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Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.
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Blotting, Western , Calcium , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Endothelial Cells , Homeostasis , Human Umbilical Vein Endothelial Cells , Tunicamycin , Unfolded Protein ResponseABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.</p><p><b>METHODS</b>Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.</p><p><b>RESULTS</b>Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.</p><p><b>CONCLUSIONS</b>As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.</p>
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AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.
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AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .
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AIM:To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium , and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts .METHODS:Masson trichrome stai-ning was performed on the myocardium of the diabetic db /db mice and the non diabetic db/m control mice .circRNA ex-pression profile in the diabetic myocardium was detected by circRNAs microarray .The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR ( RT-qPCR ) .Recombinant circRNA_000203 adenovirus was pre-pared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts.The expression of Col1a2, Col3a1andα-SMA was determined in circRNA_000203-modified cardiac fibroblasts , respectively .RESULTS:Masson trichrome stai-ning showed that fibrosis was increased in the diabetic mouse myocardium .The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium .circRNA_000203 was up-regulated in the diabetic myocardi-um.Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombi-nant circRNA_000203 adenovirus.The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly in-creased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION:circRNA_000203 is up-regu-lated in the diabetic mouse myocardium .It has pro-fibrotic effect on the cardiac fibroblasts .
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AIM:To investigate the effect of miR-214 on cardiomyocyte hypertrophy and the expression of the potential target genes . METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular car-diomyocytes (NMVCs).Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Wes-tern blotting, respectively.RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-in-duced hypertrophic cardiomyocytes .Dual luciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly in-creased in the hypertrophic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hy-pertrophy-related genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .
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AIM:MicroRNAs ( miRNAs) were recognized to play significant roles in cardiac hypertrophy .But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy .This study investigates the potential roles of microRNA-1 (miR-1) and microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy .METHODS:An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC).In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte .RESULTS:miR-1 and-16 expression were markedly de-creased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats .Overexpression of miR-1 and -16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes .Expression of cyclins D1, D2 and E1, CDK6 and phosphorylated pRb was increased in hypertrophic myocardium and hypertrophic cardiomyocytes , but could be reversed by enforced expression of miR-1 and -16.CDK6 was validated to be modulated post-transcriptionally by miR-1, and cyclins D1, D2 and E1 were further validated to be modulated post-transcriptionally by miR-16.CONCLUSION: Attenuations of miR-1 and -16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2, E1 and CDK6, and activating cyclin/Rb pathway.
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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 .
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AIM:To investigate the effect of caspase-8 small hairpin RNA ( shRNA) on attenuating apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed.Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR.The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid , which was linearized and transfected into HEK 293 cells for packaging and amplification of the recombi-nant adenovirus rAd-Cap8 shRNA.The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting .Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hM-SCs under the conditions of serum deprivation and hypoxia .The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR.RESULTS:The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR.The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia , with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2.CONCLUSION:Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia .
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The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.
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Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no acids, about 615.28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30.18% Glu in total amino acids of Ag332. Ag332 of P.falciparum isolate FCC1/HN and 3D7 exhibited 94.55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.
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[Objective] To construct a eukaryotic expression plasmid containing a gene encoding a 41-3 kilodalton blood stage antigen (p41-3) of Plasmodiu falciparum isolate FCC1/HN, and to determine the sequence of p41-3 gene and analyze the homology of the sequences of 341-3 gene of different P. falciparum isolates. [ Methods] Two pairs of primers were designed according to the known sequence of p41-3 gene. Using PCR technique, the p41-3 gene was obtained by amplification from genomic DNA of isolate FCC1/HN. By cloning target gene into a eukaryotic expression vector, pcDNA3, a recombinant plasmid pcDNA3-p41-3 was con structed and trarsferred into E. coli DH5α. The positive clones were screened and identified by agarose gel electrophoresis, endonu clease digestion and PCR technique. The correct recombinant plasmid pcDNA3-p41-3 was used as template, and the nucleotide se quence of p41-3 gene was determined by the dideoxy chain termination method. Using softwares to analyze the structure and sequence homology of p41-3 gene between isolate FCC1/HN and FCBR. [Results] The p41-3 gene was specifically amplified from genomic DNA of Plasmodiumm falciparum isolate FCC1/HN, and the correct recombinant plasmid pcDNA3-p41-3 was screened and identi fied. The result of sequence determination showed that the p41-3 gene of isolate FCC1/HN was 2 137 base pairs in full length, encod ing 375 amino acids. Isolate FCC1/HN and isolate FCBR exhibited 98.98 % homology in the nucleiotide sequences and 99.73 % ho mology in the encoded anino acids of p41-3 gene. [Conclusion] The eukaryotic expression plasmid pcDNA3-p41-3 is successfully con structed and nucleotide sequence of p41-3 gene of isolate FCC1/HN is determined. The p41-3 genes of isolate FCC1/HN and isolate FCBR share quite high homology.
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AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.
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AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis. [
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Objective] To determine the nucleotide sequence of the 3′\|termal of the RESA gene Plasmodium falciparum isolate FCC1/HN, and find out the differences of the sequences of RESA gene among isolate FCC1/HN,FC27,NF7 and Palo Alto. [Methods] 3′\|terminal fragment of RESA gene of P.falciparum isolate FCC1/HN was amplified by PCR method, then was cloned into pMD18\|T vector. The recombinant was screened and identified by BamHI+XhoI and PCR technique. The nucleotide sequence of the 3′\|terminal of the RESA gene was determined by the dideoxy chain termination method. DNASTAR and BLAST software were used to compare and analyze the RESA gene sequences among the different isolates. [Results] The 3′\|termal fragment of the RESA gene with about 846 bp was specifically amplified by PCR, the recombinant pMD18\|T\|RESA was successfully constructed. Different degrees of diversity of the RESA gene sequences were found among P.falciparum isolates FCC1/HN、FC27,NF7 and Palo Alto. [Conclusion] There were differences in the sequences of RESA gene among the P.falciparum isolate FCC1/HN and three other isolates (FC27,NF7 and Palto alto).
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AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.