MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1

BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.

Prediction of transmembrane helical segments in transmembrane proteins based on wavelet transform

Tmnsmembrane（TM） protein plays an important role in the life activity of the cells, and the prediction of transmembrane helical segments （TMHs） is an important subject in the bioinformatics research. Thus far, several prediction methods have been reported, but there are some deficiencies in prediction accuracy and adaptability in these methods. In this paper, a method based on discrete wavelet transform （DWT） was developed to predict the TMHs. Two sets of test data sets containing total 60 protein sequences were utilized to access the effect of the method. Compared with the prediction results of TMHMM2.0 and MEMSAT, the obtained results indicate that the presented method has high prediction accuracy.

Influences of the interferon induced transmembrane protein I on the proliferation, invasion, and metastasis of the colorectal cancer SW480 cell lines

Background Interferon-induced transmembrane protein 1 （IFITM1） has been identified as a molecular marker of the colorectal tumors; however its influences on the biological behaviors of the colorectal cancer cells are currently unknown.We aimed to study the influences of IFITM1 on the proliferation,invasion,and metastasis of the colorectal cancer SW480 cell lines.Methods We constructed IFITM1/pEGFP-C3 recombinant plasmids and transfected them into the colorectal cancer SW480 cell lines.IFITM1/pEGFP-C3 recombinant plasmids were identified by means of immunofluorescence,laser confocal scanning microscopy,and reverse transcription polymerase chain reaction.IFITM1/SW480 cells with stable over-expression of IFITM1 were confirmed by G418 screening.The influences of IFITM1 on the proliferation of the SW480 cell lines were investigated by MTT assay and tumor transplantation experiments in nude mice.Cell invasion experiments were performed to determine the invasion capacity of the IFITM1/SW480 cells.Matrix metalloproteinase 2 （MMP-2） and MMP-9 activities were detected by the gelatin zymographic analysis,and MMP-9 expression by the Western blotting analysis.Results IFITM1/pEGFP-C3 recombinant plasmids were successfully constructed in this study,and the IFITM1/SW480 cells with stable IFITM1 gene over-expression were confirmed by G418 screening.MTT results showed that the proliferation of the IFITM1/SW480 cells was significantly enhanced （P 〈0.01）.Tumors were harvested from four weeks old mice.Tumor volumes were （1347.00±60.94） mm3,（1032.40±111.38） mm3 and （1018.78±28.83） mm3; and tumor weights were （1522.34±62.76） mg,（1137.78±97.22） mg and （1155.76±133.31） mg for mice inoculated with the IFITM1/SW480 cells,pEGFP-C3/SW480 cells and SW480 cells,respectively.Tumor volumes and weights from mice inoculated with the IFITM1/SW480 cells were significantly increased （P 〈0.01）.In addition,the numbers of the SW480 cells and IFITM1/SW480 cells that migrated through Matrigel were 448.64±38.09 and 540.45±44.61,respectively; so the invasive ability of the SW480 cells transfected with IFITM1 gene was significantly greater than that of the SW480 cells （P 〈0.01）.Gelatin zymographic analysis showed that MMP-9 and MMP-2 protein activities in the IFITM1/SW480 cells were significantly enhanced,and Western blotting analysis showed that MMP-9 expression in the IFITM1/SW480 cells was also increased.Conclusion IFITMl can enhance the proliferation,invasion,and metastasis of the colorectal cancer SW480 cell lineS.

A Role for Transmembrane Protein 16C/Slack Impairment in Excitatory Nociceptive Synaptic Plasticity in the Pathogenesis of Remifentanil-induced Hyperalgesia in Rats

Remifentanil is widely used to control intraoperative pain. However, its analgesic effect is limited by the generation of postoperative hyperalgesia. In this study, we investigated whether the impairment of transmembrane protein 16C(TMEM16C)/Slack is required for a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor(AMPAR) activation in remifentanil-induced postoperative hyperalgesia. Remifentanil anesthesia reduced the paw withdrawal threshold from 2 h to 48 h postoperatively,with a decrease in the expression of TMEM16C and Slack in the dorsal root ganglia(DRG) and spinal cord.Knockdown of TMEM16C in the DRG reduced the expression of Slack and elevated the basal peripheral sensitivity and AMPAR expression and function. Overexpression of TMEM16C in the DRG impaired remifentanilinduced ERK1/2 phosphorylation and behavioral hyperalgesia. AMPAR-mediated current and neuronal excitability were downregulated by TMEM16C overexpression in the spinal cord. Taken together, these findings suggest that TMEM16C/Slack regulation of excitatory synaptic plasticity via GluA1-containing AMPARs is critical in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats.

Association of Lysosome Associated Protein Transmembrane 4 Beta Gene Polymorphism with the Risk of Pancreatic Cancer

Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, rapid amplification of cDNA ends （RACE） and reverse transcription polymerase chain reaction （RT-PCR）. Previous study showed that the novel gene played an important role in the occurrence, development, migration and prognosis of tumors. Pancreatic cancer is an aggressive malignancy with the majority of patients dying within one year after diagnosis. This study tries to find out the relationship between lysosome associated protein transmembrane 4 beta gene polymorphism and the susceptibility of pancreatic cancer. Methods： A case-control study was conducted in China, including 58 pancreatic cancer cases and 156 healthy controls. Human genomic DNA was used as the template, polymerase chain reaction （PCR） was used to detect the distribution of LAPTM4B genotype. Analyses Odds ratio （OR） and corresponding 95% confidence interval （95%CI） with logistic regression were performed. Results： Two alleles of LAPTM4B generated three kinds of genotypes in population, ＊1/1, ＊1/2, and ＊2/2. The genotype frequency of ＊1/1, ＊1/2 and ＊2/2 in the pancreatic cancer group were 41.4%, 44.8% and 13.8% respectively, which were not significantly different from those of healthy group （47.4%, 42.9%, 9.6%） （P=0.773, P=0.291）. Also the ＊2 allele frequency of LAPTM4B among pancreatic cancer had no significantly difference with the controls （P=0.354）. When compared to the ＊1 allele, the people with ＊2 allele had no increased risk of pancreatic cancer. Conclusion： The gene polymorphism of LAPTM4B may not influence the susceptibility of pancreatic cancer.

Crosslinking-mediated activation of the FcεRI:Does it need antigen for success?

Mast cells(MCs),hematopoietic cells of the myeloid lineage,are well-known for their pro-inflammatory nature contributing to the development of various allergic and autoimmune diseases.One of the characteristic receptors on MCs,the high-affinity receptor for IgE(FcεRI),is activated in its IgE-bound state via binding and crosslinking by polyvalent antigen.This results in its phosphorylation by the SRC family kinase LYN,initiating differential signaling pathways,eventually triggering immunological effector functions,such as degranulation and cytokine production.Few publications have reported on FcεRI-dependent but antigen-independent MC activation by antibody-mediated crosslinking of membrane molecules(e.g.,transmembrane proteins and glycosphingolipids)that are both localized in membrane rafts and in close vicinity to the FcεRI.In this Viewpoint we will briefly introduce FcεRI-mediated MC stimulation,cite examples of FcεRI-proximal molecules,the crosslinking of which can cause FcεRI-dependent MC activation,and discuss the potential of certain viruses as well as auto-antibodies to act as indirect FcεRI-crosslinking agents.In latter cases,antigen-independent FcεRI-mediated pro-inflammatory MC activation could contribute to the development of detrimental cytokine storms.

TMEM158 May Serve as a Diagnostic Biomarker for Anaplastic Thyroid Carcinoma:An Integrated Bioinformatic Analysis

Anaplastic thyroid carcinoma(ATC)is a rare but extremely lethal malignancy.However,little is known about the pathogenesis of ATC.Given its high mortality,it is critical to improve our understanding of ATC pathogenesis and to find new diagnostic biomarkers.In the present study,two gene microarray profiles(GSE53072 and GSE65144),which included 17 ATC and 17 adjacent non-tumorous tissues,were obtained.Bioinformatic analyses were then performed.Immunohistochemistry(IHC)and receiver operating characteristic(ROC)curves were then used to detect transmembrane protein 158(TMEM158)expression and to assess diagnostic sensitivity.A total of 372 differentially expressed genes(DEGs)were identified.Through protein-protein interaction(PPI)analysis,we identified a significant module with 37 upregulated genes.Most of the genes in this module were related to cell-cycle processes.After co-expression analysis,132 hub genes were selected for further study.Nine genes were identified as both DEGs and genes of interest in the weighted gene co-expression network analysis(WGCNA).IHC and ROC curves confirmed that TMEM158 was overexpressed in ATC tissue as compared with other types of thyroid cancer and normal tissue samples.We identified 8 KEGG pathways that were associated with high expression of TMEM158,including aminoacyl-tRNA biosynthesis and DNA replication.Our results suggest that TMEM158 may be a potential oncogene and serve as a diagnostic indicator for ATC.

PRRT2 Mutation and Serum Cytokines in Paroxysmal Kinesigenic Dyskinesia

Objective Paroxysmal kinesigenic dyskinesia(PKD)is a rare movement disorder PRRT2 gene mutations have been reported to cause PKD.However,the pathophysiological mechanism of PKD remains unclear,and it is unknown whether an inflammatory response is involved in the occurrence of this disease.We aimed to investigate the symptomatology,genotype,and serum cytokines of patients with PKD.Methods We recruited 21 patients with PKD,including 7 with familial PKD and 14 with sporadic PKD.Their clinical features were investigated,and blood samples were collected,and PRRT2 mutations and cytokine levels were detected.Results The mean age at PKD onset was 12.3±2.2 years old.Dystonia was the most common manifestation of dyskinesia,and the limbs were the most commonly affected parts.All attacks were induced by identifiable kinesigenic triggers,and the attack durations were brief(<1 min).Four different mutations from 9 probands were identified in 7 familial cases(71.4%)and 14 sporadic cases(28.6%).Two of these mutations(c.649dupC,c.620_621delAA)had already been reported,while other 2(c.1018_1019delAA,c.1012+1G>A)were previously undocumented.The tumor necrosis factor(TNF)-αlevel in the PKD group was significantly higher than that in the age-and sex-matched control group(P=0.025).There were no significant differences in the interleukin(IL)-1β,IL-2R,IL-6,IL-8,or IL-10 levels between the two groups.Conclusion In this study,we summarized the clinical and genetic characteristics of PKD.We found that the serum TNF-αlevels were elevated in patients clinically diagnosed with PKD,suggesting that an inflammatory response is involved in the pathogenesis of PKD.

Phosphorylation‐dependent Traffcking of Plasma Membrane Proteins in Animal and Plant Cells

In both unicellular and multicellular organisms, transmembrane （TM） proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane （PM）-localized TM proteins （PM proteins）, especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED （PIN） carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

Inner nuclear membrane protein TMEM201 maintains endothelial cell migration and angiogenesis by interacting with the LINC complex

The nuclear envelope comprises the outer nuclear membrane,inner nuclear membrane(INM),and nucleopore.Although∼60 INM proteins have been identified,only a few of them have been well characterized,revealing their crucial roles.Our group focused on the INM protein transmembrane protein 201(TMEM201),whose role in cellular function remains to be defined.In this study,we investigated the role of TMEM201 in endothelial cell migration and angiogenesis.Depletion of TMEM201 expression by short hairpin RNA-mediated interference impeded human umbilical vein endothelial cell(HUVEC)angiogenic behavior in tube formation and fibrin gel bead sprouting assays.Meanwhile,TMEM201-deficient HUVECs exhibited impaired migration ability.We next explored the underlying mechanism and found that the N-terminal of TMEM201 interacted with the linker of nucleoskeleton and cytoskeleton complex and was required for regulating endothelial cell migration and angiogenesis.These in vitro findings were further confirmed by using in vivo models.In Tmem201-knockout mice,retinal vessel development was arrested and aortic ring sprouting was defective.In addition,loss of tmem201 impaired zebrafish intersegmental vessel development.In summary,TMEM201 was shown to regulate endothelial cell migration and control the process of angiogenesis.This study is the first to reveal the role of INM proteins in the vascular system and angiogenesis.

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA_(2) subunit

Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin(HA).The restriction of IFITM3 on HAmediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses.Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA_(2) subunit but not HA_(1) subunit of H5 and H7 virus.Truncated analyses showed that the transmembrane domain of the IFITM3 and HA_(2) subunit might play an important role in their interaction.Finally,this interaction of IFITM3 was also verified with HA_(2) subunits from other subtypes of influenza A virus and influenza B virus.Overall,our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain,providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.

Effects of antiallergic herbal agents on cystic fibrosis transmembrane conductance regulator in nasal mucosal epithelia of allergic rhinitis rabbits

Background It has been found that the expression of cystic fibrosis transmembrane conductance regulator （CFTR） is closely related to allergic rhinitis （AR）. In the previous study, we have demonstrated that antiallergic herbal agents （AHA） can obviously inhibit the allergic reaction of AR. The aim of this study was to explore the expression of CFTR and the effects of AHA on CFTR to improve the allergic reaction of AR. Methods An animal model of an AR rabbit was established using ovalbumin （OVA）. The rhinitis rabbits were randomly assigned to three groups： AHA treating group （AHATG）, modeling group （MG） and healthy controlling group （HCG）. The expressions of CFTR protein were examined by immunohistochemical method. The mucosal epithelial cells of all the rabbits were primarily cultured with tissue culture method in vitro and treated with or without glibenclamide for 24 hours. The levels of monocyte chemotactic factor-l（MCP-1） and RANTES protein in supernatants of culture were measured by ELISA, and the expressions of CFTR mRNA were detected by real-time PCR. Results The expressions of CFTR mRNA and protein greatly increased in mucosal epithelial cells of MG. The protein concentrations of MCP-1, RANTES in culture supernatants of MG were significantly higher than those in the other two groups （P 〈0.01）, and they reached much higher level than those at the start points in the MG （P 〈0.05） and were significantly different compared with those in the AHATG after being cultured for 24 hours （P 〈0.01）. CFTR mRNA in MG ＋ glibenclamide were much lower than those in MG （P 〈0.05）. RANTES and CFI-R mRNA treated with glibenclamide in AHATG were significantly lower than those in the AHATG （P 〈0.01）. Minimal changes in the secretions of MCP-1 in the epithelial cells were detected between AHATG and AHATG ＋ glibenclamide （P 〉0.05）. Conclusions AHA can inhibit the secretions of CFTR, RANTES and MCP-1 in mucosal epithelia and improve inflammatory reaction of AR. CFTR may play an important role in the secretion of RANTES and mucosal inflammatory response in AR. Glibenclamide can inhibit the CFTR secretion in mucosal epithelial cells, in particular during AR process. These effects of glibenclamide on secretion of RANTES can be effectively strengthened by AHA.

OsHT, a Rice Gene Encoding for a Plasma-Membrane Localized Histidine Transporter

Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT1) from Arabidopsis, we isolated a full-length cDNA named OxHT(histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3 kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice. This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than Arabidopsis.

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. in this study, we identified that the Arabidopsis Transmembrane Protein 18 （AtTMEM18） gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins （TMEM18s） that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility.Furthermore, expression of the rice TM EM18-homologous protein （OsTMEM18） driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.

Farnesoid X receptor,the bile acid sensing nuclear receptor,in liver regeneration

The liver is unique in regenerative potential, which could recover the lost mass and function after injury from ischemia and resection. The underlying molecular mechanisms of liver regeneration have been extensively studied in the past using the partial hepatectomy(PH) model in rodents, where 2/3 PH is carried out by removing two lobes. The whole process of liver regeneration is complicated, orchestrated event involving a network of connected interactions, which still remain fully elusive. Bile acids(BAs) are ligands of farnesoid X receptor(FXR), a nuclear receptor of ligand-activated transcription factor. FXR has been shown to be highly involved in liver regeneration. BAs and FXR not only interact with each other but also regulate various downstream targets independently during liver regeneration. Moreover, recent findings suggest that tissue-specific FXR also contributes to liver regeneration significantly. These novel findings suggest that FXR has much broader role than regulating BA, cholesterol, lipid and glucose metabolism. Therefore, these researches highlight FXR as an important pharmaceutical target for potentialuse of FXR ligands to regulate liver regeneration in clinic. This review focuses on the roles of BAs and FXR in liver regeneration and the current underlying molecular mechanisms which contribute to liver regeneration.