Objective To identify the role of FOXC2 in the invasion and migration of colorectal cancer cells .Methods Stable cell lines expressing FOXC2(SW480/FOXC2) or vector (SW480/pBabe) were established using retroviral infection method .The morphology alterations of SW480 cells were observed using a microscope .Western blot analysis and immunofluorescence staining assays were performed to detect the expression of E‐cadherin ,Vimentin and N‐cadherin .The invasive and migratory abilities of colorectal cancer cells evaluated using Transwell invasion chamber experiment detection .Results The morphology of SW480 cells was significantly changed after overexpression .From the original shape typical of epithelial cells became spindle shaped growth , similar to the morphology of fibroblasts .Western blot analysis and immunofluorescence staining displayed that overexpression of FOXC2 led to significant downregulation of the epithelial marker E‐cadherin ,but upregulation of the mesenchymal markers Vimen‐tin and N‐cadherin .Transwell assay reveals that overexpression of FOXC2 strongly enhanced the migratory and invasive ability of SW480 cells .Conclusion FOXC2 induces epithelial‐mesenchymal transition and promotes the invasive ability of colorectal cancer cells .

Objective:To illustrate the role of miRNA-143 on the invasiveness of cervical cancer cells. Methods:MiRNA-143 mimics or inhibitor sequences were transiently expressed in the cervical cancer cells by liposome transfection. Transwell assay was ap-plied to test the invasive ability of cervical cancer cells after miRNA-143 over-expression or inhibition. Bioinformatics assay was used to predict the targets of miRNA-143. RT-qPCR and luciferase reporter assay were performed to detect the expression of MACC1 mRNA in the cancer cells. RT-qPCR was conducted to test the expression of miRNA-143 and MACC1 mRNA in 20 fresh primary cervi-cal cancer and their matched para-neoplastic tissues. Statistical analyses were performed to evaluate the association between the expres-sion of miRNA-143 and MACC1 mRNA in the 20 cases of cervical cancer. Results:Transwell assays revealed that the miRNA-143 over-expression inhibited the cell invasiveness, while miRNA-143 inhibition promoted the invasive ability of the cervical cancer cells. Bioinformatics analyses revealed that miRNA-143 could target the 3'-UTR of MACC1. Dual luciferase reporter assay confirmed that miRNA-143 can affect 3'-UTR sequence in MACC1 genes. RT-qPCR analyses indicated that the expression of MACC1 mRNA was ob-viously down-regulated after miRNA-143 over-expression, while significantly increased after the miRNA-143 inhibition. The migration in Caski/miRNA-143 inhibitor cells was obviously elevated after being transfected with MACC1 shRNAs. RT-qPCR analyses showed that the expression of miRNA-143 was obviously decreased in the cancer tissues compared with the normal tissues, while MACC1 mRNA was apparently decreased in cancer tissues compared with the normal ones. Statistical analyses revealed that miRNA-143 was negatively correlated with MACC1 mRNA in the 20 cases of cervical cancer. Conclusion:This study reveals that miRNA-143 is down-regulated in the cervical cancer tissues. MiRNA-143 may play an important role in the regulation of cell invasiveness by targeting MACC1 in the cervical cancer cells.

Objective:To determine the function of miR-30b in the metastasis of colorectal cancer cells. Methods:RT-qPCR was performed to test miR-30b expression in 20 fresh primary colorectal cancer tissues and their corresponding adjacent tissues. Transwell and wound healing assays were performed to test the invasion and migration of colorectal cancer cells after miR-30b overexpression or inhibition. Bioinformatics assay was performed to predict miR-30b targets. Western Blot and Dual Luciferase reporter assay were per-formed to test the expressions of Snail and downstream target genes in colorectal cancer cells. Results:The results reveal that miR-30b expression decreased in cancer tissues compared with normal tissues. Transwell and wound healing assays reveal that miR-30b overex-pression inhibited cell invasion and migration, whereas miR-30b inhibition promoted the invasion and migration of colorectal cancer cells. Bioinformatics analyses reveal that miR-30b targets the 3'-UTR of Snail. Dual Luciferase reporter assay confirms that miR-30b af-fects the 3'-UTR of Snail. Western Blot analyses show that Snail and Vimentin expressions were significantly downregulated, whereas E-cadherin expression obviously increased after miR-30b overexpression. However, Snail and Vimentin expressions increased, but E-cadherin expression decreased after miR-30b inhibition. Conclusion:The miR-30b gene is downregulated in colorectal cancer tis-sues. The miR-30b protein may be important in the regulation of cell invasion and migration by targeting Snail in colorectal cancer cells.

Objective To enhance the awareness of diagnose and treatment of venous thrombus in premature infants. MethodsThe clinical features, treatment and prognosis of lower limb vein thrombus in three case of premature infants were retrospectively analyzed.ResultsThree premature infants of gestational age 29-36 weeks were found thrombosis in 1-57 days after birth. One case received peripherally inserted central cathete (PICC). All of three cases had infection signs and trauma. One case received thrombolytic and anticoagulant therapy, one case received high dose thrombolytic therapy, and both of them were treated successfully without bleeding complication. However, another case was deteriorated after giving up treatment.ConclusionsCritically ill premature infants are at high risk of thrombosis. Infection, indwelling tube, thrombophilia constitution, and maternal risk factors were the high risk factors of thrombosis. Early detection, early diagnosis, and effective individualized treatment can improve the prognosis.

Objective To find out the association between the indicators(pulse concussion lung function test index) of bronchial hyperresponsiveness (BHR) with fractional concentration of exhaled nitric oxide (FeNO) at different control periods among preschool asthmatic children.Methods Totally 74 asthmatic children in the pediatric department of our hospital from April 2015 to February 2017 were enrolled in this study,and 25 children undergoing the lung function and FeNO examination served as the controls,aged 3-5 years old.The cases were divided into three groups according to the standard in 2016 version of the Prevention and Treament Guide of Children Bronchial Asthma:asthma control group(n =26),asthma non-control;group(n =48) and control group (n=25).All data of FeNO,resistance of the respiratory system at 5 Hz(R5),resistance of the respiratory system at 5 Hz (R20),difference of R5 and R20(R5-20),reactance area (AX),reactance of the respiratory system at 5 Hz (X5) and resonant frequency of reactance (Fres) were collected.The FeNO,pulse concussion lung function test value and their association were analyzed.Results (1) The FeNO value of asthma the non-control group was significantly higher than that of the asthma control group and the control group,which were 34.00 ± 18.17,20.23± 11.07 and 28.00± 17.30 respectively.The AX detection value of the asthma non-control group was significantly higher than that of the control group(37.29 ± 15.27 vs.30.17 ± 9.50,P＜0.05).(2)R20 had weak correlation with FeNO in the control group(P＜0.05),while R20 had no correlation with FeNO in the non-control group and control group (P＞0.05).FeNO had no obvious correlation with R5,R520,AX,X5 and Fres in the asthma non-control group,asthma control group and control group(P＞0.05).Conclusion In preschool children with asthma,FeNO can reflect the airway eosinophilic inflammation control,and does not reflect the airway hyperresponsiveness.Thereforeit ie needed to combined with FeNO and IOS indicators (airway hyperresponsiveness index AX,etc.),which can more precisely judge whether asthma being controlled.

Objective Deleting the cysteine-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its stability and expression level in E.coli and to analyze the immunogenicity of Tat protein without the cystein-rich region [Tat(△C)protein]. Methods Tat DNA deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pET-32a vector. pET-32a-Tat(△C)plasmid and the pET-32a-Tat plasmid were established and transformed into E.coli BL21(DE3) strains respectively to express and purify the protein. Three rabbits were vaccinated with pET-32a-Tat(△C)protein, then testify the reactivity of sera from rabbits by ELISA and Western blot. Results The dense of the purified pET-32a-Tat(△C)protein was 7.12 mg/ml,which was greatly more than pET-32a-Tat protein(1.50 mg/ml). Dimer of pET-32a-Tat protein can be observed just after the protein purification and stored at 25℃ and 4℃ for 7 days, but dimer of pET-32a-Tat(△C)protein was not formed at the same condition. Experimental rabbits were immunized with pET-32a-Tat(△C)protein and produced high titre of anti-pET-32a-Tat(△C)serum(1∶320 000), the antibody can react specifically with Tat(△C)protein, Tat protein (1-101 AA)and synthetic Tat(1-86 AA) protein. Deletion mutation of the cysteine-rich region of Tat protein was first performed in the study. Conclusion The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, and the protein remains good immunogenicity. The results may provide a novel antigen for further development of HIV-1 Tat vaccine.

OBJECTIVETo investigate the role of Sam68 (Src-associated substrate during mitosis 68 kD) in the occurrence and development of colorectal cancer.

METHODSColorectal cancer cell lines with stable Sam68 over-expressing and low Sam68 expression were established to test the effect of Sam68 in the proliferation, invasion, and migration of the cancer cells using colony formation, MTT and Transwell assays.

RESULTSSW480 and Ls174t colorectal cell lines over-expressing Sam68 showed significantly enhanced cell proliferation, invasion and migration (P<0.05). Conversely, the low Sam68 expression in SW620 and HCT116 colorectal cell lines significantly suppressed the cell proliferation, invasion and migration (P<0.05).

CONCLUSIONThe expression of Sam68 can promote the proliferation, invasion and migration of colorectal cancer cells lines in vitro.

Adaptor Proteins, Signal Transducing ; metabolism ; Cell Cycle Proteins ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; DNA-Binding Proteins ; metabolism ; Humans ; RNA-Binding Proteins ; metabolism

OBJECTIVETo explore the effects of emodin in myocardial protection in mice with viral myocarditis (VMC) and explore molecular mechanisms.

METHODSFifty-five male 4-week-old BALB/c mice were randomly divided into control group (n=15), model group (n=20), and emodin group (n=20). The mice in model and emodin groups were inoculated with 0.1 ml Eagle's solution containing coxsackievirus B3 intraperitoneally, and those in the control group were given only 0.1 ml Eagle's solution. From the day of inoculation, the mice in emodin group received intragastric administration with 0.1 ml of 3 mg/ml emodin solution once daily for 21 consecutive days, and those in the control and model groups received 0.1 ml distilled water only. On day 7 after inoculation, 5 mice from each group were sacrificed to determine the viral titers in the cardiac tissues. All the mice were sacrificed on day 22 for measurement of the heart weight and histopathological inspection of the heart with HE staining. The mRNA and protein expression levels of myocardial interleukin-23 (IL-23) and interleukin-17 (IL-17) were detected by real-time quantitative PCR and Western blotting, respectively, and serum IL-23 and IL-17 levels were examined using enzyme linked immunosorbent assay (ELISA). Th17 cell frequencies were analyzed by flow cytometry. The expression levels of myocardial nuclear factor-κB (NF-κB) p65 in the cardiomyocyte nuclei were examined using Western blotting, and myocardial interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) contents were detected by ELISA.

RESULTSThe mortality, myocardial histopathologic scores and virus titers in emodin group were all significantly lower than those in the model group (P<0.05). The heart-to-body weight ratio, myocardial IL-23 and IL-17 expressions, serum IL-23 and IL-17 levels, Th17 cell frequencies, cardiomyocyte nuclear NF-κB p65 expression, and myocardial contents of IL-1β, IL-6 and TNF-α were all significantly increased in the model group as compared to the control group (P<0.01) but reduced significantly in emodin group as compared to model group (P<0.05).

CONCLUSIONEmodin can protect against VMC by inhibiting IL-23/IL-17 inflammatory axis, Th17 cell proliferation and viral replication in mice.

Animals ; Coxsackievirus Infections ; immunology ; Cytokines ; immunology ; Emodin ; pharmacology ; Enterovirus ; physiology ; Interleukin-17 ; immunology ; Interleukin-23 ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; virology ; Th17 Cells ; cytology ; drug effects ; Transcription Factor RelA ; metabolism ; Virus Replication

Protein A and protein G are two well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Protein A and protein G contained several highly homologous IgG-binding domains which had been demonstrated to have function to bind to IgG. Whether combinations of Ig-binding domains of various IBPs could produce useful novel binding properties remains interesting. We constructed a combinatorial phage library which displayed randomly-rearranged A, B, C, D and E domains of protein A, B2 and B3 domains of protein G. Four rounds molecular evolution of this library directed by all four human IgG subclasses respectively generated a common arrangement of D-C respectively which didn't exist in SpA. The dynamic loss of control phages and increase of the phages displaying two or more binding domains, especially the selective enrichment of D-C and strict selection of its linking peptides demonstrated the efficient molecular evolutions and the significance of the selected D-C arrangement. The phage binding assays confirmed that D-C possessed a binding advantage with four human IgG subclasses compared to SpA. In this work, a novel combination of Ig-binding domains, D-C, was obtained and presented the novel Ig binding properties which provided a novel candidate molecule for the purification, production and detection of IgG antibodies and a new approach for the further study of structures and functions of IBPs.
Amino Acid Sequence ; Antibody Specificity ; Bacterial Proteins ; immunology ; metabolism ; Binding Sites ; Binding, Competitive ; Evolution, Molecular ; Immunoglobulin G ; immunology ; metabolism ; Molecular Sequence Data ; Peptide Library ; Sequence Alignment ; Staphylococcal Protein A ; immunology ; metabolism

OBJECTIVETo investigate the synergistic effect of oleanolic acid (OA) and cyclosporine A (CsA) on the survival of renal allografts in rats.

METHODSRenal allograft transplantation was performed using BN rats as donors and LEW rats as recipients. Forty male LEW rats were randomized into 4 equal groups for interventions with DMSO-PBS (control), OA, CsA, or CsA+OA, starting from 1 day before transplantation. Serum creatinine levels were regularly examined, and the survival of rats were recorded. On day 5 after transplantation, CD4(+) and CD8(+) T-cell infiltration in the renal grafts was analyzed by immunohistochemistry; the concentrations of the proinflammatory cytokines (IL-1β, IFN-γ, IL-2, IL-4, and IL-17), anti-inflammatory cytokine IL-10 and chemokines (IP-10, MCP-1, MIP, and Mig) were analyzed with Luminex; the T-cell phenotypes (IFN-γ, IL-10, IL-4, and IL-17) were analyzed using ELISpot.

RESULTSIn OA+CsA group, renal allograft survival was markedly prolonged and CD4(+) and CD8(+) T cell infiltration in the graft significantly decreased as compared to other groups. A significant decrease in IL-2 was observed in OA group and OA+CsA group, especially the latter. Compared with the control group, all the 3 treated groups showed significantly decreased IL-1β, IP-10 and MCP-1, increased IL-10 levels, decreased percentages of T cells secreting IFN-γ, IL-4 and IL-17, and increased percentage of T cells secreting IL-10. The increments of serum IL-10 level and T cell percentage were more prominent in OA+CsA group than in the other two intervention groups.

CONCLUSIONSOA and CsA synergistically ameliorate renal graft rejection and inflammation and promote allograft survival and function in rats.

Animals ; Cyclosporine ; pharmacology ; Cytokines ; metabolism ; Drug Synergism ; Graft Survival ; Kidney ; drug effects ; Kidney Transplantation ; Male ; Oleanolic Acid ; pharmacology ; Rats ; Rats, Inbred BN ; Rats, Inbred Lew ; T-Lymphocytes ; cytology ; Transplantation, Homologous