As a key component of cellular regulatory machinery at the transcriptional, post-transcriptional, and epigenetic levels, long non-coding RNAs(lncRNAs) participate in almost every basic biological process. Dysfunction of lncRNA often accompanies a vareity of diseases. Clarification of the biological functions of lncRNAs has become a research focus in recent years. However, the sheer magnitude of vast amounts of lncRNA sequencing data from a myriad of eukaryotes that has been produced by high-throughput sequencing techniques actually represents a significant challenge in this field. Accordingly, a number of lncRNA databases have been established. These are regularly updated by scientific research institutions and provide useful tools for researchers to share, annotate, and analyze the functions of lncRNAs. In this review, we introduce the latest features and applications of various lncRNA database resources in four sections, including: original resource integration, selection and identification, functional analysis, and association of lncRNAs with human diseases. By familiarizing investigators with the strengths and features of these various databases, our review should help expediate the identification and analysis of important lncRNA information.
Tumor metastasis is the main cause of cancerrelated death. Abnormal glucose metabolism of tumor cells plays a key role in tumor metastasis. One of the features for glucose metabolism of cancer cells is "Warburg effect", which means tumor cells mainly produce energy by glycolysis even if oxygene is abundant. It has been proved that enhanced Warburg effect was found in metastatic cancer cells. Metastatic cancer cells have faster growth rate and high proliferation ability with enormous glucose flux and high rate of glycolysis and nucleotide biosynthesis. The research focus on glucose metabolism of metastatic tumor cells will shed light on better understanding the mechanisms for tumor metastasis. Here, we reviewed the most recent research progress on the alteration of glucose metabolism in metastatic tumor cells,such as glycolysis, aerobic metabolism of mitochondria and pentose phosphate pathway (PPP). These results suggested that understanding tumor-metabolism regulation will help us to find new strategies in controlling tumor metastasis.
Exosomes are small extracellular vesicles(30～100 nm diameter) derived from the endosomal system in almost all kind of cells under physiological and pathological condition. It has shown that exosomes mediated cell-to-cell communication to modulate a variety of biological processes. Exosome can be secreted by several cell types and retrieved from body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, it also contains nucleic acids, like mRNAs and miRNAs with normal expression or targeting activities. These features have prompted extensive research to exploit exosomes as a source of biomarkers for cancer and neurodegenerative disorders. We reviewed the characteristics, component and function of the exosome according to the included proteins and nucleotides. The immunomodulatory and regenerative properties of exosome inspires the application for therapeutic purposes. Nevertheless, questions on exosome biogenesis and secretion mechanism, and physiological or pathological function remained to be fully addressed.
DNA double strand breaks (DSBs) is one of the most severe forms of DNA damages in cells. The repair of DNA DSBs involves homologous recombination (HR) and non-homologous end joining (NHEJ) process. Poly-ADP ribosylation (PARylation) is a post-translational protein modification catalyzed by poly(ADPribose)polymerases (PARPs). PARP1 is the most important member of the family for DNA damage responses. Studies have shown that PARP1 played a key role in the repair of DNA DSBs. The early specific responses of PARP1 could play different roles depending on the presence or absence of KU protein. This review summarized the research advances of PARP1 in DNA DSBs repair pathways and prospected its potential for clinical diagnosis and treatment of disease.
Recombinase polymerase amplification(RPA) is a kind of isothermal nucleic acid amplification technique rising in recent years. RPA is more rapid, convenient and efficient than the polymerase chain reaction (PCR) and other isothermal amplification techniques. In this paper, this new technique will be introduced in detail, with reviewing over the development of its relevant researches and applications until now on in medical diagnosis, agriculture, food safety, biological safety and so on. It is expected that this technique will attract more attention and be perfect in the near future making it play an important role in more fields, and what’s more, write a new start in nucleic acid detection.
Histone variants are key players in regulating fundamental cellular processes such as gene expression. In human, there are five H3 variants, H3.1, H3.2, H3.3, centromere specific histone CENP-A (centromere protein A) and testis specific histone H3t. Human H3.3 is encoded by two genes, H3F3A and H3F3B. Recurrent H3F3A mutations were identified with whole-genome sequencing of DNA in pediatric high-grade gliomas, such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG). There are over 70% of DIPG and 30% of GBM carrying K27M amino acid missense mutation (methionine replaces lysine 27) in histone variant H3.3. H3.3 K27M mutation can inhibit the enzymatic activity of the Polycomb repressive complex 2 through interaction with the H3K27 methyltransferase EZH2 subunit and globally reduce H3K27me3 levels. H3.3 K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis. K27M mutant can be used as a molecular marker to better distinguish pediatric gliomas subgroups. K27M mutant can also be used as a sensitive and specific prognostic marker for pediatric gliomas. It is a valid therapeutic strategy for treating K27M mutant glioma to increase H3K27 methylation by inhibiting H3K27 demethylase such as JMJD3. Here, we review the histone variant H3.3 H3K27 mutation pattern, molecular mechanism and clinical application in pediatric gliomas.
Alternative splicing is a ubiquitous phenomenon in eukaryote, and it plays important roles in various physiological processes of living organisms. Plant MADSbox proteins are a kind of transcription factors having crucial roles during the development of plant. Lemads1 is a SEPALLATA(SEP) subclade MADSbox gene, and it was a critical factor during the fruit ripening process of tomato. However,the transcription of this gene has never been studied. In this study, we found that alternative splicing existed in the transcription of Lemads1. Four spliced variants were discovered, and their lengths were 699 bp, 741 bp, 1 404 bp and 1 539 bp, respectively. The splicing patterns for the variants were exon skipping and intron retention. Three different proteins with the lengths of 232 aa, 246 aa, and 266 aa were encoded by the four spliced variants. Multiple protein alignment showed that the sequence variations of the three proteins were mainly located in the C domain. The phylogenetic analysis indicated that proteins encoded by the spliced variants of Lemads1 still belong to the FBP9/23 group of the LOFSEP supclade in SEP clade, in the same subgroup with SLMBP21. Through phenotype observation, we found that Lemads1 was involved in the processes of fruit maturity and sepal development. The spatial and temporal expression pattern analyses of the four spliced variants showed that their expression patterns were similar but not identical. They were all highly expressed in sepals and fruits, but had different temporal expression patterns during the development of the two organs. And the unique expression pattern for each of the four spliced variants during the development of sepals and fruits hints their different roles during these two processes. In this study, we provided important information that alternative splicing is underway during the transcription of Lemads1, and it lays a foundation for the deep exploration of the function of Lemads1.
Low-dose cisplatin induces premature senescence of cancer cells by elevating the expression of p21 and p16. However, the mechanisms underlying cisplatin-induced upregulation of p21 and p16 are not fully elucidated. In the present study, we investigated the mechanisms controlling the expression of p21 and p16 in low-dose cisplatin-induced premature senescence. Our results showed that treatment of HeLa cells by low-dose cisplatin(4 μmol/L) decreased the protein levels of DNMT1 and the promoter methylation of p21 and p16, thereby elevating the mRNA and protein levels of p21 and p16. Activation of p38MAPK is responsible for the reduction of DNMT1 in response to low-dose cisplatin, since pre-incubating cells with p38MAPK inhibitor SB203580 could partly reverse the effect of cisplatin in reducing the protein levels of DNMT1 and the promoter methylation of p21 and p16. Furthermore, inhibition of p38MAPK by SB203580 reversed cisplatin-induced premature senescence. In sum, cisplatin reduces the promoter methylation of p21 and p16 via activating p38MAPK, which in turn, increases the expression of p21 and p16 in premature senescence. These studies provide evidence for elucidating the mechanisms underlying low-dose cisplatin-induced premature senescence.
MircroRNA-125b(miR-125b) has an important role in processes of malignant neoplasm, including cell proliferation, differentiation, and apoptosis. However, less is known for the involvement of miR-125b in epithelial-mesenchymal transformation(EMT) in hepatocellular carcinoma(HCC). This study aims to exploit the effects of miR-125b overexpression on EMT regulated by TGF-β signaling pathway. A miR-125b overexpression vector pHRS-1cla-miR125b-CMV-EGFP was constructed into pHRS-1cla-EGFP lentivirus vector by double digestion with NheI and XbaI. MHCC97-H cells were infected with viruses; and GFP-positive cells were collected from a flow cytometry sorting. HCC cell stably transfected with MHCC97-H-PHRS-miR-125b-EGFP was established. The qRT-PCR results showed that the miR-125b expression stable cells was nearly 6 fold than that of the control. Western blot showed that the expression of mesenchymal cell marker α-SMA of MHCC97-H-PHRS-miR-125b-EGFP was significantly down-regulated; while the expression of epithelial cell marker E-cadherin was upregulated. The activity of TGF-β signaling pathway was decreased with down-regulated expression of Smad2 and Smad4. In apoptosis assay, a 19.66% increase in apoptosis rate was detected in MHCC97-H-PHRS-miR-125b-EGFP cells. An additional increase to 74.7% was shown after TGF-β1 treatment. In vivo experiment results showed that transfection of miR-125b mimics in mice subcutaneous tumor tissues with a positive correlation between miR-125b expression and apoptosis rate of tumor tissues(r=0.83463，P<0.01). Immunohistochemistry showed that E-cadherin was significantly up-regulated; while α-SMA, Smad2 and Smad4 were down-regulated.
DEPDC1(DEP domain containing 1) is a novel tumor-related gene and plays important roles in the development of a variety of malignant tumors. Study indicated that DEPDC1-depletion repressed cell proliferation and induced apoptosis in HNE1 nasopharyngeal carcinoma cells. This study aims to explore its role in cell migration and invasion.The results demonstrated that DEPDC1 depletion significantly inhibited the migration and invasion in HNE-1 and CNE-1 cells by wound healing and TransWell assays. The expression of Twist1 and Vimentin were downregulated as determined by qRT-PCR and Western blot. It suggested that high expression of DEPDC1 enhanced the migration and invasion in nasopharyngeal carcinoma, and the underlining molecular mechanism needed to be elucidated.
Long term and severe endoplasmic reticulum stress (ERS) can induce cellular apoptosis. Inositolrequiring protein 1α (IRE1α)-X-box binding protein 1(XBP1) signaling is the most common ERS-related pathway that may be the most promising target for tumor therapy. We have previously demonstrated that brefeldin A (BFA) enhances the cis-dichlorodiamine platinum (CDDP)-induced apoptosis in human lung cancer cells, however, its mechanism is unclear. In the study, we demonstrated the involvement of IREl-XBP1 pathway in the synergistic anti-cancer effects of BFA and CDDP on the apoptosis of human lung cancer GLC-82 cells. AnnexinV/PI double staining and flow cytometry showed that compared to BFA or CDDP single exposure, combined BFA and CDDP exposure increased GLC-82 cell apoptosis. Further evidences by RT-qPCR and Western blotting revealed that BFA plus CDDP exposure increased the transcript (mRNA) and protein levels of procaspase3 and the cleavage/activation of procaspase3 when compared to single exposure. Importantly, RTqPCR and Western blotting demonstrated that BFA increased IRE1α and XBP1 expression significantly when compared to CDDP exposure (P < 0.05). BFA plus CDDP exposure increased IRE1α and XBP1 expression markedly than BFA single treatment (P < 0.01). These findings indicate that BFA and BFA+CDDP both can activate IRE1α-XBP1 pathway under ERS and BFA enhances cisplatin-induced lung cancer cell apoptosis via activating IIER1-XBP1 signaling. Our data provide new evidence for IREl-XBP1 signaling as a promising target for chemotherapy.
Monocyte chemoattractant protein-1(MCP-1) is a member of the CC chemokine family, which is closely correlated to carcinoma cell migration and invasion. MCP-1 is highly expressed in human esophageal carcinoma EC109 cells．The migration and invasion abilities of human EC109 cells decreased after inhibition of MCP-1 expression．To explore the effects of small interfering RNA(siRNA) silencing MCP-1 gene on migration and invasion of human EC109 cells, MCP-1-specific siRNA was designed and synthesized primarily. Human EC109 cells were transfected for 120 hours. The control groups were established accordingly by using siRNA with insignificant order and no transfection. The wound healing assay showed that silencing MCP-1 gene significantly inhibited the abilities of cells migration compared with all the control groups.The transwell experiment demonstrated that silencing MCP-1 gene led to decreased invasion abilities of EC109 cells．Furthermore，Western blotting and RT-PCR assay revealed that the expressions of MCP-1,MMP-7,MMP-9,TGF-β1 and VEGF were decreased after the silencing of MCP-1 gene，and there were obvious positive correlations among MCP-1, MMP-7, MMP-9, TGF-β1 and VEGF expressions. These data suggested that RNA interference of MCP1 could inhibit carcinoma cell migration and invasion regulated by the expression of MCP-1, MMP-7, MMP-9, TGF-β1 and VEGF expressions.
The genetic engineering has become important ways to produce antibody. Due to its great molecular weight, the antibody is difficult to be produced. Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond. By cleaving the antibody into Fab and Fc fragments and fusing the fragments to the N-terminus and C-terminus of intein respectively, the antibody can be obtained by intein splicing. In order to develop antibody by intein splicing, we must find inteins with high splicing efficiency in KSCDKTH, the amino acid sequence existing in hinge region of antibody. In this article, we found Ssp DnaXS0/S1/S11 split inteins spliced KSCDKTH sequence successfully, which potentially makes it feasible to develop antibody by inteins splicing.
We previously reported that α2,3-sialic acid residues in breast cancer were associated with metastatic potential. The α2,3-sialyltransferase ST3Gal Ⅲ, which adds α2,3-sialic acids to glycoprotein, is overexpressed in breast cancer and enzyme activity is correlated with tumor cell metastasis. However, its mechanistic role has not been fully evaluated. In the present work, we investigated the influence of ST3Gal Ⅲ in key steps of the breast cancer metastasis processes. Short hairpin RNA(shRNA) targeting ST3Gal Ⅲ lentiviral vector was constructed and transfected into MDA-MB-231 cells to silence ST3Gal Ⅲ. After verification by real-time PCR and Western blot, we have established two stable cells clones shRNA-2 and shRNA-4 with ST3Gal Ⅲ down-regulated expression. α2,3-sialic acids was downstream product of ST3Gal Ⅲ enzyme, and examined by flow cytometry. The results demonstrated that the levels of α2,3-sialic acids were significantly decreased in shRNA cells(P<0.05). Adhesion assay and transwell assay revealed that the abilities of adhesion and invasion in shRNA cells were markedly reduced(P<0.05). β1 integrin expression is involved in suppression of invasive behavior. Silencing ST3Gal Ⅲ reduced β1 integrin expression(P<0.05). In summary, the expression of ST3Gal Ⅲ in breast cancer cell plays a vital role in key steps of tumor metastasis. Silencing ST3Gal Ⅲ inhibits adhesion and invasion of MDA-MB-231 cells via down-regulating β1 integrin expression. This research may clarify the mechanism of breast cancer metastasis in new angle and provide new target for the effective drug treatment in breast cancer.
Mesenchymal-epithelial signaling between the dermal papilla and the hair matrix regulates cell proliferation and differentiation during the hair follicle cycle. Insulin-like growth factor 1(IGF1) is one of the components of this signaling pathway; it is a multifunctional regulator molecule in cells. The first hair follicle cycle is the most important determinant for the normal growth and development of hair follicles, but the function of IGF1 has not been reported. The expression of IGF1 and the IGF1 receptor(IGF1R) in the skins undergoing the first hair follicle cycle was measured to investigate the function of IGF1 and IGF1R in mice. Quantitative real time PCR analysis showed that the mRNA expression level of IGF1 was at its lowest at anagen, highest at catagen, and decreased from telogen(but remained higher than that of early anagen). In comparison to the mRNA expression level of IGF1 at early anagen, the expression levels at catagen and telogen was significantly different(P＜0.01). The mRNA expression of IGF1R was highest at catagen, lowest at anagen, and increased at telogen(but remained lower than that of early anagen). In comparison to the mRNA expression levels of IGF1R at early anagen, the expression levels at catagen and telogen were significantly different(P＜0.01). Western blotting analysis showed that the immune reactive bands corresponding to IGF1 and IGF1R were detected, and the expression of IGF1 and IGF1R shared the same trends as the expression of their corresponding mRNA transcripts. Immunohistochemistry analysis showed that IGF1 was expressed mainly in the epidermis, while IGF1R was localized in the hair dermal papilla, the lower section of the hair bulb, and at the outer and inner root sheath of hair follicles. The expression and localization of IGF1 and IGF1R at the different stages of the first hair follicle cycle were significantly different, which suggested a potential role of IGF1 in regulating the transformations from anagen through to catagen and telogen may be involved in melanogenesis. However, IGF1 must not bind to IGF1R to play its roles in the first hair follicle cycle in mouse skin.
To investigate the microRNA(miRNA) expression profiles in cow mammary glands of cows with varying milk quality, high throughput sequencing technology was used to detect the expression of miRNAs in high milk quality cows mammary glands(H) and low milk quality cows mammary glands(L). Using miRNA database tools, miREvo, and mirDeep2 software analysis, 56 differentially expressed miRNAs were identified(P < 0.05, FDRq<0.05). GO(Ontology Gene) and signal pathway enrichment analysis of target genes were conducted with DAVID. Screening of the target genes indicated that four genes(CSN3, SCD, LALBA and DGAT2) have been previously reported to be associated with the synthesis of milk protein and milk fat. Other target genes were found to be involved in glucose metabolism, protein and lipid metabolism, cell development and differentiation, and immune functions(P<0.01, false discovery rate(FDR) q<0.01). Target genes were mainly enriched in mitogenactivated protein kinases(MAPK), glycerol phospholipid metabolism, hypoxiainducible factor-1(HIF-1), and the PI3K-Akt signaling pathway. The predicted target genes of the miRNAs were mainly enriched in carbohydrate metabolism, fat metabolism, protein metabolism, cell apoptosis and immune related pathways.
Microsatellites are special short repetitive DNA sequences of length variation. Microsatellite instability has been linked to the classification and prognosis of cancers. Currently, the method for detecting microsatellite lengths is limited in sensitivity and often requires large number of cells. We integrated laser microdissection, multiple annealing and looping-based amplification cycles (MALBAC), and adopted capillary electrophoresis to establish a technology allowing analysis of multiple microsatellite length using small amounts of cells. After optimizing the sample collection and cell lysis processes, the HE stained tissues were isolated with laser microdissection, subjected to amplification with MALBAC single cell whole genome assay. The lengths of microsatellite markers were determined with capillary electrophoresis. The lengths of microsatellite markers of five sub-segments (one sub-segment contains about 5 cells) from individual gastric glands indicated that the method satisfied the accuracy and reproducibility. Individual intestinal metaplastic (IM) glands at pre-cancerous stage were analyzed for variations. The detected microsatellite instability suggested defectives in the DNA mismatch repair system. Our established method is useful for microsatellite studies, and the amplified products can be further analyzed in exon or whole genome sequencing.
