AIM:To construct recombinant retroviral vector of short interfering RNAs (siRNA) specific for macrophage migration inhibitory factor (MIF) and to establish the stable knockdown of MIF cell line of mammalian cells by transfecting the recombinant retroviral vectors. METHODS: We synthesized oligo-nucleotides for MIF in vitro, and cloned them into retroviral vector pSuper.retro. Subsequently the plasmids were sequenced and digested to identify the construction of the recombinant retroviral vectors. The vectors RNAi were transfected into packing cell line PHOENIX, which was selected by puromycin later. HeLa cell line was infected by the virus supernatant of stable PHOENIX cell lines, and the stable HeLa cell line showed significantly to silence MIF was established by selecting with puromycin. We also compare the characters of HeLa-pSuper-mock to HeLa-pSuper-MIF cells by using migration assay, adhesion assay, soft agar assay and FACS analysis of the cell-cycle progression. RESULTS: The recombinant retroviral vectors were constructed successfully. The HeLa cell line infected by the supernatant containing the retrovirus of package PHOENIX cells was persistent knockdown of MIF confirmed by Western blotting. Knockdown of MIF in HeLa cells inhibited the migration and adhesion, and decreased the clone formation. FACS analysis revealed that knockdown of MIF arrested HeLa cells in G0/G1 phase. CONCLUSION: We establish the stable HeLa cell line with a persistent knockdown of MIF. Our current studies reveal that MIF is necessary for HeLa cell migration and anchorage-independent growth.

Objective To explore the effects of manumycin on U937 cells and its mechanisms.Methods Apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) after treatment with manumycin (2 μmol/L) for different time.The reactive oxygen species (ROS) was detected by flow cytometry using Reactive Oxygen Species Assay Kit.The expressions of PI3K,P-Akt and Akt were detected by Western blotting.Results Compared with the 0 h treatment,manumycin treatment resulted in apoptosis (P ＜ 0.05) and ROS generation (P ＜ 0.05) gradually in U937 cell lines.Furthermore,manumycin also inhibited the activation of phosphatidylinositol-3 kinase (PI3K)/Akt pathway.Moreover,NAC could decrease manumycin-induced ROS generation and inhibit the effects of manumycin on the PI3K/Akt pathway and protect U937 cells from apoptosis induced by manumycin.Conclusion Manumycin induces apoptosis in U937 human leukemia cells through the regulation of the PI3K/Akt pathway and ROS production plays a critical role in the progress.

OBJECTIVE: To investigate the significance of human thioredoxin-2 (TRX-2) in monitoring minimal residual disease (MRD) in acute leukemia (AL). METHODS: We used real-time quantitative PCR to serially quantitize TRX-2 expression levels in the bone marrow of AL patients at diagnosis (n=68), at complete hematologic remission (CHR, n=57) and at relapse (n=25). Another 25 normal donors served as normal controls. The upper limit of the bone marrow at 91 was regarded as the reference. TRX-2 expression level at CHR with <5% blast cells in the bone marrow of relapse patients was analyzed and compared with MRD by flow cytometry. RESULTS: The TRX-2 levels between the CHR patients and newly diagnosed patients, and between the CHR patients and the relapse patients had significant difference. TRX-2 expression level of 21(21/25) relapse patients at CHR with <5% blast cells in the bone marrow was higher than the reference (>91). TRX-2 level was correlated to the expression level of MRD. CONCLUSION TRX-2 may be the marker for AL and used in MRD monitoring.
Case-Control Studies ; Female ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; metabolism ; Male ; Neoplasm, Residual ; diagnosis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Thioredoxins ; genetics ; metabolism

AIM:To investigate whether the association of connexin 43 ( Cx43 ) and L-type calcium channel involved in the pathogenesis of atrial fibrillation ( AF) .METHODS:The biochemical assays and whole-cell patch-clamp technique were used to study the expression of Cx43 in human atrial tissue.The co-localization of Cx43 and L-type calcium channel, and the regulation of L-type calcium current in atrial myocytes were investigated.RESULTS:The expression of Cx43 at mRNA and protein levels was decreased in human atrial tissues of AF patients.In cultured atrium-derived myocytes ( HL-1 cells) , knockdown of Cx43 significantly inhibited the mRNA expression of L-type calcium channelα1c subunit, as well as L-type calcium current.Co-localization of Cx43 with L-type calcium channel α1c subunit in mouse atrial myocytes was observed.CONCLUSION:The decrease in Cx43 is involved in the pathogenesis of AF, probably through reducing the L-type calcium current in atrial myoctyes by co-localization with L-type calcium channel, thus representing the potential pathogenesis in atrial fibrillation.

AIM:Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation ( AF) .Al-though tumor necrosis factor ( TNF)-αlevels are increased in patients with AF , the role of TNF-αin the pathogenesis of AF remains unclear.Recent research has revealed that T-type Ca2+currents ( ICa,T ) play an important role in the pathogenesis of AF .METH-ODS:In this study , we used the whole-cell voltage-clamp technique and biochemical assays to explore the role of TNF-αin the regula-tion of ICa,T in atrial myocytes.RESULTS:We found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased , while TNF-αexpression levels were increased , in human atrial tissue from patients with AF .In murine atrial myocyte HL-1 cells, after cultured for 24 h, 12.5, 25 and 50 μg/L TNF-αsignificantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner .The peak current was reduced by the application of 12.5 or 25μg/L TNF-αin a concentration-dependent manner [from ( -15.08 ±1.11) pA/pF in controls to ( -11.89 ±0.83) pA/pF and (-8.54 ±1.55) pA/pF in 12.5 and 25 μg/L TNF-αgroups, respectively].TNF-αapplication also inhibited voltage-dependent inactivation of ICa,T shifted the inactivation curve to the left .CONCLUSION:These results suggest that TNF-αis involved in the path-ogenesis of AF, probably via decreasing ICa,T function in atrium-derived myocytes through impaired channel function and down -regula-tion of channel protein expression .This pathway thus represents a potential pathogenic mechanism in AF .

Objective To evaluate the diagnostic value of gray-scale ultrasound combined with contrast-enhanced ultrasound (CEUS) in thyroid imaging and reporting system ( TI-RADS) 3-5 nodules of American College of Radiology ( ACR) . Methods The 208 patients( 216 nodules) who underwent thyroid contrast-enhanced ultrasound examination from April 2014 to December 2016 were retrospectively analyzed . The ACR TI-RADS classification and contrast-enhanced mode of thyroid ultrasound were evaluated . The benign and malignant thyroid lesions were constructed through the analysis of contrast-enhanced mode of thyroid benign and malignant lesions in the past literature and the preliminary study of our group . Prediction model of gray scale combined with contrast-enhanced ultrasound for lesions were established . Besides ,the sensitivity ,specificity and accuracy of gray scale ultrasound combined with contrast-enhanced ultrasound prediction model for differential diagnosis of benign and malignant thyroid lesions were calculated based on the results of puncture biopsy or surgical pathology as the gold standard . Results Compared with pathological results , the positive predictive value , negative predictive value , accuracy , sensitivity and specificity of gray scale combined with contrast-enhanced ultrasound in evaluating thyroid nodules were 90 .8％ ,93 .3％ ,91 .2％ ,98 .8％ and 62 .2％ ,respectively . Conclusions Gray-scale ultrasound combined with contrast-enhanced ultrasound can provide clinical value in differential diagnosis of benign and malignant TI-RADS 3-5 thyroid nodules .