Construction and identification of FRT cell line stably expressing UT-A2

AIM: To obtain an FRT cell line that can stably express urea transporter A2 (UT-A2) and provide a cell model for screening UT-A2 inhibitors. METHODS: FRT cells stably expressing aquaporins 1 (AQP1) and YFP were transfected with the recombinant plasmid pUB6/V5-UT-A2 by eukaryotic expression plasmid-lipoplast mediating pathway. The stable UT-A2-FRT cell line was cloned by selection with BSD and confirmed by Western blotting and immunofluorescence staining. The urea permeability across the plasma membrane was detected by a 2 mol/L urea lysis assay. RESULTS: We have obtained a stable UT-A2-FRT cell line. Western blotting analysis showed that UT-A2 protein was expressed stably in this cell line. The immunofluorescence staining detection indicated UT-A2 expression in the plasma membrane. It was found that there was significant urea permeability in this cell line by 2 mol/L urea lysis assay. CONCLUSION: We constructed an FRT cell line that could stably express UT-A2 in plasma membrane in the non-renal epithelia cell. The cell line will be used to screen UT-A2 inhibitors.     

Establishment and application of cell model for P2ry2 modulators screening based on CaCC

AIM To construct a high-throughput screening cell model for P2Y₂ purinergic receptor （P2ry2） modulators based on calcium-activated chloride channel （CaCC）. METHODS The mRNA expression of P2ry2 in Fischer rat thyroid （FRT） cells was detected by RT-PCR， and the PCR products were collected and sequenced. The protein expression of P2ry2 in the FRT cells was also detected by Western blot. The eukaryotic expression vectors ANO1 and YFP-H148Q/I152L were constructed. The FRT cells co-expressing ANO1 and YFP-H148Q/I152L were obtained by liposome transfection， antibiotic screening and limited dilution. The expression of ANO1 and YFP-H148Q / I152L in the cells was observed under fluorescent inverted microscope. The validation of the cell model for screening P2ry2 modulators was verified by the fluorescence quenching kinetics tests， and intracellular free calcium was analyzed by Fura-2 staining to investigate the dose-dependent relationship between intracellular calcium concentration and P2ry2 modulators. Z' factor was applied to evaluate the stability and repeatability of the cell model. RESULTS P2ry2 were endogenously expressed in the FRT cells. The expression of ANO1 on the cell membrane and the expression of YFP-H148Q/I152L in the cytoplasm were observed under fluorescent inverted microscope. The cell model was successfully constructed. The fluorescence quenching kinetics test confirmed the cell model for screening P2ry2 modulators was constructed successfully， and the calcium concentration in cytoplasm was increased rapidly after the addition of a small amount of P2ry2 agonist， indicating that the cell model was sensitive for detecting the calcium concentration in cytoplasm. The calcium concentration in cytoplasm， P2ry2 modulators and the slope of fluorescence change were in a dose-dependent manner， respectively. The Z' factor was 0.75， indicating that the established cell model was able to use for high-throughput screening of P2ry2 modulators with excellent stability and repeatability. CONCLUSION A simple， economical， and efficient cell screening model of P2ry2 modulators is successfully constructed， which is suitable for the detection and evaluation of P2ry2 modulators.     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Chloride currents activated by cisplatin in poorly differentiated naso-pharyngeal carcinoma cells are not Ca2+-activated chloride currents

AIM：To investigate the type of chloride channel activated by cisplatin in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). METHODS：The technique of whole-cell patch-clamp was used to investigate the role of Ca2+ in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells. RESULTS：Chloride currents were induced when the cells were exposed to the calcium-free cisplatin solution, showing the similar density to the currents induced by cisplatin with the presence of extracellular calcium. However, the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged. The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra- and extracellular calcium. Calcium channel antagonist nifedipine had no effect on the cisplatin-activated chloride currents, while hypertonic solution completely inhibited those currents. CONCLUSION：The cisplatin-activated chloride currents are independent on intra/extracellular calcium. The chloride channels activated by cisplatin are not calcium-activated chloride channels, but are probably volume-sensitive chloride channels.     

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels. METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z). Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels. The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope. The chloride current was recorded by the patch-clamp technique. RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK1 potassium. The mRNA expression of ClC-3 was increased by the IK1 siRNA. IK1 siRNA inhibited the expression of ClC-3 protein. A chloride current was activated by hypotonic challenges, and the hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.     

miRNA-122 promotes mouse embryonic stem cell-derived hepatic precursor cells to differentiate into hepatocytes

AIM：To investigate whether miRNA-122 (miR-122) promotes the differentiation of mouse embryonic stem cell (ESC)-derived hepatic precursor cells (HPCs) into hepatocytes. METHODS：Mouse ESCs were initially induced to differentiate into HPCs by stimulating with fibroblast growth factor 4 (FGF-4), sodium butyrate and dexamethasone (Dex) sequentially. Then a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP was constructed by Gateway technology and transfected into the mouse ESC-derived HPCs 9 d after induction, so as to gain the cells with stable high expression of miR-122. The morphological changes of transfected cells were observed under inverted phase-contrast microscope. The liver-specific gene expression levels were detected by real-time RT-PCR. The liver-specific protein expression levels were also detected by immunofluorescence method. The liver functions were assessed by indocyanine green (ICG) uptake experiment, glycogen staining and urea synthesis function test. RESULTS：The mouse ESCs were successfully induced into HPCs by stimulating with FGF-4, sodium butyrate and Dex sequentially. At 6 d after transfection of miR-122, the morphology of the cells was closer to the mature hepatocytes. The mRNA levels of liver-specific genes such as albumin (ALB), transthyretion, α1-antitrypsin, glucose-6-phosphatase, cytokeration 8, cholesterol 7α-hydroxylase and cytochrome P450 3A4 were up-regulated. The expression levels of liver-specific proteins such as ALB and cytokeratin 18 were increased, while alpha-fetoprotein was decreased. The results of ICG uptake experiment, glycogen staining and urea synthesis function test indicated that the hepatocyte functions were strengthened as compared with control group. CONCLUSION：The combination of FGF-4, sodium butyrate and Dex successfully induces the mouse ESCs into HPCs. Over-expression of miR-122 effectively promotes the differentiation and maturation of mouse HPCs.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor (MIF) knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids.
METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α (cPLA2α) and phospho-cPLA2α were evaluated by Western blotting.
RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone (Dex) on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α.
CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.     

Expression of volume-activated chloride channel in rat PASMCs treated with hypoxia and hypercapnia and its relationship with MAPK pathway

AIM：To investigate the expression of volume-activated chloride channel (CLC3) in rat pulmonary artery smooth muscle cells (PASMCs) treated with hypoxia and hypercapnia and its relationship with MAPK pathway. METHODS：The method of enzyme digestion was used to isolate the PASMCs in male SD rat for cell primary culture. The cells were identified by immunofluorescence cytochemical method with mouse anti-rat α-smooth muscle actin antibody. The rat model of hypoxia and hypercapnia was established. The protein expression of CLC3 was detected by Western blotting. The mRNA expression of CLC3 was determined by RT-PCR. RESULTS：Compared with control group, the mRNA and protein expression of CLC3 in PASMCs was significantly raised in hypoxia and hypercapnia group. Compared with hypoxic and hypercapnic group, the expression of CLC3 was significantly reduced in ERK inhibitor U0126+ hypoxia and hypercapnia group, and was up-regulated in p38 inhibitor SB203580+ hypoxia and hypercapnia group. p38 activator anisomycin significantly decreased the expression of CLC3 at mRNA and protein levels in hypoxia and hypercapnia group. CONCLUSION：The expression of CLC3 at mRNA and protein levels in PASMCs increases under hypoxia and hypercapnia conditions. The ERK1/2 pathway mediates CLC3 expression in PASMCs induced by hypoxia and hypercapnia. Activation of p38 MAPK pathway down-regulates the expression of CLC3 at mRNA and protein levels induced by hypoxia and hypercapnia.     

Role of ClC-3 chloride channel in inhibition of nasopharyngeal carcinoma cell cycle by metformin

AIM: To study the roles of ClC-3 chloride channel in the inhibition of nasopharyngeal carcinoma cell cycle by metformin. METHODS: The CNE-2Z cells were treated with metformin at different concentrations. The viability of CNE-2Z cells was measured by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The protein expression of ClC-3 was determined by Western blot. The Cl^- currents was record by the patch-clamp technique. In addition, the cell cycle distribution was analyzed in the nasopharyngeal carcinoma CNE-2Z cells which over-expressed ClC-3 by pEZ-M03-ClC-3 plasmid transfection. RESULTS: Metformin inhibited the viability of CNE-2Z cells at 5, 10 and 20 mmol/L. Metformin at 10 mmol/L prevented the activation of chloride currents induced by hypotonicity, inhibited the protein expression of ClC-3 chloride channel and arrested the nasopharyngeal carcinoma CNE-2Z cells at G₀/G₁ phases. ClC-3 chloride channel protein over-expression reversed the effect of metformin on the cell cycle distribution of CNE-2Z cells. CONCLUSION: Metformin inhibits the CNE-2Z cell cycle, which may be related to the inhibition of ClC-3 chloride channel function and protein expression.     

Effects of interferon-inducible protein 16 on proliferation and migration of human brain vascular adventitial fibroblasts

AIM：To investigate the effects of interferon-inducible protein 16 (IFI16) on the proliferation and migration of human brain vascular adventitial fibroblasts (HBVAFs). METHODS：The siRNA of IFI16 gene was transfected into HBVAFs. Forty-eight hours after transfection, the cells were exposed to 2×106 U/L interferon alpha (IFN-α) for 24 h. Cell cycle was analyzed by flow cytometry. Cell migration was determined by scratch assay and transwell method. The mRNA and protein levels of IFI16, p53 and p21 were measured by real-time PCR and Western blotting, respectively. RESULTS：After transfection with IFI16 siRNA, the expression of IFI16, p53 and p21 at mRNA and protein levels was decreased in HBVAFs, and the cell cycle at G1/S transition was promoted. Meanwhile, stimulated with IFN-α up-regulated the expression of IFI16, p53 and p21 at mRNA and protein levels, and inhibited the cell cycle transition at G1/S and cell migration in HBVAFs. Such effect was restrained by transfection with IFI16 siRNA into HBVAFs. CONCLUSION：The expression of IFI16 inhibits the proliferation and migration of HBVAFs, which may be related to the activation of p53 and p21 expression.     

Cloning and expression of NK4 gene in Raji cells

AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct the recombinant eukaryotic expression vector pVITRO2-mcs-NK4. Raji cells were transfected by recombinant vector pVITRO2-mcs-NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT-PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-NK4 successfully. NK4 gene in Raji cells expresses stably.     

Effect of human xeroderma pigmentosum D on expression of Mdm2 and Mdm4 in HepG2 cells

AIM：To investigate the effects of human xeroderma pigmentosum D (XPD) on the expression of murine double minute 2 (Mdm2) and murine double minute 4 (Mdm4) in human hepatoma cells. METHODS：Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into HepG2 cells using liposome, and the cells were divided into blank control group, pEGFP-N2 group and pEGFP-N2/XPD group. The cell growth was detected by MTT assay. The cell cycle and apoptotic rate were examined by flow cytometry. The mRNA and protein expression levels of XPD, Mdm2, Mdm4 and P53 were determined by RT-PCR and Western blotting. RESULTS：The results of MTT assay showed that the cell growth was inhibited by the transfection of pEGFP-N2/XPD. The results of flow cytometry showed that the transfection of pEGFP-N2/XPD increased the cell number in G 1 phase, decreased the cell number in S phase and increased the apoptotic rate of HepG2 cells. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, decreased the expression of Mdm2 and Mdm4, and increased the expression of P53. CONCLUSION：XPD down-regulates Mdm2 and Mdm4 expression and up-regulates P53 expression in hepatoma cells. Moreover, the proliferation of hepatoma cells can be inhibited and the apoptosis can be induced by XPD.     

Effects of up-regulation of c-myc expression on U937 cell line

AIM:To establish a human monocytic　leukemia cell line U937 stably expressing c-myc gene and to investigate the biological characteristics of this cell line. METHODS:The recombinant plasmid MSCV-c-myc-IRES-GFP (MMIG) was constructed. MMIG and MSCV-IRES-GFP (MIG) were used to package the viruses for infecting U937 cells. Fluorescence-activated cell sorter (FACS) was used for sorting U937/GFP and U937/MYC cells. The GFP-positive cells were detected by fluorescence microscopy and FACS. The protein expression of c-Myc, survivin, X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 was detected by Western blotting. Cell proliferation was evaluated by MTT assay. Propidium iodide (PI) staining was used to determine the cell cycle distribution. Self-renewal ability was observed by colony- forming assay. RESULTS:The GFP expression in the cells infected with MIG or MMIG virus was observed under fluorescence microscope. The green fluorescent rate of the cells infected with MIG was 26.0%, while that of the cells infected with MMIG was 27.7%. The protein expression of c-Myc in MMIG-infected U937 cells was higher than that in MIG-infected cells. After sorting, the green fluorescent rates of U937/GFP and U937/MYC cells reached 98.7% and 93.7%, respectively. The protein expression of c-Myc in U937/MYC cells was higher than that in U937/GFP cells. In addition, survivin, a downstream protein of c-Myc, was up-regulated, while the protein expression of XIAP and Bcl-2 remained unchanged. Cell cycle analysis showed that the percentage of the cells in S phase increased in U937/MYC cells. Moreover, the proliferation and colony-forming ability of U937/MYC cells were also enhanced. CONCLUSION: U937/MYC cell line stably expressing c-myc gene was successfully established. c-Myc may increase cell viability via enhancing the expression of anti-apoptotic protein survivin, the cell cycle transition and the self-renewal ability.     

MEF2A/2D regulates depolarization induced differentiation via Rho signaling pathway in VSMCs

AIM：To investigate the role of myocyte enhance factor 2 (MEF2) in the regulation of depolarization-induced differentiation via Rho signaling pathway in vascular smooth muscle cells (VSMCs). METHODS： In primarily cultured mouse portal vein VSMCs, the techniques of immunofluorescence, Western blotting, real-time RT-PCR and siRNA transfection were applied to determine the membrane translocation of RhoA, the phosphorylation of LIM kinase (LIMK) and cofilin-2, and the mRNA expression of 4 MEF2 isoforms, myocardin and SM22α under high KCl-induced depolarization. RESULTS：RhoA membrane translocation occurred 30 s after depolarization, while phosphorylation of LIMK and cofilin-2 peaked at 10 min and 30 min, with increment of 42.20% and 32.75%， respectively. The mRNA expression of MEF2A and 2D was increased by 47.63% and 48.15%, respectively. In MEF2A/2D knockdown VSMCs, the mRNA expression of myocardin was not sensitive to depolarization, while SM22α expression was not affected. CONCLUSION：MEF2A/2D, acting on myocardin, is involved in the regulation of depolarization-induced differentiation via Rho signaling pathway in VSMCs     

Difference of ClC-3 protein expression and channel function between cisplatin-sensitive and -resistant ovarian cancer cells

AIM: To study the difference of ClC-3 chloride channel protein expression and channel function between cisplatin-sensitive (a2780) and -resistant (a2780cp) ovarian cancer cells. METHODS: The inhibition of a2780 and a2780cp cell proliferation induced by cisplatin were detected by MTT assay. The mRNA and protein expressions of ClC chloride channel families in a2780 cells and a2780cp cells were detected by real-time PCR and Western blot, respectively. The distribution of ClC-3 protein in a2780 cells and a2780cp cells were analyzed by immunofluorescence staining. The whole cell patch-clamp technique was used to record the chloride current in the cells. RESULTS: The sensitivities of a2780 cells and a2780cp cells to cisplatin were different. The IC₅₀ values of a2780 cells and a2780cp cells to cisplatin were 5 μmol/L and 20 μmol/L, respectively (P<0.01). The a2780 cells and a2780cp cells mainly expressed ClC-3 in ClC families. However, the mRNA expression of ClC-3 was much lower in a2780cp cells than that in a2780 cells (P<0.01). Compared with a2780 cells, the protein expression of ClC-3 in a2780cp cells was also significantly decreased (P<0.01). ClC-3 protein was mainly distributed on the membrane in a2780 cells, while was in cytoplasma in a2780cp cells. Cisplatin activated the chloride channel and induced the chloride current in the a2780 cells, but not in the a2780cp cells. Cisplatin did not induced the chloride current in a2780 cells treated with ClC-3 siRNA. CONCLUSION: The differences in protein distribution, expression and function of ClC-3 chloride channel were observed in cisplatin-sensitive and -resistant ovarian cancer cells, which may be one of the underlying mechanisms of cisplatin resistance.     

Expression and identification of ANO1 in mouse cardiomyocytes

AIM: To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride channels (CaCCs), in mouse cardiomyocytes and its functional properties. METHODS: The cardiomyocytes from the myocardial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method. The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells. RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes. The protein expression of ANO1 in the mouse cardiomyocytes was determined by Western blotting analysis. The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes. RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells. The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells. Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1. The functional properties were similar to the classic CaCCs. CONCLUSION: ANO1 expression was identified in the mouse myocardial cells. The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.     

Effect of sorcin expression on sensitivity of human glioma cells to cisplatin

AIM：To investigate the effect of sorcin expression on the sensitivity of human glioma cells to cisplatin. METHODS：pSilencerTM 3.1-H1-sorcin siRNA recombinant plasmid was constructed, and transfected into human glioma U251 cells. RT-PCR and Western blotting were used to analyze the expression of sorcin at mRNA and protein levels after transfection. The viability of U251 cells was measured by MTT assay. The protein expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) in U251 cells was detected by Western blotting. RESULTS：The plasmid pSilencerTM 3.1-H1-sorcin siRNA was successfully constructed, and was confirmed by restriction enzyme digestion and sequence analysis. The expression of sorcin at mRNA and protein levels was significantly decreased after sorcin siRNA was transfected into U251 cells (P<0.05). Inhibition of sorcin expression significantly decreased the viability of U251 cells treated with cisplatin (P<0.05), and the expression of P-gp and MRP1 proteins was also inhibited (P<0.05). CONCLUSION：Inhibition of sorcin expression increases the sensitivity of U251 cells to cisplatin by decreasing the expression of resistance-related proteins P-gp and MRP1, suggesting that sorcin may be associated with the resistance of glioma cells to cisplatin.