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Meg8-DMR is the first maternal methylated DMR to be discovered in the imprinted Dlk1-Dio3 domain. The deletion of Meg8-DMR enhances the migration and invasion of MLTC-1 depending on the CTCF binding sites. However, the biological function of Meg8-DMR during mouse development remains unknown. In this study, a CRISPR/Cas9 system was used to generate 434 bp genomic deletions of Meg8-DMR in mice. High-throughput and bioinformatics profiling revealed that Meg8-DMR is involved in the regulation of microRNA: when the deletion was inherited from the mother (Mat-KO), the expression of microRNA was unchanged. However, when the deletion occurred from the father (Pat-KO) and homozygous (Homo-KO), the expression was upregulated. Then, differentially expressed microRNAs (DEGs) were identified between WT with Pat-KO, Mat-KO, and Homo-KO, respectively. Subsequently, these DEGs were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) term enrichment analysis to explore the functional roles of these genes. In total, 502, 128, and 165 DEGs were determined. GO analysis showed that these DEGs were mainly enriched in axonogenesis in Pat-KO and Home-KO, while forebrain development was enriched in Mat-KO. Finally, the methylation levels of IG-DMR, Gtl2-DMR, and Meg8-DMR, and the imprinting status of Dlk1, Gtl2, and Rian were not affected. These findings suggest that Meg8-DMR, as a secondary regulatory region, could regulate the expression of microRNAs while not affecting the normal embryonic development of mice.

Mutagenic purine–pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine–pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine–pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

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Baicalin has been demonstrated to have multiple pharmacological activities but low solubility. Various baicalin hydrogels have been used to improve its solubility and break its limitation in clinical applications. However, traditional baicalin hydrogels contain numerous ingredients and usually show low baicalin loading capacity. Herein, we discovered a dynamic covalent hydrogel only consisting of baicalin and inorganic borate, in which baicalin is considered as the carrier and drug without other ingredients. The dynamic boronate bonds endow the hydrogel with excellent degradability and multi-stimuli-responsiveness. Moreover, the hydrogel displayed remarkable thixotropy, moldability, and self-healing properties. And the biocompatible baicalin hydrogel exhibited significant antibacterial activities, and can be considered as a potential drug delivery system for biomedical applications.

Increasing evidence has indicated that long noncoding RNAs (lncRNAs) play important roles in human diseases, including cancer; however, only a few of them have been experimentally validated and functionally annotated. Here, we identify a novel lncRNA that we term HITT (HIF-1α inhibitor at translation level). HITT is commonly decreased in multiple human cancers. Decreased HITT is associated with advanced stages of colon cancer. Restoration of the expression of HITT in cancer cells inhibits angiogenesis and tumor growth in vivo in an HIF-1α-dependent manner. Further study reveals that HITT inhibits HIF-1α expression, mainly by interfering with its translation. Mechanically, HITT titrates away YB-1 from the 5'-UTR of HIF-1α mRNA via a high-stringency YB-1-binding motif. The reverse correlation between HITT and HIF-1α expression is further validated in human colon cancer tissues. Moreover, HITT is one of the most altered lncRNAs upon the hypoxic switch and HITT downregulation is required for hypoxia-induced HIF-1α expression. We further demonstrate that HITT and HIF-1α form an autoregulatory feedback loop where HIF-1α destabilizes HITT by inducing MiR-205, which directly targets HITT for degradation. Together, these results expand our understanding of the cancer-associated functions of lncRNAs, highlighting the HITT-HIF-1α axis as constituting an additional layer of regulation of angiogenesis and tumor growth, with potential implications for therapeutic targeting.

Background Methionine is an essential sulfur-containing amino acid. To elucidate the influence of l-methionine on activation of the nuclear factor erythroid 2-related factor 2-antioxidant responsive element (Nrf2-ARE) antioxidant pathway to stimulate the endogenous antioxidant activity for depressing reactive oxygen species (ROS)-derived oxidative stress, male Wistar rats were orally administered l-methionine daily for 14 days. Results With the intake of l-methionine, Nrf2 was activated by l-methionine through depressing Keap1 and Cul3, resulting in upregulation of ARE-driven antioxidant expression (glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modulatory subunit, glutathione synthase (GS), catalase (CAT), superoxide dismutase (SOD), heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx)) with increasing l-methionine availability. Upon activation of Nrf2, glutathione synthesis was increased through upregulated expression of methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, cystathionine β-synthase, cystathionine γ-lyse, glutamate cysteine ligase (GCL) and GS, while hepatic expressions of methionine sulfoxide reductases (MsrA, MsrB2, MsrB3) and hepatic enzyme activities (CAT, SOD, GCL, GR, GST, GPx) were uniformly stimulated with increasing consumption of l-methionine. As a result, hepatic content of ROS and MDA were effectively reduced by l-methionine intake. Conclusion The present study demonstrates that methionine availability plays a critical role in activation of the Nrf2-ARE pathway to induce an endogenous antioxidant response for depressing ROS-derived oxidative stress, which is primarily attributed to the stimulation of methionine sulfoxide reductase expression and glutathione synthesis. © 2019 Society of Chemical Industry.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three well-characterized paternally methylated differentially methylated regions (DMRs): IG-DMR, Gtl2-DMR, and Dlk1-DMR. These DMRs control the expression of many genes involved in embryonic development, inherited diseases, and human cancer in this domain. The first maternal methylation DMR discovered in this domain was the Meg8-DMR, the targets and biological function of which are still unknown. Here, using an enhancer-blocking assay, we first dissected the functional parts of the Meg8-DMR and showed that its insulator activity is dependent on the CCCTC-binding factor (CTCF) in MLTC-1. Results from RNA-seq showed that the deletion of the Meg8-DMR and its compartment CTCF binding sites, but not GGCG repeats, lead to the downregulation of numerous genes on chromosome 12, in particular the drastically reduced expression of Dlk1 and Rtl1 in the Dlk1-Dio3 domain, while differentially expressed genes are enriched in the MAPK pathway. In vitro assays revealed that the deletion of the Meg8-DMR and CTCF binding sites enhances cell migration and invasion by decreasing Dlk1 and activating the Notch1-Rhoc-MAPK/ERK pathway. These findings enhance research into gene regulation in the Dlk1-Dio3 domain by indicating that the Meg8-DMR functions as a long-range regulatory element which is dependent on CTCF binding sites and affects multiple genes in this domain.

Arginine is a conditionally essential amino acid. To elucidate the influence of l-arginine on the activation of endogenous antioxidant defence, male Wistar rats were orally administered daily with l-arginine at different levels of 25, 50, 100 mg/100 g body weight. After 7 and 14 days feeding, the antioxidative capacities and glutathione (GSH) contents in the plasma and in the liver were uniformly enhanced with the increasing consumption of l-arginine, whereas the oxidative stress was effectively suppressed by l-arginine treatment. After 14 days feeding, the mRNA levels and protein expressions of Keap1 and Cul3 were gradually reduced by increasing l-arginine intake, resulting that the nuclear factor Nrf2 was activated. Upon activation of Nrf2, the expressions of antioxidant responsive element (ARE)-dependent genes and proteins (GCLC, GCLM, GS, GR, GST, GPx, CAT, SOD, NQO1, HO-1) were up-regulated by l-arginine feeding, indicating an upward trend in antioxidant capacity uniformly with the increasing consumption of l-arginine. The present study demonstrates that the supplementation of l-arginine stimulates GSH synthesis and activates Nrf2 pathway, leading to the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway. Results suggest the availability of l-arginine is a critical factor to suppress oxidative stress and induce an endogenous antioxidant response.

The main purpose of this study was to perform a meta-analysis to assess the interaction between smoking and nine genes (GSTM1, GSTT1, GSTP1, CYP1A1, NAT2, SULT1A1, hOGG1, XRCC1 and p53) on colorectal cancer, gastric cancer, liver cancer and oesophageal cancer. Published articles from the PubMed, ISI and EMBASE databases were retrieved. A total of 67 case-control studies or nested case-control studies were identified for the analysis. The pooled jodds ratio (OR) with 95% confidence interval (CI) was calculated using the random effect model. The overall study showed that the GSTM1 polymorphism was associated with the risk of the four digestive cancers among Asian population (OR 1.284, 95% CI: 1.122-1.470, p: 0). Subgroup analyses by cancer site showed that GSTM1 null genotype increased the gastric cancer risk in total population (OR 1.335, 95% CI: 1.145-1.556, p: 0). However, the association of GSTM1 null genotype with the oesophageal cancer risk was found in smokers (OR 1.382, 95% CI: 1.009-1.894, p:0.044), but not in non-smokers (OR 1.250, 95% CI: 0.826-1.891, p:0.290). Moreover, smokers with the CYP1A1 IIe462Val polymorphism were at an increased cancer risk in Asian population (OR=1.585, 95% CI 1.029-2.442, p: 0.037). None of the other gene-smoking interactions was observed in the above cancers. This meta-analysis reveals two potential gene-smoking interactions, one is between smoking and GSTM1 on oesophageal cancer, and the other is between smoking and CYP1A1 IIe462Val on the four cancers in Asian population. Future studies need to be conducted to verify the conclusions.

Elevated plasma free fatty acid (FFA) levels are associated with insulin resistance and can cause lipotoxicity in skeletal muscles. In response to FFAs, skeletal muscle can secrete a variety of cytokines. Irisin, one such muscle-secreted cytokine, can improve glucose tolerance, glucose uptake, and lipid metabolism. It is produced by the transmembrane protein fibronectin type Ⅲ domain containing 5 (FNDC5) by specific proteases. The purpose of this study was to investigate the regulatory mechanisms of the FNDC5 response to palmitate and their relationships with insulin resistance in C2C12 myotubes. RNA sequencing analysis results from C2C12 myotubes treated with palmitate showed that palmitate could activate the TGF-β signaling pathway. Palmitate directly affected the expression of Smad3, but not its phosphorylation level, in C2C12 myotubes. Furthermore, knockdown and knockout of Smad3 alleviated the inhibitory effect of palmitate on the expression of FNDC5. In contrast, overexpression of Smad3 aggravated the inhibition of FNDC5 expression. There is a Smad3 binding motif in the −660 bp to −649 bp region of the Fndc5 promoter. CRISPR/Cas9 knockout of this region also alleviated the inhibition of FNDC5 expression in response to palmitate. More importantly, inhibition of FNDC5 expression mediated by Smad3 led to a decrease in insulin sensitivity in C2C12 myotubes. Collectively, these findings suggest that palmitate could induce insulin resistance through Smad3-mediated down-regulation of the Fndc5 gene.

Ras-association domain family protein 6 (RASSF6) is a member of tumor suppressor RASSFs family with a wide range of function from RAS interaction, Hippo signaling involvement to cell cycle and apoptosis regulation. RASSF6 is reported inactivated in various types of cancer. However, whether RASSF6 is associated with colorectal cancer and the underlying mechanisms have yet to be investigated. In our previous exome sequencing study, we found a somatic loss-of-function (LoF) mutation in RASSF6 in one sporadic colorectal cancer (sCRC) patient, and two missense mutations in deep sequencing group of sCRC samples, implying the possibility that RASSF6 may be involved in the pathogenesis of sCRC. In this study, we demonstrate that RASSF6 acts as a tumor suppressor in colon cancer cells. Decreased level of RASSF6 was observed in adenocarcinoma compared to normal tissues, especially in advanced tumor cases. Further experiments showed exogenous introduction of RASSF6 into LoVo cells suppressed cell proliferation, migration, invasion, and induced apoptosis in vitro as well as tumor growth in vivo. In contrast, knockdown of RASSF6 in HT-29 cells showed the opposite effects. Taken together, our results suggest, in addition to epigenetics changes, functional somatic mutations may also contribute to the downregulation of RASSF6 and further participate in the pathogenesis of sCRC. RASSF6 may serve as a novel candidate against tumor growth for sCRC.

Mutations in transforming growth factor beta receptor II (TGFBR2) are detected in up to 30% of overall colorectal cancer (CRC). Dysregulation of some microRNAs participated in the CRC pathogenesis. In this study, we used the gene ontology analyses, the Kyoto Encyclopedia of Genes and Genomes pathway analyses and gene set enrichment analysis to indicate that miR-3191 was involved in the regulation of transforming growth factor beta (TGF-BETA) signal pathway in CRC. These bioinformatics results were supported by data obtained from CRC samples and experiments in vitro. The luciferase reporter assay was used to confirm that miR-3191 modulates TGF-BETA signal pathway by targeting TGFBR2. By transwell migration and invasion assays, we showed that miR-3191 promoted CRC cell migration and invasion by downregulating TGFBR2. And it may serve as a novel therapeutic strategy for treating CRC patients.

Glucosamine (GlcN) is a dietary supplement that is widely used to promote joint health. Reports have demonstrated that oral GlcN adversely affects glucose metabolism. Here, we found that oral administration of GlcN induced insulin resistance (IR) and increased plasma glucose levels in mice. Musclin is a muscle-secreted cytokine that participates in the development and aggravation of diabetes. In this study, we found that increased expression of the musclin plays a pathogenic role in GlcN-induced IR in mice. Additional in vivo and in vitro studies showed that 4-PBA inhibited GlcN-induced endoplasmic reticulum (ER) stress and reduced musclin expression, indicating that ER stress might be closely linked to musclin expression. Moreover, the inhibition of musclin gene expression was also observed when sh-RNAs and small molecular compound inhibitors inhibited ER stress-induced PERK and IRE1-associated unfolding protein response (UPR) signaling pathways, and the CRISPR/Cas9 genome editing technology knockout the ATF6-associated UPR pathway in C2C12 myotubes cells. Silencing of the expression of musclin effectively relieved GlcN-affected phosphorylation of Akt, glucose intake and glycogen synthesis. These results suggest that GlcN increased musclin gene expression though UPR, and musclin represents an important mechanism underlying GlcN-induced IR in mice.

Sporadic colorectal cancer (sCRC) is one of the leading causes of cancer death worldwide. As a highly heterogeneous complex disease, the currently reported classical genetic markers for sCRC, including APC, KRAS, BRAF, and TP53 gene mutations and epigenetic alterations, can explain only some sCRC patients. Here, we first reported a deleterious c.551C>T mutation in SARDH in sCRC. SARDH was identified as a novel tumor suppressor gene and was abnormally decreased in sCRC at both the transcriptional and the translational level. SARDH mRNA levels were also down-regulated in oesophageal cancer, lung cancer, liver cancer, and pancreatic cancer in the TCGA database. SARDH overexpression inhibited the proliferation, migration, and invasion of CRC cell lines, whereas its depletion improved these processes. SARDH overexpression was down-regulated in multiple pathways, especially in the chemokine pathway. The SARDH transcript level was positively correlated with the methylation states of CXCL1 and CCL20. Therefore, we concluded that SARDH depletion is involved in the development of sCRC.

The aim of the present study was to investigate the effect and therapeutic potential of baicalin in breast cancer. Baicalin is used to treat inflammatory diseases. The effects of baicalin were assessed in breast cancer MCF-7 and MDA-MB‑231 cells, and human breast cancer xenograft mice. Cells were treated with 0, 20 or 30 µM baicalin for 48 h, while xenograft mice were treated with intraperitoneal injection of 0, 100 or 200 mg/kg baicalin for 30 days. The results demonstrated that treatment with baicalin dose-dependently suppressed breast cancer cell invasion, migration and proliferation, and also induced G1/S-phase cell cycle arrest in vitro and in vivo. Baicalin alleviated inflammation injury and inhibited the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, thus suppressing nuclear factor (NF)-ĸB-p65 activation via inhibition of IĸB kinase. Investigation of the mechanism underlying baicalin activity indicated that it inhibited protein expression of NF-ĸB-p65, leading to NF-ĸB‑induced increased expression of CCND1, BCL2, BIRC2 and BIRC3, thus inhibiting cell proliferation, invasion and migration and suppressing anti-apoptotic factors in vitro and in vivo. In addition, baicalin did not affect non-tumorigenic normal breast epithelial cells. These results indicate that baicalin may exert therapeutic effects in breast cancer.

Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes.

Most imprinted genes are concerned with embryonic development, especially placental development. Here, we identified a placenta-specific imprinted gene Qpct. Our results show that Qpct is widely expressed during early embryonic development and can be detected in the telecephalon, midbrain, and rhombencephalon at E9.5b-E11.5. Moreover, Qpct is strikingly expressed in the brain, lung and liver in E15.5. Expression signals for Qpct achieved a peak at E15.5 during placental development and were only detected in the labyrinth layer in E15.5 placenta. ChIP assay results suggest that the modification of histone H3K4me3 can result in maternal activating of Qpct.

There have been few reports about the possible toxic effects of titanium dioxide (TiO2 ) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO2 nanoparticles (0, 64 and 320 mg kg(-1) body weight per day to control, low-dose and high-dose groups, respectively, 7 days per week for 14 weeks). TiO2 nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and reactive oxygen species (ROS)-related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO2 nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO2 nanoparticles increase ROS-induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF-α and IL-6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO2 nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice.

Figure S1. Exome capture and sequencing of 3 sporadic colorectal cancer samples. Figure S2. Detection of BMP5 somatic mutations in initial exome sequencing and expanded deep sequencing samples. Figure S3. Alignment of BMP5 in different species. The nonsynonymous mutation are marked and indicated with arrows, all seven amino acids are highly conserved among different species. Sus: Sus scrofa, Dr.: Zebra fish. Figure S4. Kaplan–Meier analysis of overall survival according to high or low BMP5 expression in six tumor types (Log rank test). Figure S5. Immunofluorescence staining of BMP5 in NCM460 (A) and SW480 (B) cells. Figure S6. Pearson correlation analysis between BMP5 and miR-32, miR-655. The data were analyzed using TCGA CRC tumor samples and stage IV samples. Figure S7. Overexpression and knockdown efficiency validation in HT-29 and SW480 cells. (A) Overexpression efficiency of BMP5 in HT-29 cells. Tumor proliferation marker PCNA was checked. (B) Overexpression and knockdown efficiency of BMP5 in SW480. Tumor proliferation marker PCNA and migration marker (MMP2 and MMP9) were detected. MMP9 showed no significant difference. Figure S8. Cell apoptosis of BMP5 in HT-29 and SW480 cells. The data are showed as mean ± sem. Figure S9. Pearson correlation analysis between BMP5 and EMT markers. (A) Correlation analysis in tumor samples. (B)Correlation of fold change (Log2(Tumor/Normal)) between BMP5 and EMT markers. (C) Correlation between BMP5 and E-cadherin in six tumor types. Figure S10. Coexpression network of control HT-29 (A) and BMP5-expressing HT-29 cells (B). Figure S11. Correlation analysis between BMP5 and SMAD or STAT signaling in lung, breast, esophagus, stomach, liver and pancreatic cancer. (DOC 10825 kb)

Table S1. Sample details. Table S2. BMP5 primer sequences. Table S3. qPCR Primers used in this study. Table S4. Tissue microarray sample details. Table S5. Primers used in BMP5 cloning. Table S6. BMP5 siRNAs used in this study. Table S7. Somatic loss of function mutations identified in exome sequencing. Table S8. 71 Gene expression analysis in normal and tumor samples. Table S9. Gene expression analysis in 3 GEO datasets. Table S10. Kaplan-Meier survival analysis of 71 genes in CRC (Data from HPA database using best seperation). Table S11. Gene function and pathway annotation. Table S12. Correlation of BMP5 expression to age, gender and clinical grade of 129 caddolorectal adenocarcinomas. Table S13. Correlation of BMP5 expression to age, gender, BMI, and recurrence of TCGA CRC samples. Table S14. Differentially expressed genes (Control vs BMP5 transfected in HT-29 cells). Table S15. Coexpression network analysis. (DOC 2017 kb)

The aim of this study was to evaluate the role of vimentin expression in the prognosis and progression of CRC. Meta-analysis was conducted to investigate the correlations between vimentin and prognosis and clinicopathological features in CRC. Literatures were searched by PubMed, Embase, ClinicalKey, CNKI, VIP, and WanFang databases. The Cancer Genome Atlas (TCGA) database was used to assess the association of vimentin expression with survival rate in CRC. Eleven reports with 1969 cases were included in the meta-analysis. The results showed that positive vimentin expression predicted a poor overall survival (OS) in the univariate analysis (HR: 2.087, 95%CI: 1.660-2.625) and multivariate analysis (HR: 1.633, 95%CI: 1.223-2.181). Vimentin overexpression also conferred worse disease-free survival (DFS) in the univariate analysis (HR: 2.069, 95%CI: 1.024-4.179) and multivariate analysis (HR: 2.802, 95%CI: 1.421-5.527). Moreover, upregulated vimentin is related to lymph node metastasis (OR: 2.288, 95%CI: 1.159-4.517), TNM stages (OR: 1.957, 95%CI: 1.333-2.873), and N stage (OR: 2.316, 95%CI: 1.482-3.620). Analysis of TCGA database indicated that elevated vimentin predicated a shorter OS (p=0.033). Our findings reveal that upregulated vimentin contributes to the progression and poor prognosis of CRC. Vimentin may be a prognostic biomarker and therapeutic target in patients with CRC.

Sporadic colorectal cancer (sCRC) is one of the most commonly diagnosed cancers worldwide, but few genetic markers have been identified and used for its early detection. MicroRNAs are diverse cellular regulators in cancer pathogenesis that bind to the 3'-untranslated region (3'-UTR) of their target mRNAs, and variants within the miRNA target sites on sCRC-related genes may influence its pathogenesis. To investigate this possibility, we used a bioinformatical method to screen SNPs for putative changes in miRNA recognition sites within the 3'-UTR of sCRC-related genes. The rs11466537 single nucleotide polymorphism was predicted to modify the regulation of hsa-miR-1193 on the Transforming Growth Factor β Receptor II (TGFBR2) gene. Additionally, luciferase reporter assays indicated that hsa-miR-1193 bound the T allele more strongly than the A allele of rs11466537 (with A being the less frequent variant), and real time-polymerase chain reaction and western blot analysis showed that TGFBR2 is significantly repressed by hsa-miR-1193. Furthermore, overexpression of hsa-miR-1193 promoted HT-29 cell proliferation, while the loss of hsa-miR-1193 inhibited the process. Finally, the rs11466537 genotyping result revealed that the frequency of A allele carriers was 1.5% in the control blood samples, but 0 in the sCRC patients' normal colon tissue samples. Our results demonstrated that hsa-miR-1193 may be involved in sCRC tumourigenesis at least in part by suppression of TGFBR2, and the A allele of rs11466537 disturbed the regulation of hsa-miR-1193 on TGFBR2.

Long non-coding RNAs (lncRNAs) have been studied extensively over the last few years. Liver is an important organ that plays a crucial role in glucose metabolism and homeostasis; however, there are few reports of the identification and functional characterization of lncRNAs with important roles in liver development. Therefore, it is necessary to systematically identify lncRNAs that are involved in liver development. In this paper, we assembled the transcriptome using published RNA-seq data across three mouse liver developmental stages and identified 4,882 putative long intergenic non-coding RNAs (lincRNAs) expressed in at least one of the investigated stages. Combining these with Ensembl lincRNAs, we established a reference catalog of 6,602 transcribed lincRNAs in the mouse liver. We then analyzed all the lincRNAs in this reference catalog systematically and revealed that liver lincRNAs carry different genomic signatures from protein-coding genes, while the putative lincRNAs are generally comparable with known Ensembl lincRNAs. In addition, putative lincRNAs are functionally associated with essential biological processes, including RNA splicing, protein localization and fatty acid metabolic process, implying that they may play an important role in regulating liver development. The validation of selected lincRNAs that are specifically expressed in developing liver tissues further suggested the effectiveness of our approach. Our study shows that lincRNAs that are differentially expressed during three liver developmental stages could have important regulatory roles in liver development. The identified putative lincRNAs are a valuable resource for further functional studies.

Mcts2 and H13 comprise an imprinted retrogene-host gene pair. Imprinted genes have been proved to be closely related with embryo development. In order to understand its expression relationship during embryo development and influence of the retrogene on the host gene, we studied expression patterns in mouse embryos and transcriptional interference in a cell culture system. The present study determined the spatio-temporal expression pattern of Mcts2 and H13 from embryonic day 9.5 to 15.5. A similar expression pattern between Mcts2 and H13 was observed in mouse embryogenesis by in situ hybridization and real-time PCR, these two genes were extensively expressed in the neural tissues at mid-embryonic stages. As the embryo development proceeded, H13 and Mcts2 were widely detected throughout the developing organism, especially highly expressed in brain. Moreover, neither over expression nor knockdown of Mcts2 has any significant detectable effect on H13 expression in NIH/3T3 cells. In addition, transcriptional up-regulation of Mcts2 caused by demethylation of DMR in the Mcts2 promoter was not directly associated with the H13 transcription in NIH/3T3 cells treated by 5-Aza-cdR. The regulatory relationship between H13 transcripts and the promoter methylation status of Mcts2 was complex, demonstrating host/retrogene relationship may not be limited to the imprinted locus.

MicroRNAs are small noncoding RNAs involved in various biological processes. We characterized the expression of miR-344-3p during mouse embryonic development. At E9.5-E10.5 and E15.5, in situ hybridization detected strong miR-344-3p signal in the central nervous system, including the cerebral cortex, hindbrain, cerebellum, thalamus, hindbrain, medulla oblongata, spinal cord, and dorsal root ganglia. Further, qRT-PCR analysis identified miR-344-3p expression at E15.5, with expression stably maintained in the brain from E12.5 to E18.5 before decreasing to relatively low levels postnatally. We also analyzed miR-344-3p expression using immunofluorescence in situ hybridization at E18.5 and within the adult brain. miR-344-3p signal was mainly detected in cortical regions surrounding the ventricular system, choroid plexus, glomerular layer of the olfactory bulb, and granular cell layer of the cerebellar cortex. Altogether, our results indicate miR-344-3p may play an important role in morphogenesis, nervous system development in the brain.

In silico prediction of genomic long non-coding RNAs (lncRNAs) is prerequisite to the construction and elucidation of non-coding regulatory network. Chromatin modifications marked by chromatin regulators are important epigenetic features, which can be captured by prevailing high-throughput approaches such as ChIP sequencing. We demonstrate that the accuracy of lncRNA predictions can be greatly improved when incorporating high-throughput chromatin modifications over mouse embryonic stem differentiation toward adult Cerebellum by logistic regression with LASSO regularization. The discriminating features include H3K9me3, H3K27ac, H3K4me1, open reading frames and several repeat elements. Importantly, chromatin information is suggested to be complementary to genomic sequence information, highlighting the importance of an integrated model. Applying integrated model, we obtain a list of putative lncRNAs based on uncharacterized fragments from transcriptome assembly. We demonstrate that the putative lncRNAs have regulatory roles in vicinity of known gene loci by expression and Gene Ontology enrichment analysis. We also show that the lncRNA expression specificity can be efficiently modeled by the chromatin data with same developmental stage. The study not only supports the biological hypothesis that chromatin can regulate expression of tissue-specific or developmental stage-specific lncRNAs but also reveals the discriminating features between lncRNA and coding genes, which would guide further lncRNA identifications and characterizations.

The major aim of this study is to elucidate the hypocholesterolemic mechanism exerted by rice protein (RP) in adult rats under cholesterol-enriched dietary condition. Compared with casein, the cholesterol levels in plasma and the liver were significantly reduced by RP, accompanying significant inhibition of cholesterol absorption. RP increased the activity and mRNA level of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase activity and gene expression were significantly depressed with consumption of RP. Neither the activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP differed from that of casein. The gene expression of the peroxisome proliferator-activated receptor α and liver X receptor α were significantly activated by consumption of RP. RP did not modify the mRNA level of sterol regulatory element-binding protein-2 with respect to casein. These results suggest RP can induce a cholesterol-lowering effect through modifying cholesterol metabolism-related gene expression and enzyme activity in adult rats.

The Dlk1-Dio3 imprinted domain on mouse chromosome 12qF1 contains three paternally expressed protein-coding genes and multiple maternally expressed long or short noncoding RNA genes. All these imprinted genes are regulated by IG-DMR located between Dlk1 and Meg3/Gtl2. Recently, several novel imprinted noncoding RNAs were identified in the intergenic region of this domain, although the exact number of imprinted genes within the region is unclear. Here, we report that a novel noncoding RNA, AK003491, located between Meg3/Gtl2 and Meg8, is maternally expressed in E15.5 brain, tongue, heart, lung, liver and kidney tissues. In situ hybridization analysis at E15.5 shows AK003491 is predominantly expressed in forebrain, tongue, thymus, somites, lung and liver. Quantitative real-time RT-PCR analysis confirms this expression pattern and detects highest expression in tongue. While the AK003491 expression pattern at postnatal day 1 is similar to E15.5, AK003491 expression at postnatal day 30 is mainly restricted to the brain. These results expand the number of known imprinted long noncoding RNAs in this domain, and contribute to further investigation of their regulatory mechanism and function.

To explore the association of the APOA5 gene c.553GT polymorphism with hypertriglyceridemia (HTG) susceptibility and altered triglyceride levels.We searched the PubMed, Google Scholar, and CNKI databases for published studies relating to analyses of these associations. Case-control and comparative studies of the association between the APOA5 c.553GT variant and altered triglyceride levels were included. In total, the meta-analysis involved 10 studies on HTG, which provided 2219 cases and 3401 controls. To measure the correlation between the c.553GT polymorphism and HTG susceptibility, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The overall OR was calculated using a random-effects model.Compared with APOA5 c.553 GG carriers, c.553T carriers displayed an increased risk of HTG in the Asian population, with an overall random effects OR of 3.55 (95% CI: 2.46-5.13) in the dominant model. There was significant heterogeneity among the studies (POur results suggest that APOA5 c. 553T is an independent risk factor for HTG and increased triglyceride levels in the Asian population. APOA5 c. 553T could be employed as a genetic risk marker for HTG and increased triglyceride levels.

As the regulatory factors, lncRNAs play critical roles in embryonic stem cells. And lincRNAs are most widely studied lncRNAs, however, there might still might exist a large member of uncovered lncRNAs. In this study, we constructed the de novo assembly of transcriptome to detect 6,701 putative long intergenic non-coding transcripts (lincRNAs) expressed in mouse embryonic stem cells (ESCs), which might be incomplete with the lack coverage of 5′ ends assessed by CAGE peaks. Comparing the TSS proximal regions between the known lincRNAs and their closet protein coding transcripts, our results revealed that the lincRNA TSS proximal regions are associated with the characteristic genomic and epigenetic features. Subsequently, 1,293 lincRNAs were corrected at their 5′ ends using the putative lincRNA TSS regions predicted by the TSS proximal region prediction model based on genomic and epigenetic features. Finally, 43 putative lincRNAs were annotated by Gene Ontology terms. In conclusion, this work provides a novel catalog of mouse ESCs-expressed lincRNAs with the relatively complete transcript length, which might be useful for the investigation of transcriptional and post-transcriptional regulation of lincRNA in mouse ESCs and even mammalian development.

Non-coding RNAs (ncRNAs) Meg8 and Irm were previously identified as alternatively splicing isoforms of Rian gene. Ascertaining ncRNAs spatiotemporal expression patterns is crucial for understanding the physiological roles of ncRNAs during tissue and organ development. In this study in mouse embryos, we focused on the developmental regulation expression of imprinted macro ncRNAs, Meg8 and Irm by using in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). The in situ hybridization results showed that Meg8 and Irm were expressed in the developing brain at embryonic day 10.5 (E10.5) and E11.5, while Irm expression signals were strikingly detected in the somite, where Meg8 expression signals were undetectable. By E15.5, they were expressed in brain, tongue, liver, lung and neuroendocrine tissues, while Irm displayed more restricted expression in tongue and skeletal muscle than Meg8. Furthermore, quantitative analysis confirmed that they were highly expressed in tongue and brain at E12.5, E15.5 and E18.5. These results indicated that Meg8 and Irm might be coordinately expressed and functionally correlated in diverse of organs. Notably, Irm was more closely associated with morphogenesis of skeletal muscle in contrast to Meg8 during embryonic development.

Dopamine receptor D4 (DRD4) is activated by the neurotransmitter dopamine and links to many neurological and psychiatric conditions because of its close relationship with prefrontal cortex and other important brain regions. To explore the possibility that genetic variants of DRD4 gene predispose to children with mental retardation (MR), five target SNPs of DRD4 were selected and genotyped in the samples of 163 MR pedigrees from the Qinba region of China. Two SNPs (rs752306 and rs3758653) showed weak association with MR (the P values were 0.022 and 0.015 for dominant model, and 0.027 and 0.015 for recessive model, respectively). Although they did not bear the multiple testing corrections, the haplotype which contained rs3758653 exhibited a significant association with MR (global P values were 0.018 for dominant model and 0.028 for recessive model, respectively). The in silico analysis also indicated that rs752306 and rs3758653 would be biologically meaningful SNPs. Therefore, the present study suggested that the genetic variants of DRD4 gene may play an important role in human MR. Further investigations, such as confirmation with other independent samples and functional studies, may elucidate their effect on gene expression and MR susceptibility.

Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.

Recent research has revealed that the maternal non-coding RNA genes (Gtl2, Rian and Mirg) from the Dlk1-Dio3 imprinted cluster are closely related to the full development potential of the induced pluripotent stem cells (iPSCs). Transcriptional silencing of these genes failed to generate all-iPSC mice, indicating their significant contribution to embryogenesis. However, except for Gtl2, little information regarding these genes has been acquired in this cluster. In the present study, we analyzed the spatiotemporal expression patterns of Mirg during mouse embryogenesis. Using in situ hybridization and quantitative PCR, we demonstrated that Mirg non-coding RNA exhibited sustained expression throughout mouse embryogenesis from E8.5 to E18.5. Strong expression was detected in the central nervous system (E9.5-E15.5) and various skeletal muscles (E13.5 and E15.5), and the subcellular localization appeared to be in the nuclei. The pituitary and adrenal gland also showed high expression of Mirg, but, unlike the skeletal muscles and the neural circuitry, the signals were not concentrated in the nuclei. In the major internal organs, Mirg maintained low expression during embryogenesis (E12.5-E18.5) whereas in the liver and the developing lung, Mirg was expressed with a gradually decreasing trend and a gradually raising trend, respectively. These findings indicate that temporal regulation of Mirg expression may be required during specific stages and in specific tissues during embryonic development.