Genomic and Transcriptomic Landscape of Tumor Clonal Evolution in Cholangiocarcinoma.

Cholangiocarcinoma remained a severe threat to human health. Deciphering the genomic and/or transcriptomic profiles of tumor has been proved to be a promising strategy for exploring the mechanism of tumorigenesis and development, which could also provide valuable insights into Cholangiocarcinoma. However, little knowledge has been obtained regarding to how the alteration among different omics levels is connected. Here, using whole exome sequencing and transcriptome sequencing, we performed a thorough evaluation for the landscape of genome and transcriptome in cholangiocarcinoma and illustrate the alteration of tumor on different biological levels. Meanwhile, we also identified the clonal structure of each included tumor sample and discovered different clonal evolution patterns related to patients' survival. Furthermore, we extracted subnetworks that were greatly influenced by tumor clonal/subclonal mutations or transcriptome change. The topology relationship between genes affected by genomic/transcriptomic changes in biological interaction networks revealed that alteration of genome and transcriptome was highly correlated, and somatic mutations located on important genes might affect the expression of numerous genes in close range.

MEIS2C and MEIS2D promote tumor progression via Wnt/ß-catenin and hippo/YAP signaling in hepatocellular carcinoma.

BACKGROUND: MEIS2 has been identified as one of the key transcription factors in the gene regulatory network in the development and pathogenesis of human cancers. Our study aims to identify the regulatory mechanisms of MEIS2 in hepatocellular carcinoma (HCC), which could be targeted to develop new therapeutic strategies. METHODS: The variation of MEIS2 levels were assayed in a cohort of HCC patients. The proliferation, clone-formation, migration, and invasion abilities of HCC cells were measured to analyze the effects of MEIS2C and MEIS2D (MEIS2C/D) knockdown with small hairpin RNAs in vitro and in vivo. Chromatin immunoprecipitation (ChIP) was performed to identify MEIS2 binding site. Immunoprecipitation and immunofluorescence assays were employed to detect proteins regulated by MEIS2. RESULTS: The expression of MEIS2C/D was increased in the HCC specimens when compared with the adjacent noncancerous liver (ANL) tissues. Moreover, MEIS2C/D expression negatively correlated with the prognosis of HCC patients. On the other hand, knockdown of MEIS2C/D could inhibit proliferation and diminish migration and invasion of hepatoma cells in vitro and in vivo. Mechanistically, MESI2C activated Wnt/ß-catenin pathway in cooperation with Parafibromin (CDC73), while MEIS2D suppressed Hippo pathway by promoting YAP nuclear translocation via miR-1307-3p/LATS1 axis. Notably, CDC73 could directly either interact with MEIS2C/ß-catenin or MEIS2D/YAP complex, depending on its tyrosine-phosphorylation status. CONCLUSIONS: Our studies indicate that MEISC/D promote HCC development via Wnt/ß-catenin and Hippo/YAP signaling pathways, highlighting the complex molecular network of MEIS2C/D in HCC pathogenesis. These results suggest that MEISC/D may serve as a potential novel therapeutic target for HCC.

Identifying key regulating miRNAs in hepatocellular carcinomas by an omics' method.

The miRNAs play important regulating roles in the pathogenesis of hepatocellular carcinoma (HCC). To uncover key regulating miRNAs in HCC that were neglected by traditional analyzing methods of transcriptomics data, we proposed a novel molecular-network-based omics' (MNBO) method. With this method, we predicted HCC-regulating miRNAs, and confirmed the role of a novel miR-590-3P/EED axis by a clinical study and in vitro, in vivo wet-experiments. The miR-590-3P is significantly down-regulated in HCC patients. And low level of miR-590-3P in HCC is associated with poor prognosis of patients. In HCC cell lines, the miR-590-3P suppressed cell proliferation by inhibiting the transformation G1 phase to S phases of the cell cycle. Moreover, the miR-590-3P inhibited migration and invasion of HCC cells. Further investigations indicated that miR-590-3P play its roles by inhibiting polycomb protein EED. The experiments in animal model implied miR-590-3P could be a potential therapeutic agent for HCC in the future. In conclusion, the discovery of miR-590-3P revealed the MNBO would be a useful strategy to uncover key regulating miRNAs in HCC.

Melatonin prevents sleep deprivation-associated anxiety-like behavior in rats: role of oxidative stress and balance between GABAergic and glutamatergic transmission.

Sleep deprivation (SD) has been shown to induce anxiety-like behavior. Melatonin, an endogenous potent antioxidant, protects neurons from oxidative stress in many disease models. Here we investigated the effect of melatonin against SD-induced anxiety-like behavior and attempted to define the possible mechanisms involved. SD was induced in rats using modified multiple platform model. Melatonin (15 mg/kg) was administered to the rats via intraperitoneal injection. The elevated plus maze test, open field test and light-dark exploration were used to evaluate anxiety-like behavior. Serum corticosterone was measured to determine stress level. Malondialdehyde (MDA) level and superoxide dismutase (SOD) enzyme activity of amygdala and serum were performed to determine the level of oxidative stress. Levels of protein were detected by means of Western blot. The results showed that SD induces anxiety-like behavior, while melatonin treatment prevented these changes. Serum corticosterone also increased with SD but its levels were normalized by melatonin. In addition, melatonin reversed SD-induced changes in MDA and SOD in both of amygdala and serum. The results of Western blot showed that melatonin attenuated the up-regulation of NR2B-containing N-methyl-D-aspartate receptors, GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor as well as phosphorylation of GluR1 at Ser831, and Ca2+/calmodulin-dependent protein kinase II-alpha in SD rats. Meanwhile, melatonin blocked the down-regulation of γ-aminobutyric acid A-alpha-2 receptor. In conclusion, our results suggest that melatonin prevents anxiety-like behavior induced by SD. The possible mechanism may be attributed to its ability to reduce oxidative stress and maintain balance between GABAergic and glutamatergic transmission.

Investigation on maternal lineage of a Neolithic group from northern Shaanxi based on ancient DNA.

A magnetic bead purification method was successfully used to extract ancient DNA from the skeletal remains of 10 specimens excavated from Wuzhuangguoliang (Wzhgl) site, which was located in northern Shaanxi. The multidimensional scaling (MDS) and analysis of molecular variance approach (AMOVA) revealed that ancient Wzhgl people bored a very high similarity to southern Han Chinese. By constructing the MJ-network of various modern people including Han Chinese and Japanese, the phylogenetic analysis indicated that the Wzhgl population had close maternal distance with ancient Shandong and Xinjiang people. These findings indicated that Wzhgl contributed to the gene pool of Han Chinese and modern Japanese. In addition, population migration and interflow between Wzhgl people and ancient Shandong or Xinjiang probably occurred in Neolithic period.

CTNNA3 is a tumor suppressor in hepatocellular carcinomas and is inhibited by miR-425.

Hepatocellular carcinoma (HCC) is a common and leading cause of death worldwide. Here, we identified that a cell-cell adhesion gene, CTNNA3, is a tumor suppressor in HCC. CTNNA3 inhibited the proliferation, migration and invasion of HCC cell lines. In these cells, CTNNA3 inhibited Akt signal, and in turn decreased the proliferating cell nuclear antigen (PCNA) and the matrix metallopeptidase MMP-9, and increased the cell cycle inhibitor p21(Cip1/Waf1). Meanwhile, CTNNA3 is inhibited by miR-425 in HCC. The miR-425 directly bound to the 3'UTR of CTNNA3 and inhibited its expression. The tumor suppressor function of CTNNA3 and the oncogenic function of miR-425 were further confirmed in HCC cell xenograft in nude mice. The miR-425/CTNNA3 axis may provide insights into the mechanisms underlying HCC, and contribute to potential therapeutic strategy of HCC.

Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma.

AIMS: CD155 is an important ligand in triggering tumour rejection by immune cells. However, the expression of CD155 and its clinical significance in hepatocellular carcinoma (HCC) remains unknown. METHODS AND RESULTS: We examined the expression level of CD155 in 174 HCC tissue samples by immunohistochemical staining and in HCC cell lines by flow cytometry; 63.8% (111 of 174) of HCC tissue samples showed negative CD155 expression. When compared with adjacent peritumour tissues, HCC tissues exhibited a significantly lower expression of CD155 (P < 0.001). Flow cytometry analysis indicated that HCC cell lines had low levels of CD155 expression. Moreover, negative CD155 expression was associated significantly with higher serum α-fetoprotein level (P = 0.016) and a higher incidence of portal vein tumour thrombus (P = 0.050). Importantly, patients with positive CD155 expression had better overall survival after surgery than those with negative CD155 expression (P = 0.005). Furthermore, Cox regression analyses showed that CD155 expression was an independent prognostic factor for HCC (P = 0.049). CONCLUSIONS: Our findings suggest that loss of CD155 expression may play an important role in the immune escape of HCC cells and thus CD155 may serve as a prognostic marker as well as a potential therapeutic target for HCC.

Melatonin Attenuates Noise Stress-induced Gastrointestinal Motility Disorder and Gastric Stress Ulcer: Role of Gastrointestinal Hormones and Oxidative Stress in Rats.

BACKGROUND/AIMS: There are increasing evidences for gastrointestinal motility disorder (GIMD) and gastric stress ulcer induced by noise stress. The present study was to investigate the reversed effect of melatonin on GIMD and gastric stress ulcer induced by noise stress and potential mechanism. METHODS: Noise stress was induced on rats, and melatonin (15 mg/kg) was administered to rats by intraperitoneal injection. Differences were assessed in gastric residual rate (GRR), small intestine propulsion rate (SPR), Guth injury score, cortisol, gastrointestinal hormones (calcitonin-gene-related peptide and motilin) and oxidative stress markers (superoxide dismutase and malondialde hyde) in blood plasma as well as gastric mucosa homogenate with or without melatonin. The pathological examination of gastric mucosa was also performed. RESULTS: The GRR and SPR were improved by noise stress compared with control (P < 0.05). The pathological examination and Guth injury score revealed gastric stress ulcer. Moreover, the levels of cortisol, motilin and malondialdehyde in blood plasma and ma-londialdehyde in gastric mucosa homogenate were increased by noise stress (P < 0.05). CGRP and superoxide dismutase activ-ity in both of blood plasma and gastric mucosa homogenate were significantly decreased (P< 0.05). Furthermore, melatonin reversed changes in GRR, SPR, pathological examination, Guth injury score, cortisol, motilin, CGRP, superoxide dismutase activity and malondialdehyde (P < 0.05). CONCLUSIONS: Melatonin is effective in reversing the GIMD and gastric stress ulcer induced by noise stress. The underlying mechanism may be involved in oxidative stress and gastrointestinal hormones.

Melatonin ameliorates cognitive impairment induced by sleep deprivation in rats: role of oxidative stress, BDNF and CaMKII.

Sleep deprivation (SD) has been shown to induce oxidative stress which causes cognitive impairment. Melatonin, an endogenous potent antioxidant, protects neurons from oxidative stress in many disease models. The present study investigated the effect of melatonin against SD-induced cognitive impairment and attempted to define the possible mechanisms involved. SD was induced in rats using modified multiple platform model. Melatonin (15 mg/kg) was administered to the rats via intraperitoneal injection. The open field test and Morris water maze were used to evaluate cognitive ability. The cerebral cortex (CC) and hippocampus were dissected and homogenized. Nitric oxide (NO) and malondialdehyde (MDA) levels and the superoxide dismutase (SOD) enzyme activity of hippocampal and cortical tissues (10% wet weight per volume) were performed to determine the level of oxidative stress. The expression of brain-derived neurotrophic factor (BDNF) and calcium-calmodulin dependent kinase II (CaMKII) proteins in CC and hippocampus was assayed by means of immunohistochemistry. The results revealed that SD impairs cognitive ability, while melatonin treatment prevented these changes. In addition, melatonin reversed SD-induced changes in NO, MDA and SOD in both of the CC and hippocampus. The results of immunoreactivity showed that SD decreased gray values of BDNF and CaMKII in CC and hippocamal CA1, CA3 and dentate gyrus regions, whereas melatonin improved the gray values. In conclusion, our results suggest that melatonin prevents cognitive impairment induced by SD. The possible mechanism may be attributed to its ability to reduce oxidative stress and increase the levels of CaMKII and BDNF in CC and hippocampus.

Effect of Feining on bleomycin-induced pulmonary injuries in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The flowers of Gentiana veitchiorum has been widely used in decoction form in the traditional medicine of Tibet against tussis, tracheitis, angina for their anti-inflammatory, antimicrobial and alexipharmic properties. AIM OF THE STUDY: The aim of current study was to evaluate the therapeutic effects of Feining, a Chinese herbal formula (national invention patent: ZL200510042636.3) against pulmonary injuries and to clarify the mechanisms involved. MATERIALS AND METHODS: Experimental pulmonary injuries were induced by bleomycin (BLM) in rats with or without subsequent treatment of Feining or prednisone as positive control. The pulmonary injuries were evaluated by histological analysis. Also, the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and hydroxyproline (Hyp) in the lung tissue were determined. To clarify one of the possible active principles responsible for Feining, high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) method was applied to identify the components of Gentiana veitchiorum, one of major ingredients of Feining. RESULTS: Feining significantly improved lung alveolitis scores and reduced the Hyp content of lungs, which is an index of collagen accumulation. Moreover, Feining played a role against the oxidative damages by decreasing the MDA level, whereas increasing SOD and GSH activity, which correlated with oxidation resistance and scavenging of free radicals. In addition, Feining alleviated inflammatory lung injury by decreasing tumor necrosis factor-α (TNF-α) expression. HPLC-DAD-MS analysis revealed that there was 1.97% gentiopicroside in Gentiana veitchiorum. CONCLUSION: Feining has certain therapeutic effects against pulmonary injuries.

[Construction and expression of a fusion gene of human heptoma peptide with human heat shock protein 70].

AIM: To construct and express a fusion gene of human heptoma peptide (EPVTKAEML) with human heat shock protein 70 (HSP70). METHODS: A cDNA fragment encoding EPVTKAEML was added to 3 terminus of human HSP70 gene by PCR amplification. The PCR products of fusion gene were cloned into pET-28a(+)vector. The recombinant plasmid pET-28a(+)/EPVTKAEML-HSP70 was identified by enzyme digestion analysis and sequencing, and then it was transformed into E.coli BL21(DE3) through IPTG induction to express the target protein bearing His tag. RESULTS: A fragment of about 2.0 kb was amplified by PCR. Sequence analysis revealed that the sequence of EPVTKAEML was connected successfully to 3 terminus of human HSP70. Enzyme digestion analysis showed the fusion gene was cloned into pET-28a(+). SDS-PAGE showed that a relative molecular mass 72 000 fusion protein was expressed. CONCLUSION: The fusion gene of EPVTKAEML-HSP70 has been successfully constructed and expressed in E.coli BL21(DE3).

Disease progression, response to ACEI/ATRA therapy and influence of ACE gene in IgA nephritis.

Various studies have shown that angiotensin-converting enzyme (ACE) gene insertion/deletion (ID) polymorphism may play a role in the progression to end stage renal failure (ESRF) in patients with IgA nephritis (IgAN). In this randomized controlled trial, patients were followed up for 5 years to determine their long-term renal outcome to ACEI/ATRA therapy and to ascertain if their ACE gene profile could play a role in determining their response to therapy. Seventy-five patients with IgAN were enlisted. Thirty-seven were on ACEI/ATRA therapy for 62+/-5 months and thirty-eight were untreated and served as controls. All patients had their ACE gene ID polymorphism genotyped. Compared to controls, treated patients had lower serum creatinine (p < 0.001), lower proteinuria (p < 0.002) and fewer numbers progressing to ESRF (p < 0.002). Among patients with genotype II, there were less ESRF in the treatment group when compared to the untreated control group (p < 0.02). The advantage of therapy was not seen in patients with ID or DD genotypes. ACEI/ATRA therapy was found to be effective in retarding disease progression in IgAN with years to ESRF significantly extended in patients at all levels of renal function, including patients whose outcome were ESRF. Genotyping showed better response to therapy only for those with genotype II. The common mechanism is probably through lower levels of ACE, glomerular pressure and proteinuria resulting in reduced renal damage and retardation of progression to ESRF.

[Homologous amelogenin gene test of archaeological samples].

OBJECTIVE: Based on the sequence differences of Amelogenin homologous gene in the X and Y chromosomes, a pair of specific primers was designed to identify the sex of archaeological samples. METHODS: Ancient DNA fragments were extracted from the bones and teeth of sacrificial slaves with an improved method that combines phenol-chloroform extraction, silicon dioxide adsorption with ultrafiltration concentration. The polyacrylamide gel electrophoresis (PAGE) was used to detect PCR products. RESULTS: Seven in sixteen samples from eight graves showed positive results and the targeted segments were visible: a male with two bands of 106bp (Amel-X) and 112 bp (Amel-Y), while a female with only one band of 106 bp (Amel-X). Ancient DNA analyzing results from tooth samples are more marked than that from bones. CONCLUSION: The improved extraction method is more effective for ancient DNA extraction, which reduced the PCR inhibitors and lowered experimental costs. The sex determination technology based on Amelogenin homologous gene is an important and feasible method in the molecular archaeological research.