High-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139.

AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139.

Design, synthesis, and biological evaluation of a potential long-acting glucagon-like peptide-1 (GLP-1) analog.

By binding to its receptor, glucagon-like peptide-1 (GLP-1) plays various physiological roles, including activating glucose-dependent insulin secretion, inhibiting gastric emptying, and reducing appetite. This suite of activities makes GLP-1 and its analogs an attractive choice for treating type 2 diabetes mellitus in the context of overweight or obesity. This study used different types and lengths of fatty acids to design dual fatty acid side chains for GLP-1 receptor agonists including decanoic, dodecanoic, tetradecanoic, hexadecanoic, dodecanedioic, tetradecanedioic, hexadecanedioic, and octadecanedioic acids. Sixteen GLP-1 receptor agonists (conjugates 13-28) with dual fatty acid side chains were obtained by liquid-phase synthesis. After structural confirmation using high-resolution mass spectrometry, peptide mapping, and circular dichroism, the biological activities of the conjugates were screened. First, the conjugates were screened for albumin binding and activity in GLP-1R-CRE-bla CHO-K1 cells. Albumin binding results suggested a synergistic effect between the two fatty acids in the conjugates. Next, conjugates 18, 19, and 21 selected after primary screening were assessed for receptor affinity, activity in INS-1 cells, plasma stability across different species, and efficacy and pharmacokinetics in normal and db/db mice. One candidate (conjugate 19) was found to have albumin binding of >99 %, good receptor affinity, activities of INS-1 cells, and plasma stability. We found that cellular activities in GLP-1R-CRE-bla CHO-K1 cells and pharmacodynamics and pharmacokinetics in normal and db/db mice for conjugate 19 were superior to those of semaglutide.

Exosomal lncRNA HOTAIR induce macrophages to M2 polarization via PI3K/ p-AKT /AKT pathway and promote EMT and metastasis in laryngeal squamous cell carcinoma.

Exosomes are a new way of the communication between the tumor cell and macrophage in the micro-environment. The macrophage can be induced to different phenotypes according to the different tumors. In the present study, long-chain noncoding RNA HOTAIR (lncRNA HOTAIR) was highly expressed in LSCC and exosomes. The pathway of exosomal lncRNA HOTAIR inducing macrophage to M2 polarization in the LSCC was investigated. The carcinoma tissues and adjacent tissues were collected from 104 LSCC cases, and the positive relationship between CD163-/CD206-M2 macrophage infiltration and clinical phase, lymph node spreading and pathological phase in LSCC was observed. To examine the role of exosomal lncRNA HOTAIR, macrophages were co-cultured with LSCC-exosomes of high lncRNA HOTAIR expression or transferred with HOTAIR mimics. It was suggested that exosomal lncRNA HOTAIR can induce macrophages to M2 polarization by PI3K/p-AKT/AKT signaling pathway. Furthermore, exo-treated M2 macrophages facilitate the migration, proliferation, and EMT of LSCC.

Long-Acting Human Interleukin 2 Bioconjugate Modified with Fatty Acids by Sortase A.

Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.

A Novel Method for Intelligent Single Fault Detection of Bearings Using SAE and Improved D-S Evidence Theory.

In order to realize single fault detection (SFD) from the multi-fault coupling bearing data and further research on the multi-fault situation of bearings, this paper proposes a method based on features self-extraction of a Sparse Auto-Encoder (SAE) and results fusion of improved Dempster-Shafer evidence theory (D-S). Multi-fault signal compression features of bearings were extracted by SAE on multiple vibration sensors' data. Data sets were constructed by the extracted compression features to train the Support Vector Machine (SVM) according to the rule of single fault detection (R-SFD) this paper proposed. Fault detection results were obtained by the improved D-S evidence theory, which was implemented via correcting the 0 factor in the Basic Probability Assignment (BPA) and modifying the evidence weight by Pearson Correlation Coefficient (PCC). Extensive evaluations of the proposed method on the experiment platform datasets showed that the proposed method could realize single fault detection from multi-fault bearings. Fault detection accuracy increases as the output feature dimension of SAE increases; when the feature dimension reached 200, the average detection accuracy of the three sensors for bearing inner, outer, and ball faults achieved 87.36%, 87.86% and 84.46%, respectively. The three types' fault detection accuracy-reached to 99.12%, 99.33% and 98.46% by the improved Dempster-Shafer evidence theory (IDS) to fuse the sensors' results-is respectively 0.38%, 2.06% and 0.76% higher than the traditional D-S evidence theory. That indicated the effectiveness of improving the D-S evidence theory by evidence weight calculation of PCC.

An Ensemble Deep Convolutional Neural Network Model with Improved D-S Evidence Fusion for Bearing Fault Diagnosis.

Intelligent machine health monitoring and fault diagnosis are becoming increasingly important for modern manufacturing industries. Current fault diagnosis approaches mostly depend on expert-designed features for building prediction models. In this paper, we proposed IDSCNN, a novel bearing fault diagnosis algorithm based on ensemble deep convolutional neural networks and an improved Dempster-Shafer theory based evidence fusion. The convolutional neural networks take the root mean square (RMS) maps from the FFT (Fast Fourier Transformation) features of the vibration signals from two sensors as inputs. The improved D-S evidence theory is implemented via distance matrix from evidences and modified Gini Index. Extensive evaluations of the IDSCNN on the Case Western Reserve Dataset showed that our IDSCNN algorithm can achieve better fault diagnosis performance than existing machine learning methods by fusing complementary or conflicting evidences from different models and sensors and adapting to different load conditions.

Spatial-temporal graph neural ODE networks for skeleton-based action recognition.

In the field of skeleton-based action recognition, accurately recognizing human actions is crucial for applications such as virtual reality and motion analysis. However, this task faces challenges such intraindividual action differences and long-term temporal dependencies. To address these challenges, we propose an innovative model called spatial-temporal graph neural ordinary differential equations (STG-NODE). First, in the data preprocessing stage, the dynamic time warping (DTW) algorithm is used to normalize and calculate 3D skeleton data to facilitate the derivation of customized adjacency matrices for improving the influence of intraindividual action differences. Secondly, a custom ordinary differential equation (ODE) integrator is applied based on the initial conditions of the temporal features, producing a solution function that simulates the dynamic evolution trend of the events of interest. Finally, the outstanding ODE solver is used to numerically solve the time features based on the solution function to increase the influence of long-term dependencies on the recognition accuracy of the model and provide it with a more powerful temporal modeling ability. Through extensive experiments conducted on the NTU RGB+D 60 and Kinetics Skeleton 400 benchmark datasets, we demonstrate the superior performance of STG-NODE in the action recognition domain. The success of the STG-NODE model also provides new ideas and methods for the future development of the action recognition field.

Design, synthesis and antibacterial activity of novel colistin derivatives with thioether bond-mediated cyclic scaffold.

The escalating crisis of multidrug resistance is raising the fear of untreatable Gram-negative infections and killing a substantial number of patients. The underpopulated antibiotic drug development pipelines drive polymyxins (polymyxin B and colistin) as crucial therapeutic options. However, the cumbersome synthesis process and inefficient cyclization method limit the efficient preparation of polymyxin core scaffolds in the development of polymyxin derivatives. Here, we innovatively applied a substitution reaction between bromobenzene and sulfhydryl to cyclize colistin core scaffolds. The reaction was mild and efficient, improving the total yield of the compound from less than 10% to 55.90%. Nearly 30 novel derivatives with thioether bond-mediated cyclic scaffolds were designed and synthesized. Evaluation of antibacterial activities and biological properties revealed that many new compounds that are stable in mouse plasma possess high antimicrobial potency against Gram-negative bacteria and display no hemolytic toxicity. Our optimal peptide PE-2C-C8-DH eradicated Acinetobacter baumannii within 24 h in vitro, and had lower acute toxicity and significant therapeutic effects on mice infected with Pseudomonas aeruginosa, which deserves further development.

A quantitative method for determination of colistin E2 methanesulphonate in human plasma by 15N-labeled colistin E2.

The single-component colistin E2, with superior antibacterial activity and lower toxicity, was being developed as the latest generation of polymyxin drugs. However, colistin E2 has not been tested quantitatively in biological matrices. In this study, based on the quantitative detection of colistin methanesulphonate (CMS) and colistin by Zhao et al., 15N-labeled colistin E2 was used as an internal standard (IS) for a more accurate quantitative detection of CMS E2 in human plasma. A rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS E2 and colistin E2 in human plasma. After pretreatment of plasma samples by 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the formed colistin E2 was detected and quantified by UHPLC-MS/MS system. All plasma lots were found to be free of interferences with the analyte. The matrix has no effect on the quantitation of the analyte. No significant effect of the carryover was observed. The dilution integrity was demonstrated in plasma samples without the loss of accuracy and precision. The lower limit of quantification (LLOQ) was 0.0300 mg/L for colistin E2 in plasma with accuracy (relative error, 5.1-12.7%) and precision (relative standard deviation, - 5.7-9.3%). Stability of CMS E2 and colistin E2 was demonstrated in biological samples before and during sample treatment, and in the extract. Furthermore, this method was successfully applied to the analysis of plasma samples obtained from Chinese healthy volunteers receiving a single intravenous CMS E2 dose of 5 mg/kg. In conclusion, the detection method was characterized by speed and high accuracy, which laid a solid foundation for the subsequent development of CMS E2 drug.

Down-regulation of cyclin D1 by small interfering RNA inhibits cell growth and induces apoptosis of laryngeal squamous cell carcinoma.

OBJECTIVE: The objective of the study was to explore the inhibitive role of cyclin D1 gene silence in laryngeal squamous cell carcinoma by lentivirus-mediated RNA interference. MATERIALS AND METHODS: Cd1-RNAi-Lentivirus and the control lentivirus (GFP-Lentivirus) were transfected into Hep-2 cells. Reverse transcriptase polymerase chain reaction was performed to explore the cyclin D1 expression level in Cd1-RNAi-Lentivirus-transfected Hep 2 cells. The apoptosis and viability of Cd1-RNAi-Lentivirus-treated Hep-2 cells were measured with flow cytometry and methyl thiazolyl tetrazoliym assay. In an animal experiment, 10 mice bearing Hep-2 cell tumor were intratumorally injected with Cd1-RNAi-Lentivirus; and the other 10 mice were injected with GFP-Lentivirus. Terminal deoxytransferase-mediated dUTP nick end labeling stains and transmission electron microscope were used to observe the apoptosis in the xenografts. RESULTS: Cyclin D1 was knocked down after Cd1-RNAi-Lentivirus was transfected into Hep-2 cells. The proliferative ability of Hep-2 cells was significantly inhibited by Cd1-RNAi-Lentivirus, and a significant apoptosis of Hep-2 cells was also observed after Cd1-RNAi-Lentivirus transfection. The average weight and volume of tumors in the Cd1-RNAi-Lentivirus-treated group were significantly lower than those in the control group (P < .01). The significant apoptosis was detected with terminal deoxytransferase-mediated dUTP nick end labeling stain and transmission electron microscope. CONCLUSIONS: The present findings suggest that cyclin D1 gene silence by lentivirus-mediated RNA interference can inhibit growth and promote apoptosis of laryngeal squamous cell carcinoma.

Design and biological evaluation of novel long-acting adalimumab Fab conjugated with the albumin binding domain.

Antigen-binding fragments (Fabs) are preferred alternatives to antibodies for medical application, whereas their short half-lives limit therapeutic effectiveness. Albumin binding domain (ABD) with high affinity for albumin possesses a great potential in enhancing in vivo performance of biotherapeutics. In this study, to mitigate the poor pharmacokinetics of adalimumab Fab targeting tumor necrosis factor-α (TNFα), an ABD fusion strategy was applied innovatively using GA3, ABD035, ABD094 and ABDCon with high affinities for albumin. The prokaryotic expression, bioactivities and half-lives of those novel Fab-ABD fusions were investigated in vitro and in vivo. All Fab-ABD fusions were successfully purified, and they retained similar TNFα-binding activities with the unmodified Fab control, also presented high affinities for human/mouse serum albumin (HSA/MSA). Additionally, the simultaneous binding of the difunctional Fab-ABD fusions to TNFα and albumin was verified, and ABD fused to Fab neither interfered with Fab-TNFα binding nor impaired the association between Fc fragment of IgG receptor and transporter (FcRn) and albumin. Based on the highest binding affinity for HSA and maximal yield, Fab-ABDCon was selected for further evaluation. Fab-ABDCon showed similar thermostability with the Fab control and robust stability in human and mouse plasma. Most notably, the pharmacokinetics of Fab-ABDCon in mice was significantly improved with a 22-fold longer plasma half-life (28.2 h) compared with that of Fab control (1.31 h), which have contributed to its satisfactory therapeutic efficacy in murine TNFα-induced hepatonecrosis model. Thus, Fab-ABDCon could be a promising long-acting candidate suitable for drug development targeting TNFα-mediated inflammatory disease.

D- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics.

The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo, indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.

Nanodetection of Head and Neck Cancer on Titanium Oxide Sensing Surface.

Head and neck cancer is a heterogeneous disease, originating in the squamous cells lining the larynx (voice box), mouth, pharynx (throat), nasal cavity and salivary glands. Head and neck cancer diagnosis at the later stage is greatly influencing the survival rate of the patient. It makes a mandatory situation to identify this cancer at the earlier stages of development with a suitable biomarker. Squamous cell carcinoma antigen (SCC-Ag) is a circulating serum tumour biomarker, and the elevated level has been found in the head and neck cancer patients and highly correlated with the tumour volume. The present research was carried out to detect and quantify the level of SCC-Ag on titanium oxide (TiO2)-modified interdigitated electrode sensor (IDE) by SCC-Ag antibody. The detection of SCC-Ag was found at the level of 100 fM, while it was improved to 10 fM when the antibody was conjugated with gold nanostar, representing a 10-fold improvement. Interestingly, this enhancement in sensitivity is 1000-folds higher than other substrates. Moreover, the specificity analysis was carried out using two different control proteins and noticed that the antibody only recognised SCC-Ag, indicating the specific detection on IDE-TiO2 sensing surface.

MMP-14 can serve as a prognostic marker in patients with supraglottic cancer.

The purpose of this study was to examine the expression of MMP-14, 15 and 16 (MT1, MT2 and MT3-MMP) in supraglottic carcinoma and to explore their clinical significance. Expression of MMP-14, 15 and 16 was examined using RT-PCR and immunohistochemistry methods in samples from 85 cases of supraglottic carcinoma. Results showed that both mRNA and protein expression of MMP-14, 15 and 16 were increased in supraglottic carcinoma tissues compared to expression in control adjacent non-neoplastic tissues (P < 0.05). Expression of MMP-14, but not MMP-15 and MMP-16, was significantly increased in the T3 and neck nodal metastasis groups compared with the T1-2 group and the group without nodal metastasis at both mRNA and protein levels (P < 0.05). Expression of MMP-14 mRNA and protein was also higher in tumors of patients with stage III-IV disease compared to patients with clinical stage I-II tumors (P < 0.05). Kaplan-Meier survival curves showed that the groups with high MMP-14 protein expression had a poorer prognosis than patients in the groups with weak or negative expression of MMP-14 protein (P < 0.05). In conclusion, MMP-14 may play an important role in the progression of supraglottic carcinoma and may be a novel prognostic factor for patients with supraglottic carcinoma.

Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2).

Biosynthesis of Zinc oxide nanoparticles (ZnONPs) from natural plants stands as a promising nanodrug delivery system in cancer therapeutics. Marsdenia tenacissima (M.t), a Chinese medicinal plant has been extensively used as clinical remedy for treating several types of cancer. In this present study, ZnONPs were synthesized from Marsdenia tenacissima and its anti cancer potency was assessed against in vitro laryngeal cancer cell line Hep-2. The biosynthesized Marsdenia tenacissima Zinc Oxide Nanoparticles [M.t-ZnONPs] was characterized using UV-visible Spec, SEM, TEM and EDAX analysis. The cytotoxic and apoptotic inducing potential of M.t-ZnONPs was assessed by MTT assay and staining such as DCFH-DA, AO/EtBr, Rhodamine 123, DAPI and comet assay. The anticancer potential of M.t-ZnONPs was analysed by Real time PCR analysis of proapoptotic, antiapoptotic and caspases proteins. Our present findings showed characteristic and morphological representation of synthesized M.t-ZnONPs by UV-visible Spec, SEM, TEM and EDAX analysis. M.t-ZnONPs exhibits its cytotoxicity by inhibiting the viability of Hep-2 cells and IC50 value was obtained by MTT assay. The results of apoptotic staining techniques in M.t-ZnONPs treated Hep-2 cells confirm with excess ROS generation, disruption of mitochondrial membrane potential and nuclear damage. The apoptotic inducing potential of M.t-ZnONPs was also evidenced by upregulation of proapoptotic proteins Bax, Caspase 3 & 9 and downregultion of antiapoptotic protein Bcl-2 by RT-PCR analysis. Finally, these results suggested that biosynthesized M.t-ZnONPs is an effective anticancer agent which induces apoptosis in Hep-2 laryngeal cell line and thus conclude that M.t-ZnONPs, a valid anticancer strategy in treating various cancer.

Inhibition of laryngeal cancer cell invasion and growth with lentiviral-vector delivered short hairpin RNA targeting human MMP-9 gene.

The purpose of this study was to explore the inhibiting role of MMP-9 gene silence in the invasive ability and growth of laryngeal squamous cell carcinoma (LSCC) by lentivirus mediated RNA interference. MMP-9-RNAi-lentivirus and the control lentivirus (GFP-lentivirus) were transfected into Hep-2 cells. Gelatin zymography showed the proteins expression of MMP-9 were knockdown in the MMP-9 siRNA transfected Hep-2 cells. The invasive activity and viability of MMP-9 siRNA treated Hep-2 cells were decreased than the control cells measured with modified Boyden chamber assay and MTT assay. In animal experiment, 20 nude mice bearing Hep-2 cell tumor were randomly separated into the experimental and the control groups. The former were intratumorally injected with MMP-9-RNAi-lentivirus, and the later were injected with equivalent dose of GFP-lentivirus. Results showed the average weight and volume of tumor in MMP-9-RNAi-lentivirus treated group were significantly lower than those in the control group (P < .01). The protein expressions of MMP-9 were downregulated in tumors of MMP-9-RNAi-lentivirus treatment. The PCNA index was obviously lower in the tumors of treated group than that in the control group (P < .01). These results suggest that MMP-9 gene silence by lentivirus mediated RNA interference can inhibit invasion and growth of LSCC.

Co­inhibition of miRNA­21 and miRNA­221 induces apoptosis by enhancing the p53­mediated expression of pro­apoptotic miRNAs in laryngeal squamous cell carcinoma.

Dysregulation of a numerous microRNAs (miRNAs) has been implicated in laryngeal squamous cell carcinoma (LSCC). Among those miRNAs, miR­21 and miR­221 are co­overexpressed and commonly target the phosphatase and tensin homolog protein (PTEN) that is located in the PTEN­Akt signaling pathway. The present study investigated whether co­inhibition of miR­21 and miR­221 induced synergistic apoptosis of human LSCC cells. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl­transferase­mediated deoxynucleotide triphosphate nick end labeling (TUNEL) assays were used to observe the potential effect of miR­21 and miR­221 on cell viability and apoptosis in cells co­transfected with anti­miRNA oligonucleotide (AMO)­21 and AMO­221. The protein expression levels of PTEN, Akt and p53 were determined by western blotting. The cellular abundance of 6 pro­apoptotic miRNAs transcribed by p53 mediation, consisting of miR­15a, miR­16­1, miR­26a, miR­34a, miR­143 and miR­203, was measured with using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). MTT results indicate that in vitro co­transfection of AMO­21 and AMO­221 leads to a decline in cell viability, compared with the transfection of AMO alone. This result was verified by the detection of apoptosis using TUNEL assays. Co­transfection of AMO­21 and AMO­221 resulted in a marked reduction in Akt phosphorylation and enhanced expression of PTEN and p53 were observed; consequently, leading to an amplification of the transcription of 6 pro­apoptotic miRNAs. The present findings confirmed that co­inhibition of miR­21 and miR­221 synergistically triggers cell apoptosis in vitro. The altered PTEN­Akt signaling and p53­mediated amplification of the transcription of pro­apoptotic miRNAs may be involved in the observed synergistic effect. The present study provides novel insights into the mechanism underlying apoptosis­associated miRNA­miRNA mutual regulation in LSCC.

Cyr61/CCN1 overexpression induces epithelial-mesenchymal transition leading to laryngeal tumor invasion and metastasis and poor prognosis.

BACKGROUND: To examine the expression of cysteine-rich 61 (Cyr61/CCN1) protein in laryngeal squamous- cell carcinoma (LSCC) tissues, and its relationship with the tumor epithelial-mesenchymal transition (EMT), invasion, metastasis, and prognosis. MATERIALS AND METHODS: Immunohistochemistry was used to detect the expressions of Cyr61, Vimentin (Vim), and E-cadherin (E-cad) in 88 cases of LSCC tissues and 30 cases of tumor-adjacent normal tissues. Vim and E-cad were used as mesenchymal and epithelial markers, respectively, to determine the relationship between Cyr61 expression and the EMT of LSCC cells. In addition, clinical and histopathological data were combined to analyze the relationship between the positive-expression rates of Cyr61, Vim and E-cad and LSCC invasion, metastasis and prognosis. RESULTS: In LSCC tissues, Vim expression rate was significantly higher than that of the tumor-adjacent tissues, whereas E-cad expression rate was significantly lower than that of the tumor-adjacent tissues. The Vim expression rate was significantly higher in stages T3 and T4 than in stages T1 and T2 LSCC tissues, whereas E-cad expression rate was significantly lower in stages T3 and T4 than in stages T1 and T2 LSCC tissues. Compared to the group without lymph node metastasis, the Vim expression rate was significantly higher and the E-cad expression rate was significantly lower in the group with lymph node metastasis. The expression rate of Cyr61 was significantly higher in LSCC tissues than in the tumor-adjacent normal tissues. In addition, the Cyr61 expression rate was higher in stages T3 and T4 than in stages T1 and T2 LSCC, and higher in the group with lymph node metastasis than in the group without lymph node metastasis. The Vim expression rate was significantly higher in the Cyr61 positive group than in the Cyr61 negative group, whereas the E-cad expression rate was significantly higher in the Cyr61 negative group than in the Cyr61 positive group. Survival analysis indicated that survival rates of Cyr61 positive, Vim positive and E-cad negative groups were significantly lower than that of Cyr61 negative, Vim negative and E-cad positive groups, respectively. CONCLUSIONS: Cyr61 expression is closely associated with LSCC invasion and lymph node metastasis. Overexpression of Cyr61 may induce EMT and therefore leads to LSCC invasion and metastasis and poor prognosis. Cyr61 may become a new maker for clinical prediction of LSCC invasion and metastasis and a new target for LSCC treatment.

Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma.

Serum exosomes containing noncoding RNA (ncRNA) play an important role in both physiological and pathological conditions. However, biological function of exosomal ncRNA remains unclear. The aim of the study was to investigate the prognostic and diagnostic values of exosomal ncRNA by comparing the amounts of exosomal miR-21 and HOTAIR in serum of laryngeal squamous cell carcinoma (LSCC) patients with those of polyps of vocal cords, and by determinating whether combined detection of the two molecules could provide useful information in the diagnosis of LSCC. Exosomes were isolated from the serum samples of 52 LSCC patients and those of 49 patients with polyps of vocal cords. TEM and Western blot were applied for the confirmation of isolated exosomes by observing the ultra structure and testing CD63 marker protein, respectively. RT-PCR was performed to detect the expression of miR-21 and HOTAIR in the exosomes. The receiver-operating characteristic (ROC) curve was generated to examine the prognostic value of the two molecules. The expression of exosomal miR-21 and HOTAIR was significantly higher in patients with LSCC than those with vocal cord polyps. There were significant differences of serum exosomal miR-21 and HOTAIR expressions between the advanced T classifications (T3/T4) or clinical stages (III/IV) and the early stages. The patients with lymph node metastasis had higher serum exosomal miR-21 and HOTAIR expressions than those without. There were no differences between patient sex, tumor locations and differentiations. The area under the ROC curve of combined examination of exosomal HOTAIR and miR-21 for diagnosing LSCC was 87.6 %, which was significantly higher than 80.1 % of miR-21 (p = 0.0359) or 72.7 % of HOTAIR (p = 0.0012), showing 94.2 and 73.5 % of sensitivity and specificity, respectively, in differentiating the malignant from benign laryngeal disease. Serum exosomal miR-21 and HOTAIR were significantly correlated with clinical parameters of LSCC, and combined evaluation of their serum expressions may be a valuable biomarker to screen LSCC and might be a promising predicting tool for LSCC patient.

MicroRNA-205 suppresses proliferation and promotes apoptosis in laryngeal squamous cell carcinoma.

MicroRNAs were reported to be involved in the modulation of tumor development. The aim of our study was to investigate the effect of miR-205 on proliferation and apoptosis of laryngeal squamous cell carcinoma (LSCC) and seek associations between miR-205 and Bcl-2 using in vitro and in vivo methods. Real-time qPCR was used to analyze the expression of miR-205 in LSCC samples and Hep-2 cell line. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to test the apoptosis and proliferation of LSCC cells after miR-205 transfection. Bcl-2 expression in cells was assessed with Western blotting. The tumorigenicity of LSCC cells was evaluated in nude mice model. MiR-205 was significantly down-regulated in LSCC tissues compared to adjacent normal tissues. Lower expression of miR-205 was indicated to be statistically related with advanced clinical stage and T3-4 grades. We found that restoration of miR-205 down-regulated the proliferative markers of dihydrofolate reductase and proliferating cell nuclear antigen and apoptotic regulator of Bcl-2. The findings in vitro and in vivo showed miR-205 could suppress cell proliferation and induce cell apoptosis. In addition, Bcl-2 was identified as one of the direct targets of miR-205 in LSCC cells. These results suggest that miR-205 may play as a tumor suppressor in LSCC, probably by targeting Bcl-2 and serve as a potential target for therapeutic intervention.