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  • 2021 Volume 31 Issue 06
    Published: 25 November 2021
      
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  • Expression of circular RNA hsa_circ_0079557 in gastric cancer and its clinical significance
    2021, 31(06): 461-466.
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    ［Abstract］Objective: To investigate the expression level and clinical value of hsa_circ_0079557 in gastric cancer.Methods: Five pairs of gastric cancer tissues and adjacent tissues were collected and detected by high-throughput sequencing technology, combined with the data of gastric cancer in circbase and other databases, to screen for differentially expressed circRNAs.In 62 pairs of samples, hsa_circ_0079557 was detected by qRT-PCR, and its relationship with clinicopathological characteristics was analyzed.The expression levels of hsa_circ_0079557 in gastric cancer cells and gastric mucosal epithelial cells, preoperative and postoperative serums of gastric cancer patients, and serums of gastric cancer patients and healthy people were detected.After hsa_circ_0079557 was knocked down by siRNA, cell clone formation assay, plate scratch assay, and cell cycle flow cytometry assay were used to detect the changes in cell cloning, migration and cell cycle of gastric cancer cells, respectively.Western blotting was used to determine cyclin dependent kinase 3 （CDK3） expression level.Results: The expression level of hsa_circ_0079557 in human gastric cancer tissues was significantly higher than that in adjacent tissues（P<0.05）, and its expression level was related to the tumor TMN stage（P<0.05）; the expression level of hsa_circ_0079557 in gastric cancer cells was significantly higher than that in human gastric mucosal epithelial cells（P<0.05）; the expression level of hsa_circ_0079557 in serum of gastric cancer patients before operation was significantly higher than that after operation （P<0.01）; hsa_circ_0079557 knockdown in gastric cancer cell lines resulted in decreased proliferation and migration ability of some gastric cancer cells and decreased CDK3 expression level（P<0.05）.In gastric cancer cell lines, hsa_circ_0079557 knockdown resulted in cell arrest in G0-G1 phase.Conclusion: Hsa_circ_0079557 is highly expressed in gastric cancer tissues, which might be used as a potential biomarker for early diagnosis and prognosis of gastric cancer.
    ［Key words］gastric cancer; circRNA; hsa_circ_0079557
  • Expression profile of peripheral blood mononuclear cells derived circular RNA in patients with Graves′ disease
    2021, 31(06): 467-472，500.
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    Objective： To explore the expression profile of circular RNA （circRNA） in peripheral blood mononuclear cells （PBMCs） of patients with Graves′ disease and its potential diagnostic value. Methods： Five patients with Graves′disease and five healthy controls were selected to analyze the expression profile of circRNA in PBMCs by next generation highthroughput sequencing. Another 40 patients with Graves′ disease and 40 healthy controls were selected to verify the differential expression of circRNA; MicroRNAs （miRNAs） that may bind to differentially expressed circRNA were predicted according to CircInteract database; Bioinformatics was used to analyze the potential biological functions of differentially expressed circRNA; ROC curve analysis of the differential expression of circRNA in Graves′disease. Results： Next generation highthroughput sequencing data showed that， compared with healthy controls， there were 23 215 up-regulated and 13 695 down-regulated circRNA in patients with Graves′disease， of which 669 were significantly differentially expressed， including 360 up-regulated and 309 down regulated circRNA （difference multiple≥2， P≤0.05）. Hsa_circ_0114427，hsa_circ_0007470，hsa_circ_0048232 and hsa_circ_0002672 were confirmed by real-time PCR， and the expression level was consistent with that of high-throughput sequencing， and there were statistical significance between the two groups （P<0.01 and P<0.05）， but there was no difference in expression of hsa_circ_0039161 and hsa_circ_0002600.GO enrichment analysis showed that the up-regulated circRNA was closely related to polymer metabolism， cell membrane binding organelles， and transferase activation. KEGG analysis showed that the differentially expressed circRNA might be involved in thyroid hormone signaling， phosphatidylinositol signaling system， non-small cell lung cancer and other related signaling pathways. ROC curve showed hsa_circ_0114427 has the biggest area under the curve. Conclusion： There are clearly differentially expressed circular RNAs in PBMCs of patients with Graves′disease. Hsa_circ_0114427 may be related to the occurrence and development of Graves′disease.
    ［Key words］Graves′disease; circular RNA; nextgeneration high-throughput sequencing; biomarkers; PBMCs
  • Expression and clinical significance of MDM2 circular RNA
    in bone marrow of patients with acute myeloid leukemia
    2021, 31(06): 473-478.
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    Objective: To explore the expression level and potential clinical significance of murine double minute 2 (MDM2) circular RNA (circ-MDM2) in bone marrow specimens of patients with acute myeloid leukemia (AML). Methods: Expression level of circMDM2 in bone marrow mononuclear cells (BMMNCs) of 92 AML patients and 22 controls was detected by quantitative realtime PCR. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of circ-MDM2 expression. The association between the expression level of circ-MDM2 and the clinical parameters of AML patients was analyzed. Kaplan-Meier curve was used to analyze the influence of circ-MDM2 on overall survival and leukemia-free survival. Results: Compared with the control group, circ-MDM2 was significantly increased in AML patients (P<0.05). According to the ROC curve result, the area under curve of circ-MDM2 for diagnosing AML was 0.649, and the expression level of circ-MDM2 could distinguish AML patients from controls (P=0.03). Mutation rate of CEBPA in circ-MDM2 low expression group was significantly higher than that in circ-MDM2 high expression group (P=0.039). Overall survival and leukemia-free survival of patients in high-expressed circ-MDM2 group and low-expressed circ-MDM2 group were not statistically significant (P>0.05). Conclusion:  circ-MDM2 was significantly up-regulated in AML patients and could be used as a potential biomarker for the diagnosis of AML.
    ［Key words］acute myeloid leukemia; MDM2; circular RNA; bone marrow mononuclear cells
  • Fe 3+  inhibits the killing effect of gossypol acetate on glioma cells#br#
    2021, 31(06): 484-488.
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    Objective: To investigate the effect of iron ion (Fe 3+ ) in human glioma microenvironment on gossypol acetate (GAA) killing glioma cells. Methods: Human glioma cells LN229, SW1783, U87MG and U251MG were treated with different concentrations of GAA (0, 5, 10, 15, 20, 25 μmol/L). CCK-8 assay was used to detect the cell viability and calculate the half inhibitory concentration (IC50). The 4 types of glioma cells were divided into 4 treatment groups: control group, Fe3+ group, GAA group, Fe3++GAA group, treated with medium, FeCl3·6H2O, GAA, FeCl3·6H2O+GAA separately. The concentration of GAA was selected based on the IC50 of each glioma cell, and the concentration of FeCl3·6H2O was the same as that of GAA. CCK-8 assay was used to detect the cell proliferation ability. Transwell assay was used to detect cell migration ability, and the flow cytometry was used to analyze the rate of apoptosis in the glioma cells. Results: GAA significantly inhibited the proliferation of glioma cells, and the IC50 of GAA to LN229, SW1873, U87MG, U251MG was 9.38, 9.56, 14.53, 10.39 μmol/L. Compared with the control group, the proliferation of glioma cells was not significantly changed in the Fe3+ group, while the proliferation and migration of cells were significantly inhibited in the GAA group(P<0.05). Compared with GAA group, the activity and migration of tumor cells were significantly enhanced in the Fe3++GAA group(P<0.05), and the level of apoptosis was decreased obviously(P<0.05). Conclusion: Fe 3+ could inhibit the killing effect of GAA on human glioma cells.
    ［Key words］glioma; iron metabolism; gossypol acetate; apoptosis; transferrin receptor c
  • Expression of lncRNA SPATA3-AS1 in gastric cancer and its significance
    2021, 31(06): 489-495.
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    Objective:  To investigate the expression of long non-coding RNA(lncRNA) SPATA3-AS1 in gastric cancer tissues and plasma and its clinical significance, and to further explore its effect on gastric cancer cells and its potential mechanism. Methods: Real-time quantitative fluorescence PCR(qRT-PCR) was used to detect the expression level of SPATA3-AS1 in plasma of 80 pairs of gastric cancer and adjacent tissues, 29 gastric cancer patients and 29 healthy subjects. The difference of SPATA3-AS1 expression and clinicopathological characteristics of gastric cancer patients were statistically analyzed. Cell proliferation, clone formation, transwell assay and flow cytometry were used to analyze the effects of SPATA3-AS1 knockdown on gastric cancer cells(AGC and HGC-27).The effect of SPATA3-AS1 knockdown on the expression level of tumor-related molecules was detected by qRT-PCR. Results: SPATA3-AS1 was highly expressed in gastric cancer tissues, and its expression level was correlated with lymph node metastasis, TNM stage, depth of tumor invasion and prognosis. Plasma SPATA3-AS1 was also highly expressed in patients with gastric cancer, and its expression was positively correlated with lymph node metastasis, tumor diameter, and TNM stage of gastric cancer. The area under the receiver operating characteristic curve (ROC curve) for the diagnosis of gastric cancer by the expression level of SPATA3-AS1 in plasma was 0.788. After SPATA3-AS1 knockdown, the proliferation and mobility of AGS and HGC-27 cells were decreased, and apoptosis was enhanced. Meanwhile, the mRNA expression of N-cadherin, Twist1 and Snail were down-regulated while the mRNA expression of E-cadherin was up-regulated in SPATA3-AS1 knockdown cells. Conclusion: SPATA3-AS1 is highly expressed in cancer tissues and plasma of patients with gastric cancer and is associated with prognosis. After SPATA3-AS1 knockdown, the proliferation, migration and invasion of gastric cancer cells were inhibited, and the cell apoptosis was enhanced, which may be related to the regulation of the process of epithelial-mesenchymal transformation.
    ［Key words］gastric cancer; long noncoding RNA; SPATA3-AS1; epithelial-mesenchymal transition
  • Establishment of the mouse model of suture-induced inflammatory corneal neovascularization and its significance
    2021, 31(06): 496-500.
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    Objective: To evaluate the efficacy of the mouse model of suture-induced inflammatory corneal neovascularization and its significance. Methods: The cornea of BALB/c mice was sutured by “8” stitch with 11-0 nylon thread to establish the mouse model of suture-induced inflammatory corneal neovascularization. Lymphangiogenesis and angiogenesis were observed by slit-lamp at 3, 7, 12 days after suturing and by immunofluorescence at 5, 7, 14 days after suturing. Corneal inflammation was observed by HE staining at 3, 7, 14 days after suturing. The expression of TNF-α mRNA in inflammatory cornea was detected by qRT-PCR at 1, 3, 7, 11 and 14 days after suturing. Results:  The results of slitlamp observation and immunofluorescence showed that significant lymphangiogenesis and angiogenesis were observed on the 3rd day after suturing, and the growth peaked on the 7th day after suturing. HE staining showed obvious corneal edema and inflammatory cell infiltration on the 3rd day after suturing, which began to subside on the 7th day after suturing. TNF-α mRNA expression in the sutured corneas persisted in increasing during the 7th day after suturing, and then began to decline, and kept at a low level (P<0.05). Conclusion:  The mouse model of suture-induced inflammatory corneal neovascularization is a typical inflammatory model, which conforms to regular pattern of inflammation. Thus it maybe an ideal tool to explore the growth mechanism of inflammatory lymphangiogenesis.
    ［Key words］cornea inflammation model; mice; lymphangiogenesis; angiogenesis
  • The anti-glycolysis effect of toosendanin in gastric cancer cells and mechanism
    2021, 31(06): 501-505，510.
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    Objective: To observe the anti-glycolysis effect of toosendanin (TSN) in gastric cancer cells and its effect on the regulatory factors of glucose metabolism related Sirtuin-3 (SIRT3), signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor-1α(HIF-1α)， and to explore the potential mechanism of anti-glycolysis effect of TSN in gastric cancer cells.Methods: The expression of SIRT3, STAT3 and HIF-1α in 31 paraffin gastric cancer samples were detected by immunohistochemistry, and the correlation between SIRT3 and the expression level of STAT3 and HIF-1α were analyzed. The gastric cancer cell lines AGS and HGC-27 were treated with 100 nmol/L TSN, 250 nmol/L TSN or equivoluminal solvent for 24 hours. The effect of TSN on glucose uptake and lactate production in gastric cancer cells were detected by using glucose metabolism kits. The mRNA expression level of key glycolysis-related genes were tested by real-time quantitative PCR(qRT-PCR). The transcriptional regulation of SIRT3 was analyzed by using the dual luciferase reporter system. Results:  The immunohistochemistry results showed that the expression of SIRT3 was negatively correlated with STAT3 and HIF-1α, and STAT3 was positively correlated with HIF-1α in gastric cancer tissue (all P<0.05). Compared with the control group, TSN significantly inhibited the glycolytic effect of gastric cancer cells and inhibited the ability of tumor cells to absorb glucose and secrete lactic acid (all P<0.05). After treating with TSN for 24 hours, the expression of key glycolytic enzymes glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) in gastric cancer cells were significantly reduced (all P<0.05). The expression level of STAT3 and HIF-1α decreased after TSN treatment, while the expression level of SIRT3 increased. TSN could enhance the luciferase activity in the promoter region of SIRT3, while overexpression of STAT3 could partly weak the role of SIRT3 (all P<0.01). Conclusion: TSN could induce the transcriptional expression of SIRT3 by inhibiting STAT3 expression, thereby inhibiting the expression of HIF-1α, inhibiting the glycolytic effect of gastric cancer cells, and causing the transformation of the glucose metabolism of gastric cancer cells.
    ［Key words］gastric cancer; toosendanin; antitumor; glycolysis effect; HIF-1α
  • Analysis on risk factors of first-line antiretroviral treatment failure among HIV/AIDS patients in Nanjing
    2021, 31(06): 506-510.
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    Objective: To analyze the risk factors of the firstline antiretroviral treatment failure in patients with HIV/AIDS in Nanjing area in order to reduce the failure rate of firstline regimen. Methods: The evaluation of demographic information, follow-up data and risk factors of treatment failure was conducted retrospectively in HIV/AIDS patients who failed to receive free first-line antiretroviral therapy and changed the free second-line regimen containing lopinavir/ritonavir (LPV/r) in Nanjing Second Hospital from January 2016 to September 2019, followed up to March 2020. Results:  Among 1 739 HIV/AIDS patients, 44 (2.5%) switched to second-line treatment with LPV/r due to virologic failure. Baseline CD4+T lymphocyte counts <200 cells/μL (OR=13.105, 95% CI: 5.025-34.176) and baseline viral load ≥1×105copies/mL (OR=2.491, 95% CI: 1.210-5.128) were risk factors of first-line treatment failure. First-line treatment failure was not associated with gender, age, marital status, body mass index, route of transmission, interval from the confirmation to initial treatment, or access to essential medical insurance. Conclusion:  First-line regimen failure was associated with lower baseline CD4+ T lymphocyte counts and higher baseline viral load. We should focus on patients with these risk factors.
    ［Key words］HIV；AIDS；antiretroviral therapy；first-line antiretroviral treatment；virologic failure；risk factors; CD4+ T lymphocyte；viral load
  • Difference analysis and risk prediction of lung function and plasma
    IL-5 and IL-8 among different phenotypes of asthma
     
    2021, 31(06): 511-516.
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    Objective: To analyze the differences of various airway inflammatory phenotypes in clinical features, pulmonary function and plasma inflammatory factors in patients with acute bronchial asthma, and to explore the risk factors for phenotypic classification. Methods: A total of 109 patients with acute asthma who met the inclusion and exclusion criteria from May 2019 to July 2020 in Luoyang Central Hospital were prospectively selected as asthma group. According to induced sputum, the subjects were divided into eosinophilic asthma （EA）, neutrophil asthma （NA）, mixed granulocytic asthma （MA） and oligocytic asthma （PA）. Meanwhile, 30 healthy persons were included as the control group. The general condition, asthma control test （ACT）, pulmonary function, and exhaled nitric oxide （FeNO） of subjects were recorded, and plasma IL-5 and IL-8 levels were measured by ELISA. Results:  There were significant difference in allergies, lung function, and plasma inflammation factors between control group and asthma group（all P<0.05）. The proportion of EA with allergic history and FeNO level was higher than those in NA group, but the proportion of smoking patients was lower （all P<0.05）. The scores of PA pulmonary function （FEV1% and PEF%） and ACT were higher than those of the other three groups （all P<0.05）. The pulmonary function （FEV1/FVC, FEV1%, PEF%） in NA and MA groups was worse than that in EA and PA groups. The content of plasma IL-5 in eosinophilic inflammatory asthma group （EA and MA） was higher than that in non-eosinophilic group （NA and PA）, and the level of plasma IL-8 in neutrophilic inflammatory asthma group （NA and MA） was higher than that in non-neutrophilic group （EA and PA, all P<0.05）. Logistic regression was performed in asthma group according to whether it was eosinophilic inflammation or neutrophilic inflammation. The results suggest that the increase of FeNO and serum IL-5 level and allergy are the risk factors for eosinophilic inflammatory asthma, and the severity of lung function, the increase of plasma IL-8 level and the decrease of ACT score are the risk factors of neutrophilic inflammatory asthma （all P<0.05）. Conclusion:  Pulmonary function and plasma IL-5 and IL-8 were significantly different among asthma phenotypes. Pulmonary function grade, IL-5, IL-8, FeNO, history of allergy and ACT score were risk factors for phenotype variation.
    ［Key words］asthma；inflammatory phenotype; IL-5; IL-8; FeNO; pulmonary function
  • Effect of kaempferol on ethanol induced gastric ulcer in mice and its mechanism
    2021, 31(06): 517-521,540.
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    Objective： To explore the effect of kaempferol on ethanol-induced gastric ulcer in mice and its potential mechanism. Methods： Sixty SPF Kunming mice were randomly divided into 6 groups with 10 mice in each group： normal control group, model group, positive control group, kaempferol low, medium and high groups, respectively. Normal control group and model group were given 0.5% sodium carboxymethyl cellulose, and other groups were given ranitidine (100 mg/kg), kaempferol low-dose (31.7 mg/kg), kaempferol medium-dose (63.4 mg/kg) and kaempferol high-dose (126.8 mg/kg), respectively. The gavage volume of all mice was 20 mL/kg, and the mice were continuously given the drug for 7 days. Then the acute gastric ulcer model was established by gavage with 10 mL/kg anhydrous ethanol. The gastric mucosal injury was assessed by ulcer index. The contents of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), TNF-α, IL-1β, IL-6 and NO in serum were detected by ELISA and the expression of COX-2, inducible nitric oxide synthase (iNOS) and NF-κB p65 protein in gastric tissue were detected by Western blotting. Results： Compared with the normal control group, the gastric mucosa of mice in the model group showed obvious blood spots and epithelial cells shedding. Compared with model group, kaempferol significantly decreased gastric ulcer index (P<0.05). Compared with normal control group, the contents of inflammatory mediators COX-2, PGE2, TNF-α, IL-1β, IL-6 and NO in model group were significantly increased, and the protein expressions of COX-2, iNOS and NF-κB p65 were significantly increased (P<0.05). Compared with model group, the protein expressions of COX-2, iNOS and NF-κB p65 and the content of inflammatory mediators in serum in kaempferine group were significantly decreased (P<0.05 or P<0.01). Conclusion：Kaempferol exerts a protective effect on ethanol-induced gastric ulcer, which may be related to down-regulating the contents of downstream inflammatory mediators TNF-α, IL-6 and IL-1β, reducing the activity of iNOS and inhibiting the production of NO by inhibiting NF-κB/COX-2 pathway.
    ［Key words］kaempferol; ethanol-type gastric ulcer; NF-κB/COX-2 pathway; inflammatory factor; gastric protection
  • Preparation and quality evaluation of fisetin-loaded liposomes
    2021, 31(06): 522-527.
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    Objective: To develop and optimize the preparation protocols of fisetin liposomes, and to evaluate in vitro and in vivo the properties of liposomes obtained. Methods: Fisetin liposomes were prepared by film dispersion method. Taking the particle size as the index,fisetin liposomes with different total phospholipid and cholesterol, phospholipid cholesterol ratio and different drug lipid ratio were prepared to determine the optimal formulation through single factor analysis. The particle size, polydispersity coefficient and zeta potential of fisetin liposomes were measured by laser scattering particle size meter. The entrapment efficiency and drug loading were determined by ultrafiltration centrifugation.The stability, in vitro release in three media (pH 1.2 hydrochloric acid, double distilled water, pH 7.4 phosphate buffer), cytotoxicity and pharmacokinetics were also evaluated. Results:  The average particle size of fisetin liposomes prepared with the optimal formulation (fisetin 22.2 mg, phospholipid 133.3 mg, cholesterol 16.7 mg, sodium cholate 110 mg and isopropyl myristate 60 mg) was about (60.32±1.08)nm, the polydispersity coefficient was 0.198±0.011, the entrapment efficiency was (94.37±0.62)%, and the drug loading was (4.500±0.021)%.The results of transmission electron microscope showed that the liposomes were round and uniformly distributed in vitro; The solubility, in vitro release rate and relative bioavailability of fisetin could be improved in prepared liposomes; Fisetin liposomes showed good stability within 30 days. Furthermore, fisetin liposomes significantly inhibited the proliferation of human hepatoma HepG2 cells in a dose-dependent manner. Conclusion:  Fisetin liposomes can significantly improve the solubility and bioavailability of fisetin.
    ［Key words］fisetin；liposomes；in vitro and in vivo evaluation；insoluble drug；formulation development
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    2021, 31(06): 541-548.
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