Shiyang Pan

Early second-trimester serum miRNA profiling predicts gestational diabetes mellitus.

Background Gestational diabetes mellitus (GDM) is one type of diabetes that presents during pregnancy and significantly increases the risk of a number of adverse consequences for the fetus and mother. The microRNAs (miRNA) have recently been demonstrated to abundantly and stably exist in serum and to be potentially disease-specific. However, no reported study investigates the associations between serum miRNA and GDM. Methodology/Principal Findings We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to screen miRNAs in serum collected at 16–19 gestational weeks. The expression levels of three miRNAs (miR-132, miR-29a and miR-222) were significantly decreased in GDM women with respect to the controls in similar gestational weeks in our discovery evaluation and internal validation, and two miRNAs (miR-29a and miR-222) were also consistently validated in two-centric external validation sample sets. In addition, the knockdown of miR-29a could increase Insulin-induced gene 1 (Insig1) expression level and subsequently the level of Phosphoenolpyruvate Carboxy Kinase2 (PCK2) in HepG2 cell lines. Conclusions/Significance Serum miRNAs are differentially expressed between GDM women and controls and could be candidate biomarkers for predicting GDM. The utility of miR-29a, miR-222 and miR-132 as serum-based non-invasive biomarkers warrants further evaluation and optimization.

Potentially functional polymorphisms in DNA repair genes and non-small-cell lung cancer survival: A pathway-based analysis†

To assess systematically whether potentially functional polymorphisms in DNA repair genes influence the clinical behavior of non‐small‐cell lung cancer (NSCLC), we examined the impact of a comprehensive panel of 218 signal nucleotide polymorphisms (SNP) in 50 candidate DNA repair genes on overall survival of NSCLC in a case‐cohort of 568 lung cancer patients. SNPs associated with lung cancer prognosis primarily mapped to 14 genes in different repair pathways, and 6 SNPs were remained in the final model after multivariate stepwise Cox regression analysis: ATM rs189037; MRE11A rs11020802; ERCC2 rs1799793; MBD4 rs140693; XRCC1 rs25487, and PMS1 rs5742933. In the combined analysis of these 6 SNPs, an increasing number of unfavorable loci was associated with a poorer prognosis (P for trend: <0.0001) and patients having 2–4 unfavorable loci had a 1.99‐fold elevated risk of death 95% confidence interval (CI) = 1.58–2.50, compared with those carrying 0–1 unfavorable loci, and this elevated risk was more evident among stages I–II patients (hazard ratio = 3.04, 95% CI = 1.86–4.98, P for heterogeneity: 0.07). Furthermore, a significant effect of SNPs in nucleotide excision repair pathway on lung cancer survival was observed among 185 stages III–IV patients treated with platinum‐based chemotherapy without surgical operation: XPC rs2228000 (Ala499Val; P = 0.002) and ERCC1 rs11615 (Asn118Asn; P = 0.012). Our data indicate that potentially functional polymorphisms in DNA repair genes may serve as candidate prognostic markers of clinical outcome of NSCLC.

Genetic polymorphisms in the precursor MicroRNA flanking region and non-small cell lung cancer survival.

RATIONALE Previously, we reported that common variants in precursor microRNA (pre-miRNA) sequences played a role in the prediction of non-small cell lung cancer (NSCLC) survival. OBJECTIVES To assess whether variants in the pre-miRNA flanking region can influence the clinical behavior of NSCLC. METHODS We conducted a two-stage study to examine the impact of a panel of 85 single-nucleotide polymorphisms on the overall survival of 923 patients with NSCLC (568 in the screening set and 355 in the validation set) in China. MEASUREMENTS AND MAIN RESULTS Eleven single-nucleotide polymorphisms were primarily associated with NSCLC survival in the univariate analysis. However, in the validation set, only miR-30c-1 rs928508 was consistently an NSCLC survival predictor and the protective role of rs928508 AG/GG genotypes was more pronounced among early-stage (stage I/II) patients and patients treated with surgery. The area under the curve at Year 5 was significantly increased from 0.658 to 0.741 after adding the miR-30c-1 rs928508 risk score to the traditional clinical risk score (stage and surgery). Furthermore, in the genotype-phenotype correlation analysis, rs928508 AG/GG genotypes were associated with a significantly decreased expression of precursor and mature miR-30c (P = 0.009 and 0.011), but not with that of its primary miRNA. The expression of the host nuclear transcription factor Y gene was correlated with pri-mir-30c-1, but not with rs928508 genotypes, implicating the coregulation of the transcription of nuclear transcription factor Y and pri-mir-30c-1. CONCLUSIONS Our data indicated, for the first time, that genetic polymorphisms in the pre-miRNA flanking region may be prognostic biomarkers of NSCLC, and rs928508 is such a potential candidate.

Characterization of Osterix protein stability and physiological role in osteoblast differentiation.

Osterix (Osx/SP7) is a C2H2 zinc finger-containing transcription factor of the SP gene family. Osx knockout mice indicate that the gene plays an essential role in osteoblast differentiation and bone formation. However, the mechanisms involved in the regulation of Osx are still poorly understood. Here, we report a novel post-translational mechanism for the regulation of Osx in mammalian cells. We found that the stability of endogenous and exogenous Osx reduced after cycloheximide treatment. In cells treated with the proteasome inhibitors MG-132 or lactacystin, both endogenous and exogenous Osx protein expression increased in a time-dependent manner. Co-immunoprecipitation (Co-IP) assays showed that both endogenous and exogenous Osx were ubiquitinated. Six lysine residues of Osx were identified as candidate ubiquitination sites by construction of point mutant plasmids and luciferase reporter assays. Furthermore, we confirmed that K58 and K230 are the ubiquitination sites of Osx by Co-IP assays and protein stability assays. Moreover, the Osx K58R and K230R mutations promoted the expression of osteoblast differentiation markers (alkaline phosphatase, collagen I and osteocalcin) and enhanced osteogenic differentiation in C2C12 cells. Taken together, our data indicate that Osx is an unstable protein, and that the ubiquitin-proteasome pathway is involved in the regulation of Osx and thereby regulates osteoblast differentiation.

Analysis of CD8+ Treg cells in patients with ovarian cancer: a possible mechanism for immune impairment.

Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment that blunts successful anti-tumor immunotherapy. Recent studies show that CD8+ Treg cells might impede effective immune responses to established tumors. However, there is limited research regarding CD8+ Treg cells in ovarian cancer (OC) patients. Here, we investigated CD8+ Treg cells in OC patients and their in vitro induction. The immunohistochemistry of tumor-infiltrating lymphocytes revealed a significant correlation between the intratumoral CD8+ T cells and the forkhead box p3 (Foxp3)+ cells in the intraepithelial and stromal areas of advanced OC tissues. We examined the expression of Treg markers in CD8+ T cells from the peripheral blood and fresh tumor tissues of OC patients using flow cytometry. Our results indicated an increase in the CD8+ Treg cell subsets of OC patients compared with those in patients with benign ovarian tumors and healthy controls, including an increased expression of CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Foxp3 and decreased CD28 expression. To demonstrate whether the tumor microenvironment could convert CD8+ effector T cells into suppressor cells, we used an in vitro transwell culturing system. Compared with the CD8+ T cells cultured alone, the CD8+ Treg cells induced in vitro by coculture with SK-OV-3/A2780 showed increased CTLA-4 and Foxp3 expression and decreased CD28 expression. In addition, the in vitro-induced CD8+ Treg cells inhibited naı¨ve CD4+ T-cell proliferation, which was partially mediated through TGF-β1 and IFN-γ. Our study suggests that CD8+ Treg cells were increased in OC patients and could be induced in vitro, which may be the way that tumors limit antitumor immunity and evade immune surveillance.

Kaposi's sarcoma-associated herpesvirus (KSHV) vIL-6 promotes cell proliferation and migration by upregulating DNMT1 via STAT3 activation.

Kaposis sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposis sarcoma (KS), the most common AIDS-related malignancy. KSHV vIL-6 promotes KS development, but the exact mechanisms remain unclear. Here, we reported that KSHV vIL-6 enhanced the expression of DNA methyltransferase 1 (DNMT1) in endothelial cells,increased the global genomic DNA methylation, and promoted cell proliferation and migration. And this effect could be blocked by the DNA methyltransferase inhibitor, 5-azadeoxycytidine. We also showed that vIL-6 induced up-regulation of DNMT1 was dependent on STAT3 activation. Therefore, the present study suggests that vIL-6 plays a role in KS tumorigenesis partly by activating DNMT1 and inducing aberrant DNA methylation, and it might be a potential target for KS therapy.

Potentially functional polymorphisms in cell cycle genes and the survival of non-small cell lung cancer in a Chinese population

The cell cycle governs the proliferation and growth of cells and is strictly controlled by some regulators including cyclins, CDKs and CKIs. Germ-line and somatic mutations in cell cycle genes were frequently observed in a subset of cancers including non-small cell lung cancer (NSCLC). In this study, we hypothesized that potentially functional single nucleotide polymorphisms (SNPs) in cell cycle genes may contribute to the prognosis of NSCLC in China. 54 potentially functional polymorphisms in key cell cycle genes (CDK1, CDK2, CDK4, CDK6, CDK7, CCND1, CCND2, CCND3, CCNE1, CCNA1, CCNA2, CCNB1, CCNH, p15, p16, p18, p19, p21, p27, Cdc25A and Cdc25B) were genotyped by using Illumina SNP genotyping platform to evaluate their associations with survival of NSCLC in a clinical cohort of 568 patients. We found that p18 rs3176447 variant genotypes were significantly associated with the decreased risk of death of NSCLC patients (adjusted HR=0.74, 95% CI=0.57-0.97 in an additive model; adjusted HR=0.76, 95% CI=0.55-0.97 in a dominant model); however, p21 rs2395655 variant genotypes were significantly associated with the increased risk of death (adjusted HR=1.21, 95% CI=1.02-1.42 in an additive model; adjusted HR=1.38, 95% CI=1.07-1.78 in a recessive model). Furthermore, the combined effect of unfavorable genotypes for these two SNPs was more prominent in patients with squamous cell carcinoma, late stage and without chemo- or radio-therapy. Although the exact biological function remains to be explored, our findings suggest possible association of polymorphisms of p18 and p21 with the prognosis of NSCLC in a Chinese population. Further large and functional studies are needed to confirm our findings.

High prevalence of hypovitaminosis D among pregnant women in southeast China.

Vitamin D is essential to maintain bone health, playing a key role in calcium absorption and bone mineralization (1). In addition, it may confer protection against type 1 diabetes mellitus, hypertension, multiple sclerosis and cancer (2). As stores of vitamin D in newborns are reliant on maternal vitamin D status, vitamin D deficiency during pregnancy leads to infant vitamin D deficiency and thus to increased risk of diseases, especially rickets (3). Moreover, because vitamin D can regulate placental development and function, maternal vitamin D deficiency is associated with adverse outcomes of pregnancy, such as miscarriage, preterm birth and preeclampsia (4). Given the importance of vitamin D, the aim of this study is to investigate vitamin D levels of pregnant women in southeast China. One hundred and fifty-two pregnant women of Han race who visited the First Affiliated Hospital of Nanjing Medical University for routine medical checkup for pregnancy agreed to participate in this study. The study population aged 19–41 years (mean ± SD, 27.4 ± 4.15). Samples were collected at 24–28 weeks of gestation. Half of the samples were collected in winter, and the other half were collected in summer. All the participants were urban residents at Nanjing (latitude 31 N, longitude 118 E) in southeast China. Patients with chronic renal failure, liver diseases or therapy with vitamin D were strictly excluded from this study. The hospital’s ethics committee approved this study. And informed written consent was obtained from each subject. Daily intake of dietary vitamin D was calculated from a food-frequency questionnaire. Daily sun exposure was assessed by taking a detailed history of the daily routine including the time of outdoor activities and the type of clothing worn. Sunshine exposure was calculated as hours of exposure ⁄ day · percentage of body surface area exposed. Blood was collected in the fasting condition, and sera were immediately isolated by centrifugation and stored at )70 C for future analysis. Serum 25-hydroxyvitamin D [25(OH)D] level was measured by using a commercial enzyme linked immunosorbent assay (ELISA) kit (IDS, Boldon, UK). The ELISA was performed according to the manufacturer’s protocol and all samples were assayed in triplicate. The ELISA could detect 25(OH)D in the range of 2–152 ng ⁄ mL. And the inter-assay and intra-assay coefficient of variation was 10% and 8%, respectively. For our analysis, we classified the vitamin D status into three groups: vitamin D deficiency [25(OH)D < 10 ng ⁄ mL], vitamin D insufficiency [25(OH)D 10–20 ng ⁄ mL] and vitamin D sufficiency [25(OH)D > 20 ng ⁄ mL]. Difference between the two groups was analysed by unpaired t-test. The significance level was set to <0.05. Data were analysed using SPSS, version 13.0 (SPSS Inc, Chicago, IL, USA). No difference between summer and winter groups was observed in age, body mass index and daily vitamin D intake. However, sun exposure was significant lower in winter group than in summer group (p < 0.01) (Table 1). Mean 25(OH)D concentrations of 152 pregnant women were 10.9 ± 4.78 ng ⁄ mL. The seasonal distribution of serum 25(OH)D levels were shown in Figure 1. Mean serum 25(OH)D level in winter (9.07 ± 5.07 ng ⁄ mL) was Acta Pædiatrica ISSN 0803–5253

Plasma Vitamin D Levels And Vitamin D Receptor Polymorphisms Are Associated with Survival of Non-small Cell Lung Cancer

ObjectiveVitamin D and its receptor (VDR) involve in multiple cellular processes and play an important role in the initiation and progression of malignancy. Thus we hypothesized that plasma vitamin D levels and single nucleotide polymorphisms (SNPs) in VDR may be of prognostic significance in non-small cell lung cancer (NSCLC).MethodsWe examined plasma 25-hydroxyvitamin D [25(OH)D] levels in 87 patients diagnosed with NSCLC using enzyme-linked immunosorbent assay (ELISA) and genotyped seven potentially functional SNPs in VDR in 568 NSCLC patients on Illumina Golden Gate platform.ResultsPatients with higher plasma 25(OH)D levels had worse survival than patients with lower ones (P for trend = 0.048). The SNPs of rs1544410 and rs739837 were independently associated with NSCLC survival (adjusted HR = 1.61, 95% CIs = 1.06-2.45 for rs739837 AA vs AC/CC and adjusted HR = 1.51, 95% CIs = 1.06-2.16 for rs1544410 AG/AA vs GG). A joint effect was observed between rs1544410 and rs739837 and the risk of death elevated as the number of unfavourable genotypes patients carried increased (P for trend = 0.003). There were no significant associations between VDR polymorphisms and plasma 25(OH)D levels.ConclusionOur findings indicate that plasma 25(OH)D levels and genetic variants of VDR may serve as prognostic markers for NSCLC in this Chinese population.

miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy.

MicroRNAs (miRNAs), a class of small non-coding RNAs, mediate gene expression by either cleaving target mRNAs or inhibiting their translation. They have key roles in the tumorigenesis of several cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to investigate the clinical significance of miR-638 in the evaluation of NSCLC patient prognosis in response to chemotherapy. First, we detected miR-638 expression levels in vitro in the culture supernatants of the NSCLC cell line SPC-A1 treated with cisplatin, as well as the apoptosis rates of SPC-A1. Second, serum miR-638 expression levels were detected in vivo by using nude mice xenograft models bearing SPC-A1 with and without cisplatin treatment. In the clinic, the serum miR-638 levels of 200 cases of NSCLC patients before and after chemotherapy were determined by quantitative real-time PCR, and the associations of clinicopathological features with miR-638 expression patterns after chemotherapy were analyzed. Our data helped in demonstrating that cisplatin induced apoptosis of the SPC-A1 cells in a dose- and time-dependent manner accompanied by increased miR-638 expression levels in the culture supernatants. In vivo data further revealed that cisplatin induced miR-638 upregulation in the serum derived from mice xenograft models, and in NSCLC patient sera, miR-638 expression patterns after chemotherapy significantly correlated with lymph node metastasis. Moreover, survival analyses revealed that patients who had increased miR-638 levels after chemotherapy showed significantly longer survival time than those who had decreased miR-638 levels. Our findings suggest that serum miR-638 levels are associated with the survival of NSCLC patients and may be considered a potential independent predictor for NSCLC prognosis.