only 19 gene pairs conserved between rice and Arabidopsis, accounting only for a few percent of AFE-containing clusters. CONCLUSION: Our analysis of AFE-containing genes in rice and Arabidopsis indicates that AFEs have multiple functions, from regulating gene expression to generating protein diversity. Comparisons of AFE clusters revealed different features in the two plant species, which indicates that AFEs may have evolved independently after the separation of rice (a model monocot) and Arabidopsis (a model dicot).

Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization.

AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients. METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system. The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and R110 or TAMRA labeled acyclo-terminator was added on the 3' end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed. RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9%), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD phenotype (7.1%). CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.