Integrated single-cell and bulk RNA sequencing reveals CREM is involved in the pathogenesis of ulcerative colitis.

Background: Ulcerative colitis (UC) is an inflammatory bowel disease characterized by persistent colonic inflammation. Here, we performed a systematic analysis to gain better insights into UC pathogenesis. Methods: We analyzed two UC-related datasets extracted from the gene expression omnibus database using several bioinformatics tools. The primary cell types and key subgroups of primary cells associated with UC and differentially expressed genes (DEGs) between UC and control samples were identified. The molecular regulation of the key genes was also predicted. The gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses of marker genes of key cell subgroups and model genes were performed. The expression of key enriched genes was validated in 10 clinical samples using real-time quantitative polymerase chain reaction (RT-qPCR). Results: Monocytes were identified as the major cell type. Ten differentially expressed marker genes were obtained by intersecting the 3121 DEGs, 38 marker genes in major cell types, and 104 marker genes in key cell subgroups. Four essential genes, associated with immune response, were obtained using support vector machine recursive feature elimination and least absolute shrinkage and selection operator analyses. The four essential genes were highly expressed in Cluster 0 during differentiation. Validation of the four key genes in colonic mucosal biopsy specimens from 10 normal and 10 UC patients revealed that CREM was highly expressed in both the lesion-free sites and lesion sites colonic mucosa of UC patients compared with normal adults. Conclusions: We identified CREM involved in UC pathogenesis, which is expected to provide a new therapeutic target for UC.

Role of MAPK and PI3K-Akt signaling pathways in cuprizone-induced demyelination and cognitive impairment in mice.

This study aimed to find the genes and signaling pathways underlying cuprizone-induced demyelination and cognitive impairments in mice. We used the cuprizone-exposed mice as an animal model of schizophrenia and assessed cognitive function in mice. Total RNA was extracted from mouse brain tissues for RNA sequencing. The DESeq2 R package was utilized to analyze the differentially expressed genes (DEGs). Functional and pathway enrichment analyses were performed simultaneously. We also constructed a protein-protein interaction (PPI) network to screen potential hub genes, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to validate the screened genes. After 6 weeks of cuprizone treatment, the cognitive function of mice was impaired. Compared to the controls, the cuprizone-exposed mice contained 351 DEGs, including 167 upregulated and 184 downregulated genes. Enrichment analysis showed that the DEGs were enriched in some biological processes involved in demyelination, including the MAPK pathway. Functional pathway analysis revealed that the DEGs were significantly enriched in the PI3K-Akt signaling pathway, which may be associated with cognitive impairments. MBP, IGF1, GFAP, PTPRC, CD14, CD68, ITGB2, LYN, TLR2, TLR4, VAV1, and PLEK were considered as potential hub genes. Except for MBP, all genes were upregulated in the cuprizone models, as verified by qRT-PCR. We suggest that the MAPK and PI3K-Akt signaling pathways may be associated with demyelination and cognitive impairments, respectively. GFAP and IGF-1 expression levels increased in cuprizone-exposed mice, suggesting that astrocytes may play a role in protecting the myelin sheath following treatment with cuprizone.

NCAPD3 enhances Warburg effect through c-myc and E2F1 and promotes the occurrence and progression of colorectal cancer.

BACKGROUND: NCAPD3 is one of the three non-SMC subunits of condensin II complex, which plays an important role in the chromosome condensation and segregation during mitosis. Notably, elevated levels of NCAPD3 are found in many somatic cancers. However, the clinical role, biological functions of NCAPD3 in cancers especially in colorectal cancer (CRC) and the underlying molecular mechanisms remain poorly elucidated. METHODS: Clinical CRC and adjacent normal tissues were used to confirm the expression of NCAPD3. The association of NCAPD3 expression with clinicopathological characteristics and patient outcomes were analyzed by using online database. In vivo subcutaneous tumor xenograft model, NCAPD3 gene knockout following azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced tumor mouse model, Co-IP, western blot, qRT-PCR, IHC, ChIP assays and cell functional assays were used to investigate the biological functions of NCAPD3 in CRC and the underlying molecular mechanisms. RESULTS: NCAPD3 was overexpressed in CRC tissues and positively correlated with poor prognosis of CRC patients. NCAPD3 knockout suppressed CRC development in AOM/DSS induced and xenograft mice models. Moreover, we found that NCAPD3 promoted aerobic glycolysis in CRC. Mechanistically, NCAPD3 up-regulated the level of c-Myc and interacted with c-Myc to recruit more c-Myc to the gene promoter of its downstream glycolytic regulators GLUT1, HK2, ENO1, PKM2 and LDHA, and finally enhanced cellular aerobic glycolysis. Also, NCAPD3 increased the level of E2F1 and interacted with E2F1 to recruit more E2F1 to the promoter regions of PDK1 and PDK3 genes, which resulted in the inhibition of PDH activity and TCA cycle. CONCLUSIONS: Our data demonstrated that NCAPD3 promoted glucose metabolism reprogramming and enhanced Warburg effect in colorectal tumorigenesis and CRC progression. These findings reveal a novel mechanism underlying NCAPD3 mediated CRC cell growth and provide new targets for CRC treatment.

Sphincter-Preserving Fistulectomy Is an Effective Minimally Invasive Technique for Complex Anal Fistulas.

Background: The optimal treatment of complex anal fistulas remains unclear, though many different sphincter-preserving procedures have been described. A minimally invasive technique with a better outcome is desired. The purpose of this study was to present a new technique-sphincter-preserving fistulectomy (SPF) and its clinical outcomes. Materials and Methods: A retrospective study was performed to compare the efficacy and outcomes of SPF with ligation of the intersphincteric fistula tract (LIFT) in the management of complex anal fistulas in regards to postoperative pain, complications, wound healing time, recurrence, overall success rate, fecal continence function, and quality of life. Continence function was evaluated using the Wexner incontinence scale and anal manometry. The fecal incontinence quality of life (FIQL) scale was used to assess patients' quality of life. Results: From June 2020 to July 2021, 41 patients with 43 SPF procedures and 35 patients with 35 LIFT procedures were included. Postoperative pain was comparable between two groups. The morbidity rate and the mean wound healing time in the SPF group were lower than those in the LIFT group (2.3% vs. 48.6%, p < 0.001; 1.4 ± 0.3 vs. 1.7 ± 0.4 months, p = 0.001). At a mean follow-up duration of 11.4 ± 3.5 months in the SPF group and 10.7 ± 4.3 months in the LIFT group, SPF achieved a better overall success rate than LIFT (97.7% vs. 77.1%, p = 0.014). Three patients in the SPF group and 4 patients in the LIFT group who all underwent a simultaneous fistulotomy procedure complained new incontinence of flatus. There was no statistical difference between the two groups in regards to the Wexner scores (p = 0.790), the maximum resting anal canal pressure (p = 0.641), the maximum squeeze pressure (p = 0.289), and the FIQL scores including lifestyle (p = 0.188), coping (p = 0.188), depression (p = 0.850), and embarrassment (p = 0.910). Conclusions: SPF is a novel, safe, and effective minimally invasive technique for the management of complex anal fistulas, with a promising success rate and negligible impairment on continence. Future prospective studies are needed to evaluate the long-term outcomes of SPF.

Proteomics identifies a novel role of fibrinogen-like protein 1 in Crohn's disease.

BACKGROUND: Crohn's disease (CD) is an incurable intestinal disorder with unclear etiology and pathogenesis. Currently, there is a lack of specific biomarkers and drug targets for CD in clinical practice. It is essential to identify the precise pathophysiological mechanism of CD and investigate new therapeutic targets. AIM: To explore a new biomarker and therapeutic target for CD and verify its role in the CD pathological mechanism. METHODS: Proteomics was performed to quantify the protein profile in the plasma of 20 CD patients and 20 matched healthy controls. Hub genes among the selected differentially expressed proteins (DEPs) were detected via the MCODE plugin in Cytoscape software. The expression level of one hub gene with an immunoregulatory role that interested us was verified in the inflamed intestinal tissues of 20 CD patients by immunohistochemical analysis. After that, the effects of the selected hub gene on the intestinal inflammation of CD were identified in a CD cell model by examining the levels of proinflammatory cytokines by enzyme-linked immunosorbent assays and the expression of the NF-κB signalling pathway by quantitative real-time PCR analysis and Western blot assays. RESULTS: Thirty-five DEPs were selected from 393 credible proteins identified by proteomic analysis. Among the DEPs, fibrinogen-like protein 1 (FGL1), which attracted our attention due to its function in the regulation of the immune response, had 1.722-fold higher expression in the plasma of CD patients and was identified as a hub gene by MCODE. Furthermore, the expression of FGL1 in the intestinal mucosal and epithelial tissues of CD patients was also upregulated (P < 0.05). In vitro, the mRNA levels of FGL1 and NF-κB; the protein expression levels of FGL1, IKKα, IKKß, p-IKKα/ß, p-IκBα, and p-p65; and the concentrations of the proinflammatory cytokines IL-1ß, IL-6, IL-17, and TNF-α were increased (P < 0.05) after stimulation with lipopolysaccharide, which were reversed by knockdown of FGL1 with siRNA transfection (P < 0.05). Conversely, FGL1 overexpression enhanced the abovementioned results (P < 0.05). CONCLUSION: FGL1 can induce intestinal inflammation by activating the canonical NF-κB signalling pathway, and it may be considered a potential biomarker and therapeutic target for CD.

Formation rate of secondary anal fistula after incision and drainage of perianal Sepsis and analysis of risk factors.

BACKGROUND: The choice of surgery for perianal sepsis is currently controversial. Some people advocate one-time radical surgery for perianal sepsis, while others advocate incision and drainage. The objective of this study is to observe the formation probability of secondary anal fistula after incision and drainage in patients with perianal sepsis and determine factors that contribute to secondary anal fistula after incision and drainage. METHODS: A retrospective descriptive analysis was conducted in 288 patients with perianal sepsis who were treated with anorectal surgery in the Suzhou Hospital of Traditional Chinese Medicine from January 2016 to June 2018. The patients were followed by telephone, physical examination, and pelvic MRI examination for at least 1 year after surgery. RESULTS: Three patients were not followed, 98 patients did not receive surgical treatment or one-time radical surgery for perianal sepsis, and 187 patients were ultimately identified for the study. Anal fistula was present in 105 patients, and the rate of formation of secondary anal fistula was 56.15%. There was no statistically significant difference in the fistula formation rate between different types of sepsis (P>0.05). And, in patients with secondary anal fistula, there was no significant correlation between the location of sepsis and the type of secondary anal fistula (P>0.05). CONCLUSIONS: The incidence of secondary anal fistula after incision and drainage of perianal sepsis is 56.15%, which is lower than the incidence found in previous study. Young is a risk factor for secondary anal fistula after incision and drainage of perianal sepsis. There is no significant correlation between the location of sepsis and the type of secondary anal fistula. Simple incision and drainage is a suitable choice for patients with acute perianal sepsis.

Magnetic resonance imaging findings for differential diagnosis of perianal plexiform schwannoma: Case report and review of the literature.

Plexiform schwannoma is an extremely rare variant of schwannoma, accounting for approximately 5% of cases. Due to the rarity and lack of typical symptoms, signs and radiological images, a definite diagnosis of plexiform schwannoma may not be made by clinicians prior to biopsy. In the present study, we report the first case (to our knowledge) of perianal plexiform schwannoma arising from the overlapped skin of the ischioanal fossa, and we propose an intratumorally nonenhanced circumferential capsule dividing the tumour into multiple homogeneously enhanced nodules as a magnetic resonance imaging feature to aid in the differential diagnosis of plexiform schwannoma from ancient schwannoma, cavernous haemangioma, liposarcoma and plexiform neurofibroma.

The Aqueous Fraction of Areca catechu Nut Ameliorates Demyelination in Prefrontal Cortex-Induced Depressive Symptoms and Cognitive Decline through Brain-Derived Neurotrophic Factor-Cyclic Adenosine Monophosphate Response Element-Binding Activation.

OBJECTIVE: To investigate if Areca catechu L. treatment could ameliorate depressive symptoms and cognitive decline by facilitating myelination processes in prefrontal cortex. METHODS: A mouse model of cuprizoneinduced demyelination was used to mimic demyelinating disease. Two concentrations of A. catechu nut extract (ANE; 1% and 2%) were administered orally in the diet for 8 weeks. Depressive symptoms and cognition-associated behaviors were evaluated in tests of locomotor activity, tail suspension, and forced swimming; spatial memory was tested with the Y-maze. Expression of myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), glutathione S-transferases pi (GSTpi), brain-derived neurotrophic factor (BDNF), and the transcription factor cyclic adenosine monophosphate (cAMP) response element-binding (CREB) were evaluated by western blot. RESULTS: Animals subjected to demyelination showed hyperactivity (P<0.01), impaired spatial memory (P<0.01), and depressive behaviors (P<0.05). Internally, they displayed signifificant myelin damage in the cortex, lower expression of CNPase and GSTpi, slightly decreased BDNF (P>0.05), and signifificantly reduced p-CREB (P<0.05). Nevertheless, ANE treatment demonstrated signifificant anti-depressant activity and enhancement of working memory (P<0.05 or 0.01). In addition, ANE treatment increased MBP, CNPase and GSTpi protein expression in prefrontal cortex (P<0.05). Concomitant with increased BDNF production (P<0.05), ANE treatment up-regulated phosphorylated CREB, but without statistical signifificance (P>0.05). CONCLUSION: ANE treatment might ameliorate depressive symptoms and cognitive decline by facilitating myelination processes in prefrontal cortex via induction of BDNF-CREB activation.

The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice.

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

MicroRNA miR-92a-1 biogenesis and mRNA targeting is modulated by a tertiary contact within the miR-17~92 microRNA cluster.

While functional mature microRNAs (miRNAs) are small ∼22 base oligonucleotides that target specific mRNAs, miRNAs are initially expressed as long transcripts (pri-miRNAs) that undergo sequential processing to yield the mature miRNAs. We have previously reported that the pri-miR-17∼92 cluster adopts a compact globular folded structure that internalizes a 3' core domain resulting in reduced miRNA maturation and subsequent mRNA targeting. Using a site-specific photo-cross-linker we have identified a tertiary contact within the 3' core domain of the pri-miRNA between a non-miRNA stem-loop and the pre-miR-19b hairpin. This tertiary contact is involved in the formation of the compact globular fold of the cluster while its disruption enhances miR-92a expression and mRNA targeting. We propose that this tertiary contact serves as a molecular scaffold to restrict expression of the proposed antiangiogenic miR-92a, allowing for the overall pro-angiogenic effect of miR-17∼92 expression.

Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks.

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage.

Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.

Quantitative proteomics for cancer biomarker discovery.

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), ¹8O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.

The C-terminal proteolytic fragment of the breast cancer susceptibility type 1 protein (BRCA1) is degraded by the N-end rule pathway.

The breast cancer susceptibility type 1 gene product (BRCA1) is cleaved by caspases upon the activation of apoptotic pathways. After proteolysis the C-terminal fragment has been reported to translocate to the cytoplasm and promote cell death. Here we report that the C-terminal fragment is unstable in cells as it is targeted for degradation by the N-end rule pathway. The data reveals that mutating the wild type N-terminal aspartate, of the C-terminal fragment, to valine stabilizes the fragment. If the N terminus is mutated to another N-terminal destabilizing residue, like arginine, the C-terminal fragment remains unstable in cells. Last, the C-terminal fragment of BRCA1 is stable in cells lacking ATE1, a component of the N-end rule pathway.

Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis.

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation.

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.

Comparisons of three polyethyleneimine-derived nanoparticles as a gene therapy delivery system for renal cell carcinoma.

BACKGROUND: Polyethyleneimine (PEI), which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone) grafted PEI (PCFC-g-PEI), folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI), were evaluated in terms of their cytotoxicity and transfection efficiency in vitro and in vivo in order to ascertain their potential application in gene therapy. METHODS: MTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI in vitro. Renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL) in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry. RESULTS: Each of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight in vitro. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes. CONCLUSIONS: The three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency in vitro and obviously lower toxicities compared with their precursor PEI molecules. The FA-PEAs probably provide a potential gene delivery system to treat RCC even other cancers in future.

Downregulated expression of HSP27 in human low-grade glioma tissues discovered by a quantitative proteomic analysis.

BACKGROUND: Heat shock proteins (HSPs), including mainly HSP110, HSP90, HSP70, HSP60 and small HSP families, are evolutionary conserved proteins involved in various cellular processes. Abnormal expression of HSPs has been detected in several tumor types, which indicates that specific HSPs have different prognostic significance for different tumors. In the current studies, the expression profiling of HSPs in human low-grade glioma tissues (HGTs) were investigated using a sensitive, accurate SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative proteomic strategy. RESULTS: The five HSP family members were detected and quantified in both HGTs and autologous para-cancerous brain tissues (PBTs) by the SILAC-based mass spectrometry (MS) simultaneously. HSP90 AB1, HSP A5(70 KDa), and especially HSP27 were significantly downregulated in HGTs, whereas the expression level of HSPA9 (70 KDa) was little higher in HGTs than that in PBTs. It was noted that the downregulation ratio of HSP27 was 0.48-fold in HGTs versus PBTs, which was further validated by results from RT-PCR, western blotting and immunohistochemistry. Furthermore, we detected HSP27 expression changes along with cell growth under heat shock treatment in glioma H4 cells. CONCLUSION: The SILAC-MS technique is an applicable and efficient novel method, with a high-throughput manner, to quantitatively compare the relative expression level of HSPs in brain tumors. Different HSP family members have specific protein expression levels in human low-grade glioma discovered by SILAC-MS analysis. HSP27 expression was obviously downregulated in HGTs versus PBTs, and it exhibited temporal and spatial variation under heat shock treatment (43 degrees C/0-3 h) in vitro. HSP27's rapid upregulation was probably correlated with the temporary resistance to heat shock in order to maintain the survival of human glioma cells.

Quantitative protein expression profiling of 14-3-3 isoforms in human renal carcinoma shows 14-3-3 epsilon is involved in limitedly increasing renal cell proliferation.

14-3-3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14-3-3 isoforms have different expression level and isoform-specific roles in different tumors. However, the biological functions of 14-3-3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14-3-3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14-3-3epsilon was up-regulated with 1.44-fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14-3-3sigma was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT-PCR, Western blot and immunohistochemistry analysis for specific 14-3-3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14-3-3epsilon in vitro can limitedly prompt the abnormal growth of renal tumor cells.