ASIC1 promotes migration and invasion of hepatocellular carcinoma via the PRKACA/AP-1 signaling pathway.

Hepatocellular carcinoma (HCC) exhibits a high mortality rate due to its high invasion and metastatic nature, and the acidic microenvironment plays a pivotal role. Acid-sensing ion channel 1 (ASIC1) is upregulated in HCC tissues and facilitates tumor progression in a pH-dependent manner, while the specific mechanisms therein remain currently unclear. Herein, we aimed to investigate the underlying mechanisms by which ASIC1 contributes to the development of HCC. Using bioinformatics analysis, we found a significant association between ASIC1 expression and malignant transformation of HCC, such as poor prognosis, metastasis and recurrence. Specifically, ASIC1 enhanced the migration and invasion capabilities of Li-7 cells in the in vivo experiment using an HCC lung metastasis mouse model, as well as in the in vitro experiments such as wound healing assay and Transwell assay. Furthermore, our comprehensive gene chip and molecular biology experiments revealed that ASIC1 promoted HCC migration and invasion by activating the PRKACA/AP-1 signaling pathway. Our findings indicate that targeting ASIC1 could have therapeutic potential for inhibiting HCC progression.

Linc00662 m6A promotes the progression and metastasis of pancreatic cancer by activating focal adhesion through the GTF2B-ITGA1-FAK pathway.

Recurrence and metastasis are major factors associated with the poor prognosis of pancreatic cancer (PC). Previous studies have indicated that METTL3-mediated N6-methyladenosine (m6A) modification is closely associated with PC progression and prognosis. However, its underlying regulatory mechanisms remain unclear. In this study, we found that METTL3 was upregulated in PC tissues and cells and was associated with malignant tumor progression and poor progression-free survival in PC. Linc00662 was screened as a m6A-enriched RNA that promoted tumor growth and metastasis in PC cells and mouse models and was associated with poor clinical prognosis. Four m6A motifs were identified in Linc00662, which maintained the stability of Linc00662 in an IGF2BP3-coupled manner and were closely associated with the pro-tumor properties of Linc00662 in vitro and in vivo. ITGA1 was identified as a downstream gene regulated by Linc00662. Linc00662 recruites GTF2B to activate the transcription of ITGA1 in a m6A-dependent manner and initiates the formation of focal adhesions through the ITGA1-FAK-Erk pathway, thereby promoting malignant behavior in PC cells. The FAK inhibitor-Y15 obviously repressed tumor progression in Linc00662-overexpressing PC cells in vitro and in vivo. This study proposes a novel regulatory mechanism of Linc00662 in oncogene activation in PC and indicates that Linc00662 and its downstream genes are potential targets for PC therapy.

ACTR3 promotes cell migration and invasion by inducing epithelial mesenchymal transition in pancreatic ductal adenocarcinoma.

BACKGROUND: Recurrence and metastasis are the major causes of pancreatic ductal adenocarcinoma (PDAC) mortality after treatment. The underlying molecular mechanism is poorly understood. Actin-related protein 3 (ACTR3) is an important component of the actin-related protein 2/3 complex, which is involved in the regulation of cell motility and epithelial mesenchymal transition (EMT) process. Previously published studies have indicated that ACTR3 expression is upregulated in several types of cancers, and promotes tumor development, including gastric cancer and hepatocellular carcinoma. However, to date, the expression levels and the role of ACTR3 in PDAC are not well understood. METHODS: In the present study, the expression levels of ACTR3 in PDAC tissue and the relationship of ACTR3 expression with clinical prognosis were analyzed by mRNA microarray and bioinformatics. The biological functions and underlying mechanism of ACTR3 in PDAC were examined by a series of assays, including Cell Counting Kit-8 (CCK-8), transwell assay, and Western blotting. RESULTS: We found that the expression of ACTR3 was significantly increased in PDAC tissues and cell lines. A higher expression of ACTR3 was predictive of poor outcome for patients with PDAC. In vitro, the knockdown of ACTR3 expression significantly inhibited the invasive and migratory capacity of PDAC cells, and altered the distribution of F-actin and the expression of EMT markers. CONCLUSIONS: The findings of our study indicated that ACTR3 promotes cell migration and invasion by inducing EMT in PDAC, which may be a potential therapeutic target and prognostic indicator for PDAC patients.

NEAT1/miR-101-dependent Up-regulation of DNA-PKcs Enhances Malignant Behaviors of Pancreatic Ductal Adenocarcinoma Cells.

Background: Although we previously revealed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and important for gemcitabine resistance, the role of DNA-PKcs in the progression and metastasis of PDAC remain unclear. To date, the upstream signaling events stimulating DNA-PKcs overexpression in PDAC are still not well characterized. Methods: Expression of DNA-PKcs was measured by western blot. The levels of miRNA-101 and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) were detected by real-time PCR. Cell viability was determined by CCK-8. Cell migration and cell invasion were measured by transwell assay. The regulatory relationship between NEAT1 and miR-101 was determined by a luciferase assay. Results: DNA-PKcs expression was significantly elevated in human PDAC tissues and cells. DNA-PKcs overexpression was correlated with TNM stage and lymph node metastasis. Higher expression of DNA-PKcs was closely correlated with patients of worse overall survival (OS). DNA-PKcs knockdown suppresses malignant behaviors of PDAC cells. Further study showed that miRNA-101 level was decreased in PDAC tissues and cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in PDAC cells. Moreover, NEAT1 functions as an oncogene influencing cell proliferation, migration and invasion in part by serving as a competing endogenous RNA (ceRNAs) modulating miR-101 expression, leading to up-regulation of DNA-PKcs. Conclusion: These findings suggest that NEAT1/miR-101-dependent up-regulation of DNA-PKcs promotes the malignant behaviors of PDAC cells. The NEAT1/miR-101/DNA-PKcs axis may serve as a viable prognostic marker and therapeutic target for PDAC.

[On-line coupling of microcolumn electrophoresis and UV-Vis spectrophotometry].

In the present paper, a laboratory-made high-performance electrophoresis microcolumn unit was prepared for UV-Vis spectrophotometer. X-ray diffraction was used in the preparation of electrophoretic microcolumns. And an analytical technique of microcolumn electrophoresis coupled with UV-Vis spectrophotometry was introduced. Uniform quartz microncrystals were prepared by hydrothermal synthesis. Their crystalline phase and morphology were identified by X-ray diffraction and scanning electron microscope, respectively. The quartz microncrystals were packed into a 2-mm i. d. fused-silica tube to prepare the electrophoretic microcolumn. With 1.5 mmol x L(-1) disodium phosphate buffer solution (pH 11.5) containing 25% (phi) methanol and 10% (phi) acetonitrile, tryptophan, phenylalanine and tyrosine were on-line separated on line and detected by microcolumn electrophoresis coupled with UV-Vis spectrophotometry without derivatization. The limits of detection were 0.037, 0.20 and 0.20 micromol x L(-1), respectively. The separation efficiency of tryptophan was 4.5 x 10(4) plates/m. The sample capacity of the electrophoretic microcolumn achieved 35 microL. It was found that the electrophoretic microcolumn packed with quartz microncrystals was able to limit Joule heat, increase sample capacity and enhance detection sensitivity. The laboratory-made electrophoretic microcolumn could be a high-performance separation unit for conventional UV-Vis spectrophotometer. The on-line coupling of microcolumn electrophoresis and UV-Vis spectrophotometry could separate and determine samples with complicated matrices, reduce zone broadening and enhance separation efficiency, so expand the analytical function of spectrophotometer in the trace analysis of mixed components with overlapped spectra.

Determination of kanamycin A, amikacin and tobramycin residues in milk by capillary zone electrophoresis with post-column derivatization and laser-induced fluorescence detection.

An analytical method for kanamycin A, amikacin and tobramycin of aminoglycoside (AG) antibiotics in milk samples was proposed using capillary zone electrophoresis (CZE) with post-column derivatization and laser-induced fluorescence detection. A simple and convenient homemade coaxial-gap reactor was adopted in the post-column derivatization of the AGs with 1.0 mmol/L naphthalene-2,3-dicarboxaldehyde and 8.0 mmol/L 2-mercaptoethanol in 35 mmol/L sodium tetraborate buffer (pH 10.0) of 30% (v/v) methanol. 50 mmol/L sodium acetate (pH 5.0) containing 0.5 mmol/L cetyltrimethyl ammonium bromide was used as the separation buffer. The linear calibration concentrations and detection limits were from 2.1 x 10(-5) to 5.0 x 10(-2)g/L and in the range of 7 x 10(-6) to 2 x 10(-5)g/L, respectively. The recoveries of the AGs in milk samples were from 81.6 to 93.1% (n=3).

MicroRNA-181a inhibits autophagy by targeting Atg5 in hepatocellular carcinoma.

Available evidence suggests that autophagy may serve as a tumor suppressor in cases of chronic liver disease and liver cirrhosis and that autophagic deficiency may lead to hepatocellular carcinoma (HCC). Recent studies suggested that the development of several tumor types could be regulated by microRNA-181a. However, the role of miR-181a in the autophagy of HCC remains unclear. In this study, we aimed to investigate the role of miR-181a in the autophagy of HCC. We found that the mRNA expression of miR-181a is higher but the level of autophagy is lower in human HCC compared to normal liver tissue. A luciferase assay confirmed that Atg5 is the target gene of miR-181a. Moreover, the results showed that an miR-181a sponge increased apoptosis in HepG2 cells and reduced the growth of tumors in a HepG2 cell xenograft tumor model. In conclusion, these results suggest that miR-181a can inhibit autophagy in HCC by targeting Atg5, resulting in decreased apoptosis of HCC cells and increased tumor growth.

Post-column reactor of coaxial-gap mode for laser-induced fluorescence detection in capillary electrophoresis.

A post-column reactor with coaxial-gap mode is developed for laser-induced fluorescence detection (LIF) in capillary electrophoresis (CE). The reactor can be assembled simply and conveniently, in which a thin polyimide sleeve of 10-mm length obtained from the capillary coating is used to align separation and reaction capillary with a 20 microm gap. Naphthalene-2,3-dicarboxaldehyde and 2-mercaptoethanol are used as derivatization reagents and delivered into the reaction capillary through the annulus between the separation capillary and polyimide sleeve and the gap of two capillaries by gravity. A reaction distance from the gap to detection point is 5mm. For the post-column reactor of CE-LIF, several configuration parameters are optimized, including liquid level difference between the derivatization solution and outlet buffer, annular dimension between the outer diameter of etched separation capillary and the inner diameter of polyimide sleeve, and reaction distance, etc. The detection limits in the range from 8.0x10(-8) to 1.0x10(-6) mol/L and linear calibration range more than two orders of magnitude are obtained for amino acids. The separation efficiency ranges from 1.35x10(5) to 1.67x10(5) theoretical plates.

Funnelform single-drop microextraction for gas chromatography-electron-capture detection.

A funnelform single-drop microextraction was developed for gas chromatography-electron-capture detection. A solvent microdrop of 4 microL was formed at the tip of a microsyringe needle assembled with a small brass funnel in the microextraction. In the funnel, the restricted microdrop was shaken gently by circular motion in an aqueous sample solution. Eleven organochlorine and two pyrethroid pesticides were used as the model compounds for evaluating the microextraction. The parameters affecting the enrichment factor of the microextraction were investigated, including the funnel inner angle, solvent component, and microdrop volume, etc. With the optimized microextracting conditions, the enrichment factors of organochlorines and pyrethroids were 272-875 and 147-183, respectively. The detection limit was in the range of 1-12 ng/L (S/N = 3). The relative standard deviation (RSD) of peak height was less than 10.2% (n = 8). This proposed technique is simple, convenient, and efficient.

On-line sequential preconcentration of inorganic anions by anion-selective exhaustive injection and base-stacking in capillary zone electrophoresis.

A sequential electrostacking method based on anion-selective exhaustive injection (ASEI) and base-stacking (BS) is presented for the preconcentration and determination of inorganic anions by capillary zone electrophoresis (CZE) in this paper. Tetradecyltrimethylammonium bromide as an electroosmotic flow (EOF) modifier was added into the buffer to suppress EOF of the capillary. Firstly, a water plug was hydrodynamically injected into the capillary. During ASEI under negative high voltage, the sample anions migrated quickly towards the boundary between the water plug and buffer in the capillary. Then an alkaline zone was injected electrokinetically to concentrate the anions further. With the sequential electrostacking method, the preconcentration factor of (0.8-1.3) x 10(5) was obtained compared with the conventionally electrokinetic injection and the relative standard deviation of peak area was 3.3-5.3% (n = 5). The detection limits of ASEI-BS-CZE for six inorganic anions were 6-14 ng/L. The proposed method has been adopted to analyze six anions in cigarette samples successfully.

Determination of aflatoxins in high-pigment content samples by matrix solid-phase dispersion and high-performance liquid chromatography.

A fast, efficient, and cost-effective method was developed for the analysis of aflatoxins in farm commodities with high-pigment content, such as chili powder, green bean, and black sesame. The proposed method involved matrix solid-phase dispersion (MSPD) and high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) with postcolumn electrochemical derivatization in a Kobra cell. The MSPD procedure combined the extraction with neutral alumina and pigment cleanup with graphitic carbon black (GCB) in a single step. The recoveries of aflatoxins ranged from 88% to 95% with the relative standard deviations (RSD) less than 6% (n = 6). The limits of detection (LODs) were 0.25 ng/g aflatoxin B1, G1, and 0.10 ng/g aflatoxin B2, G2, respectively. The analytical results obtained by MSPD were compared to those of the immunoaffinity column (IAC) cleanup method. No significant differences were found between the two methods by t-test at the 95% confidence level.

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.

On-line cation-exchange preconcentration and capillary electrophoresis coupled by tee joint interface.

An on-line preconcentration method based on ion exchange solid phase extraction was developed for the determination of cationic analytes in capillary electrophoresis (CE). The preconcentration-separation system consisted of a preconcentration capillary bonded with carboxyl cation-exchange stationary phase, a separation capillary for zone electrophoresis and a tee joint interface of the capillaries. Two capillaries were connected closely inside a 0.3 mm i.d. polytetrafluoroethylene tube with a side opening and fixed together by the interface. The preparations of the preconcentration capillaries and interface were described in detail in this paper. The on-line preconcentration and separation procedure of the analysis system included washing and conditioning the capillaries, loading analytes, filling with buffer solution, eluting analytes and separating by capillary zone electrophoresis (CZE). Several analysis parameters, including sample loading flow rate and time, eluting solution and volume, inner diameter and length of preconcentration capillary etc., were investigated. The proposed method enhanced the detection sensitivity of CE-UV about 5000 times for propranolol and metoprolol compared with normally electrokinetic injection. The detection limits of propranolol and metoprolol were 0.02 and 0.1 microg/L with the proposed method respectively, whereas those were 0.1 and 0.5 mg/L with conventional electrokinetic injection. The experiment results demonstrate that the proposed technique can increase the preconcentration factor evidently.

Response surface optimization for determination of pesticide multiresidues by matrix solid-phase dispersion and gas chromatography.

An optimized multiresidue analysis method based on matrix solid-phase dispersion (MSPD) and gas chromatography (GC) is proposed for the determination of organochlorines and pyrethroids in the tea samples. Response surface methodology (RSM) was used to optimize the extraction conditions of MSPD, such as the sorbent type, eluent composition, dichloromethane concentration and eluting volume. Desirability function approach was employed to optimize the pesticide recoveries and matrix cleanup. Compromising the recoveries and cleanup degree, MSPD was carried out with Florisil as the sorbent and n-hexane-dichloromethane (1:1, v/v) as the eluent. The pesticide recoveries in tea samples were better than 80% spiked in the concentration range of 0.01-0.05mg/kg and the relative standard deviations were lower than 7%. The quantification limits of the pesticides were in the range of 0.002-0.06mg/kg, which were lower than the maximum residue limits of the pesticides in tea samples established by the European Union.

Electrochromatography with a 2.7 mm inner diameter monolithic column.

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. monolithic column.

Investigations on factors that influence the moving neutralization reaction boundary method for capillary electrophoresis and isoelectric focusing.

We investigated several factors, such as temperature, current intensity (i), time (t) and the product (mA min mm(-2), viz., C mm(-2)) of i and t, etc., that obviously affect the moving neutralization reaction boundary method (MNRBM). The results manifest that the temperature and the product ti have a strong influence on the movement rate of the boundary. The data prove that about 0.6 C mm(-2) (being equivalent to 10 mA min mm(-2)) is a critical point. If the product ti is lower than the critical point, a good quantitative agreement exists between the observed and theoretical values, but if it is higher than the critical point, the agreements are poor. The optimized experimental conditions are: (1) 18-20 degrees C room temperature, (2) 0.6-0.8 mA mm(-2), (3) less than 10 mA min mm(-2), (4) 1% agarose gel, (5) daily prepared solution and gel containing NaOH. The optimized MNRBM is of benefit for the studies on MNRB itself, isoelectric focusing and capillary zone electrophoresis as will be partially shown in this paper.

Improving separation efficiency of capillary zone electrophoresis of tryptophan and phenylalanine with the transient moving chemical reaction boundary method.

A simple and convenient mode--moving chemical reaction boundary method-capillary zone electrophoresis (MCRBM-CZE)--was designed for the enhancement of separating efficiency of CZE. In this mode, the transient MCRBM is used for the on-line pre-treatment of sample. By analyses of tryptophan (Trp) and phenylalanine (Phe) as an example, the experiments by MCRBM-CZE were carried out and further compared with those by normal CZE without the transient MCRBM. The results reveal that by carefully selected appropriate electrolytes, a strong condensation effect can be achieved by using MCRBM-CZE; this effect can greatly improve the separation efficiency, resolution and peak height of Trp and Phe in CZE as compared with those of normal CZE of Trp and Phe. Even if the sample comprises high concentrations of salt, such as 80 mM NaCl (concentration of sodium ion up to 145.6 mM), the same condensation effect can also been observed; this implies obvious significance for biological samples like urine and serum. However, if the electrolytes was chosen inappropriately only a poor compression effect of sample was observed in the MCRBM-CZE runs.

Electrochemical hydride generation atomic fluorescence spectrometry for detection of tin in canned foods using polyaniline-modified lead cathode.

An electrochemical hydride generation system with polyaniline-modified lead cathode was developed for tin determination by coupling with atomic fluorescence spectrometry. The tin fluorescence signal intensity was improved evidently as the polyaniline membrane could facilitate the transformation process from atomic tin to the SnH(4) and prevent the aggradation of Sn atom on Pb electrode surface. The effects of experimental parameters and interferences have been studied. The limit of detection (LOD) was 1.5 ng mL(-1) (3σ) and the relative standard deviation (RSD) was 3.3% for 11 consecutive measurements of 50 ng mL(-1) Sn(IV) standard solution.

In-line coupling headspace liquid-phase microextraction with capillary electrophoresis.

An analytical technique of in-line coupling headspace liquid-phase microextraction (HS-LPME) with capillary electrophoresis (CE) was proposed to determine volatile analytes. A special cover unit of the sample vial was adopted in the coupling method. To evaluate the proposed method, phenols were used as model analytes. The parameters affecting the extraction efficiency were investigated, including the configuration of acceptor phase, kind and concentration of acceptor solution, extraction temperature and time, salt-out effect, sample volume, etc. The optimal enrichment factors of HS-LPME were obtained with the sample volume of about half of sample vials, which were confirmed by both the theoretical prediction and experimental results. The enrichment factors were obtained from 520 to 1270. The limits of detection (LODs, S/N=3) were in the range from 0.5 to 1 ng/mL each phenol. The recoveries were from 87.2% to 92.7% and the relative standard deviations (RSDs) were lower than 5.7% (n=6). The proposed method was successfully applied to the quantitative analysis of the phenols in tap water, and proved to be a simple, convenient and reliable sample preconcentration and determination method for volatile analytes in water samples.

Determination of preservatives by integrative coupling method of headspace liquid-phase microextraction and capillary zone electrophoresis.

An integrative coupling method of headspace liquid-phase microextraction (HS-LPME) and capillary zone electrophoresis (CZE) was proposed in this paper. In the method, a separation capillary was used to create a microextraction droplet of the running buffer solution of CZE, hold the droplet at the capillary inlet, extract analytes of sample solutions in the headspace of a sample vial, inject concentrated analytes into the capillary and separate the analytes by CZE. The proposed method was applied to determine the preservatives of benzoic acid and sorbic acid in soy sauce and soft drink samples, in which the running buffer solution of 50 mmol/L tetraborate (pH 9.2) was directly used to form the acceptor droplet at the capillary inlet by pressure, and the preservatives in a 6-mL sample solution containing 0.25 g/mL NaCl were extracted at 90°C for 30 min in the headspace of a 14-mL sample vial. Then the concentrated preservatives were injected into the capillary at 10 cm height difference for 20 s and separated by CZE. The enrichment factors of benzoic acid and sorbic acid achieved 266 and 404, and the limits of detection (LODs) were 0.03 and 0.01 µg/mL (S/N=3), respectively. The recoveries were in the range of 88.7-105%. The integrative coupling method of HS-LPME and CZE was simple, convenient, reliable and suitable for concentrating volatile and semi-volatile organic acids and eliminating matrix interferences of real samples.