BackgroundTumor suppressive let-7 miRNAs are universally down-regulated in human hepatocellular carcinoma (HCC) versus normal tissues; however, the roles and related molecular mechanisms of let-7 in HCC stem cells are poorly understood.MethodsWe examined the inhibitory effect of let-7 miRNAs on the proliferation of MHCC97-H and HCCLM3 hepatic cancer cells by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was further confirmed by apoptosis and cell cycle studies. The sphere-forming assay was used to study the effects of let-7a on stem like cells. Through western blot, immunofluorescence and the luciferase-reporter assay, we explored the activity of epithelial-mesenchymal transition (EMT) signaling factors in HCC cells. qRT-PCR was applied to detect miRNA expression levels in clinical tissues.ResultsLet-7a effectively repressed cell proliferation and viability, and in stem-like cells, also let-7a decreased the efficiency of sphere formation.in stem-like cells. The suppression of EMT signaling factors in HCC cells contributed to let-7’s induced tumor viability repression and Wnt activation repression. Besides, Wnt1 is critical and essential for let-7a functions, and the rescue with recombinant Wnt1 agent abolished the suppressive roles of let-7a on hepatospheres. In clinical HCC and normal tissues, let-7a expression was inversely correlated with Wnt1 expression.ConclusionsLet-7 miRNAs, especially let-7a, will be a promising therapeutic strategy in the treatment of HCC through eliminating HCC stem cells, which could be achieved by their inhibitory effect on the Wnt signaling pathway.

BackgroundSLC52A3 was recently identified as a susceptibility gene for esophageal squamous cell carcinoma (ESCC). However, associations between the single nucleotide polymorphisms (SNPs) rs13042395 (C > T) and rs3746803 (G > A) in SLC52A3 and risk, tumor characteristics and survival of ESCC patients remain inconclusive and of unknown prognostic significance.MethodsAnalyses of the association between SNPs in SLC52A3 and ESCC risk were performed on 479 ESCC cases, together with 479 controls, in a case-control study. Blood samples for cases and controls were collected and genotyped by real-time polymerase chain reaction (PCR) using TaqMan assays. Among the 479 ESCC cases, 343 cases with complete clinical data were used to investigate the association between SNPs and ESCC clinical characteristics; 288 cases with complete clinical data and 5-year follow-up data were used to analyze the association between SNPs and prognosis. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSAs) were used to investigate the biological function of rs13042395.ResultsNo association was found between SLC52A3 rs3746803 and susceptibility, tumor characteristics or survival of ESCC patients. For rs13042395, TT genotype carriers were likely to have reduced lymph node metastasis (odds ratio (OR) = 0.55, 95 % confidence interval (CI), 0.31–0.98) and longer relapse-free survival time (P = 0.03) . Also, both rs13042395 (hazard ratio (HR) = 0.62, 95 % CI, 0.38–0.99) and regional lymph node metastasis (HR = 2.06, 95 % CI, 1.36–3.13 for N1 vs. N0; HR = 2.88, 95 % CI, 1.70–4.86 for N2 vs. N0; HR = 2.08, 95 % CI, 1.01–4.30 for N3 vs. N0) were independent factors affecting relapse-free survival for ESCC patients who underwent surgery. Dual luciferase reporter assays and EMSAs suggested that the CC genotype of rs13042395 enhanced SLC52A3 expression, probably via binding with specific transcription factors.ConclusionsThe rs13042395 polymorphism in SLC52A3 is associated with regional lymph node metastasis and relapse-free survival in ESCC patients.

BackgroundMitogen/extracellular signal-regulated kinase kinase-5 (MEK5) has been confirmed to play a pivotal role in tumor carcinogenesis and progression. However, few studies have investigated the role of MEK5 in colorectal cancer (CRC).MethodsMEK5 expression was determined by immunohistochemistry (IHC) in tissue microarrays (TMAs) containing 2 groups of tissues, and western blotting was used to confirm MEK5 expression in 8 cases of primary CRC tissues and paired normal mucosa. RNA interference was used to verify the biological function of MEK5 gene in the development of CRC.ResultsIHC revealed the expression of MEK5 was higher in tumor tissues (38.1 %), compared with adjacent normal tissue (8.3 %). Western blot showed that, MEK5 expression was upregulated in CRC tumor tissues compared with normal tissue. Analysis of clinical pathology parameters indicated MEK5 overexpression was significantly correlated with the depth of invasion, lymph node metastasis, distant metastasis and histological grade. Survival analysis revealed that MEK5 overexpression negatively correlated with cancer-free survival (hazard ratio 1.64, P = 0.017). RNA interference-mediated knockdown of MEK5 in SW480 colon cancer cells decreased their proliferation, division, migration and invasiveness in vitro and slowed down tumors growth in mice engrafted with the cells.ConclusionMEK5 plays an important role in CRC progression and may be a potential molecular target for the treatment of CRC.

BackgroundJuvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children, the etiology of which involves a strong genetic component, but much of the underlying genetic determinants still remain unknown. Our aim was to identify novel genetic variants that predispose to JIA.MethodsWe performed a genome-wide association study (GWAS) and replication in a total of 1166 JIA cases and 9500 unrelated controls of European ancestry. Correlation of SNP genotype and gene expression was investigated. Then we conducted targeted resequencing of a candidate locus, among a subset of 480 cases and 480 controls. SUM test was performed to evaluate the association of the identified rare functional variants.ResultsThe CXCR4 locus on 2q22.1 was found to be significantly associated with JIA, peaking at SNP rs953387. However, this result is subjected to subpopulation stratification within the subjects of European ancestry. After adjusting for principal components, nominal significant association remained (p < 10−4). Because of its interesting known function in immune regulation, we carried out further analyses to assess its relationship with JIA. Expression of CXCR4 was correlated with CXCR4 rs953387 genotypes in lymphoblastoid cell lines (p = 0.014) and T-cells (p = 0.0054). In addition, rare non-synonymous and stop-gain sequence variants in CXCR4, putatively damaging for CXCR4 function, were significantly enriched in JIA cases (p = 0.015).ConclusionOur results suggest the association of CXCR4 variants with JIA, implicating that this gene may be involved in the pathogenesis of autoimmune disease. However, because this locus is subjected to population stratification within the subjects of European ancestry, additional replication is still necessary for this locus to be considered a true risk locus for JIA. This cell-surface chemokine receptor has already been targeted in other diseases and may serve as a tractable therapeutic target for a specific subset of pediatric arthritis patients with additional replication and functional validation of the locus.

BackgroundRecently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known.ResultsHere, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5–10 molecules.ConclusionsBased on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

BackgroundThe exact cerebral structural and functional mechanisms under the auditory verbal hallucinations (AVHs) in schizophrenia are still unclear. The Deutsch “high-low” word illusion might trigger attentional responses mimicking those under AVHs.MethodsWe therefore have invited 16 patients with first-episode, paranoid schizophrenia, and 16 age- and gender-matched healthy volunteers to undergo the “oddball” event-related potentials elicited by the illusion. The clinical characteristics of patients were measured with the positive and negative symptom scale.ResultsBesides the longer reaction time to the illusion, the standard P2 latency was shortened, the N2 latency was prolonged, and both N1 and P3 amplitudes were reduced in patients. The P3 source analyses showed the activated bilateral temporal lobes, parietal lobe and cingulate cortex in both groups, left inferior temporal gyrus in controls, and left postcentral gyrus in schizophrenia. Moreover, the N1 amplitude was positively correlated with the paranoid score in patients.ConclusionsOur results were in line with previous neurophysiological and neuroimaging reports of hallucination or auditory processing in schizophrenia, and illustrated a whole process of cerebral information processing from N1 to P3, indicating this illusion had triggered a dynamic cerebral response similar to that of the AVHs had engaged.