1.Prokaryotic expression of human mitochondrial transcription termination factor 3 and preparation of its polyclonal antibody
Yongqin YANG ; Chenggui ZHANG ; Meitao SUN ; Yun WANG ; Zefang YANG ; Xiaojuan ZHANG ; Wei XIONG
Journal of Medical Postgraduates 2016;29(10):1020-1025
Objective Human mitochondrial transcription termination factor 3 ( MTERF3 ) is a negative regulator of mito?chondrial gene expression and energy metabolism. This study was to construct a prokaryotic expression system for MTERF3 in Esche?richia coli ( E. coli ) and prepare its mouse?anti?human polyclonal antibody. Methods The complete open reading frame ( ORF) of human MTERF3 cDNA was amplified by RT?PCR and subcloned into prokaryotic expression vector pET28b. Then the recombinant plasmid pET28b?MTERF3 was transformed into competent E.coli BL21(DE3) and IPTG induced the expression of 6×his fusion protein. The recom?binant human MTERF3 protein was purified through Ni2+?NTA agar?ose gel column affinity chromatography and the purified recombinant protein was used as immunogen to immunize the BALB/c mice to pre?pare its specific polyclonal antibody. The titer and specificity of the antibody were analyzed by ELISA, Western blot and cellular immuno?fluorescence, respectively. Results The recombinant human MTERF3 protein was successfully expressed in E. coil and the mouse?anti?human MTERF3 polyclonal antibody with high quality was successfully prepared. ELISA showed that the titer of the antibody was 1:105 . Western blot and immunofluorescence detection revealed that the mouse?anti?human MTERF3 antibody could recognize the native MTERF3 antigen specifically. Conclusion Human MTERF3 expressed in the prokaryotic system has strong immunogenicity and the polyclonal antibody obtained from immunizing mice has high titer and specificity. The prokaryotic expression of human MTERF3 and the preparation of its antibody lay the foundation for further function research of human MTERF3.
2.Vasodilatory effect of Ferulic acid on in-vitro rat coronary artery
Longmei FANG ; Xiaomin HOU ; Rong YANG ; Fangwen FAN ; Zefang HE ; Meng SHI ; Mingsheng ZHANG
Chinese Pharmacological Bulletin 2016;32(4):554-558
Aim To investigate the vasodilatory effect of Ferulic acid on in vitro rat coronary artery and its possible mechanism. Methods By using the mi-crovessel tension recorder system, the vasodilatory effect of FA on resting and contractin-vitro rat coronary artery was determined;the influence of endothelial in-tegrity to FA-induced vasorelaxation was observed; the relationship of FA on [ Ca2+] ex-influx-induced and [ Ca2+] in-efflux-induced contractions was discussed;the mechanism of vasodilatory effect of FA was ex-plored by applying the inhibitors of KCa(TEA),KATP channel ( Gli ) , KIR channel ( BaCl2 ) , KV ( 4-AP ) , NOS( L-NAME) and COX( Indo) . Results FA had no effect on the resting tension of in vitro rat coronary artery. FA dilated the in-vitro rat coronary artery pre-treated with KCl ( 60 mmol · L-1 ) , U46619 ( 1 μmol · L-1 ) and PE ( 10μmol · L-1 ) in a concentration-dependent fashion ( P < 0. 05 ) . FA inhibited the [ Ca2+] ex-influx-induced and [ Ca2+] in-efflux-in-duced contractions significantly ( P <0. 05 ) . 4-AP ( 1 mmol· L-1 ) restrained the diastolic function of FA, while TEA, Gli, BaCl2、L-NAME, Indo had no obvious effect ( P >0. 05 ) . Conclusion The diastolic func-tion could be related to the activation of KV channel on vascular smooth muscle cells, the free Ca2+ from Sar-coplasmic reticulum cells and blockade extracellular Calcium channel do not depend on KCa, KATP, KIR channel, nor the endothelial function.
3.Mechanisms underlying contraction of rat isolated coronary artery induced by acidosis
Zefang HE ; Xiaomin HOU ; Rong YANG ; Fangwen FAN ; Pengmei GUO ; Yu LIU ; Mingsheng ZHANG
Chinese Journal of Pathophysiology 2017;33(5):838-842
AIM:To explore the mechanisms underlying contraction induced by extracelluar acidosis (pHex6.8) in rat isolated coronary artery (RCA).METHODS:Using the microvessel tension recorder system, the effects of acid-base transporters on RCA contraction induced by pHex6.8 were explored by applying the selective pharmacological inhibitors of Na+-H+ exchanger 1 (NHE-1) and Na+-HCO-3 cotransporter (NBC), HOE-642 and S0859, respectively.The effects of chloride channel on RCA contraction induced by pHex6.8 were explored by applying the inhibitors of chloride channel (NPPB and NFA), and by replacing the extracellular NaCl with equimolar NaBr.RESULTS:pHex6.8 augmented the resting tension of RCA, and the maximum contraction was (3.90±0.95) mN.HOE-642 at 30 μmol/L and S0859 at 100 μmol/L both inhibited the contraction of RCA induced by pHex6.8 (P<0.01).NPPB and NFA both inhibited the contraction of RCA induced by pHex6.8 or KCl (60 mmol/L) in a concentration-dependent manner.NPPB and NFA (100 μmol/L) both inhibited the contraction of RCA induced by U46619 (1 μmol/L).Replacing the extracellular NaCl with equimolar NaBr almost completely inhibited RCA contraction induced by pHex6.8 (P<0.01), but had no obvious effect on the contraction induced by KCl (60 mmol/L) or U46619 (1 μmol/L).CONCLUSION:Extracellular acidosis-induced contraction in RCA may be related to the activated NHE-1 and NBC, and it may be also related to the enhanced chloride transport across the membrane.
4.Regulation of nm23-H1 transfection on expression of integrins in human high-metastasis large cell lung cancer cell line L9981.
Haixia ZHENG ; Qinghua ZHOU ; Zhilin SUN ; Yang QIN ; Wen ZHU ; Yanping WANG ; Zefang SUN ; Guowei CHE
Chinese Journal of Lung Cancer 2004;7(2):108-111
BACKGROUNDTo study the regulation of nm23-H1 gene on the expression of integrins in human high-metastasis large cell lung cancer cell line L9981 by transfecting nm23-H1 into cell.
METHODSLipofect was used to transfect nm23-H1 into the L9981 cell line; semi-RT-PCR was used to detect the difference of integrin β1 and integrin β3 mRNA expressions between tranfected and non-transfected cell lines; flow cytometry was used to detect the difference of integrin β1 and integrin β3 protein expressions between transfected and non-transfected cell lines.
RESULTS(1) A transgenic cell line L9981-nm23-H1 was obtained, which could stably and effectively express nm23-H1. The mRNA expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were remarkably lower than those in L9981 cell line. (2) The protein expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were significantly lower than those in L9981 cell line too (P < 0.01).
CONCLUSIONSTransfection of nm23-H1 gene into the L9981 cell line can significantly down-regulate the mRNA and protein expressions of integrin β1 and β3 in this cell line, which indicates that nm23-H1 may reverse the metastasis potential of L9981 cell line through modulation of integrin expression.
5.Transfection of the tumor metastasis suppressor gene nm23-H1 can targetly suppress the activity of extracellular signal-regulated protein kinase (ERK) in human high-metastasis large cell lung cancer cell line L9981.
Yin LI ; Qinghua ZHOU ; Zhilin SUN ; Yang QIN ; Wen ZHU ; Yanping WANG ; Lunxu LIU ; Xiaohe CHEN ; Zefang SUN
Chinese Journal of Lung Cancer 2004;7(1):8-11
BACKGROUNDTo investigate the influence of the tumor metastasis suppressor gene nm23 H1 on the activity of extracellular signal-regulated protein kinase (ERK) in human high metastasis large cell lung cancer cell line L9981.
METHODSThe levels of total ERK1/2 and phospho-pERK1/2 were determined with p44/42 MAP kinase antibody and dually phosphospecific phospho-44/42 MAP kinase antibody in human high-metastasis large cell lung cancer cell lines L9981 (cell line with nm23-H1 gene deletion), L9981-nm23-H1 (cell line with nm23-H1 transfected ) and L9981-PLXSN (cell line with vector transfected) by Western blot method, respectively. The activity of phospho-ERK1/2 was determined with an ERK1/2 assay kit by immunopreciptation and Western blot analysis.
RESULTSThe expression levels of phospho-ERK1/2 kinase and the activity of phospho-ERK1/2 in the lung cancer cell line L9981-nm23-H1 were remarkably higher than those of the L9981 cell line and L9981-PLXSN cell line ( P < 0.01), but no significant difference in both the phospho-ERK1/2 expression and phospho-ERK1/2 activity was observed between the L9981 and L9981-PLXSN cell lines ( P > 0.05). There was no significant difference in the total ERK1/2 level among the three cell lines.
CONCLUSIONSnm23-H1 gene can obviously targetly suppress the activity of ERK1/2 in human high metastasis large cell lung cancer cell line L9981. This suggest that the mechanisms of nm23-H1 gene as a tumor metastasis suppressor gene may be related to its suppression to the MAPK/ERK signal transduction pathway.
6.Construction of adenoviral vector carrying Smad3D or Smad7.
Jianjun QIN ; Qinghua ZHOU ; Yang QIN ; Zhilin SUN ; Feng ZHAO ; Zefang SUN ; Yanping WANG ; Cheng YI ; Wen ZHU
Chinese Journal of Lung Cancer 2003;6(3):169-171
BACKGROUNDTo construct recombinant adenoviral vector carrying Smad3D or Smad7 by a simplified means.
METHODSBased on AdEasy System, adenoviral backbone plasmid vector and shuttle vector carrying the gene of interest were transferred into E.coli BJ5183 by chemical transformation methods in special order. The homologous recombination was performed.
RESULTSRecombinant adenoviral vector pAd-Smad3D and pAd-Smad7 were constructed successfully, which were confirmed by restriction enzyme digesting.
CONCLUSIONSRecombinant adenoviral vector may be constructed quickly and efficiently in E.coli by sequential chemical transformation methods.
7.Experimental study on molecular mechanism of nm23-H1 gene transfection reversing the malignant phenotype of human high-metastatic large cell lung cancer cell line.
Yin LI ; Qinghua ZHOU ; Zhilin SUN ; Zefang SUN ; Yanping WANG ; Yang QIN ; Wen ZHU ; Xiaohe CHEN
Chinese Journal of Lung Cancer 2006;9(4):307-311
BACKGROUNDnm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.
METHODSThe expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.
RESULTSThe phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01).
CONCLUSIONSBlocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.
8.Effect of nm23-H1 on reversing malignant phenotype on human lung cancer cell line L9981.
Guowei CHE ; Qinghua ZHOU ; Yanping WANG ; Lunxu LIU ; Yang QIN ; Zilin SUN ; Zefang SUN ; Xiaohe CHEN
Journal of Biomedical Engineering 2005;22(3):530-534
nm23-H1 is a proven tumor metastasis suppressive gene, tumor metastasis phenotype could be reversed by transfected nm23-H1 cDNA. This study was conducted to transfect nm23-H1 cDNA into L9981 cells and to explore the function of nm23-H1 in reversing the malignant phenotype of L9981 cells. The plasmid of pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and the transfected nm23-H1 cDNA cell lines of L9981-nm23-H1 was established. The protein expression of nm23-H1 was detected by Western blot. The biologic features of L9981-nm23-H1 cells were studied in vitro and in vivo. The results showed that the fusion protein of nm23-H1-EGFP was stable, continuous and expressed with high efficiency in L9981-nm23-H1 cells. The cell proliferation, colon formation and invasive ability are significantly lowered in L9981 cells transfected nm23-H1 cDNA (P < 0.01); the tumorgenesis and the lung metastasis incidence was lower in tranfected nm23-H1 cells than in L9981 and L9981-Plxsn in nude mice (P < 0.01); the rate for inhibiting tumorgenesis of nm23 -H1 was 82.56%. These data suggest that the malignant phenotype could be reversed by wild nm23-H1 gene in L9981 cells.
Animals
;
Cell Line, Tumor
;
Cell Proliferation
;
Humans
;
Lung Neoplasms
;
pathology
;
Mice
;
Mice, Nude
;
NM23 Nucleoside Diphosphate Kinases
;
genetics
;
Neoplasm Metastasis
;
Neoplasm Transplantation
;
Recombinant Fusion Proteins
;
genetics
;
Transfection
;
Tumor Suppressor Proteins
;
genetics
9.Construction of mutant k-ras gene recombinant adenovirus.
Feng ZHAO ; Qinghua ZHOU ; Yang QIN ; Zhiling SUN ; Zefang SUN
Chinese Journal of Lung Cancer 2002;5(1):14-17
BACKGROUNDTo construct the recombinant adenovirus of mutant k-ras by using the method of homogenous recombination in bacteria.
METHODSMutant k-ras gene was liberated from the vector of pcDNA3-k-ras 12(Val) via KpnI+XhoI digestion, and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack CMV/k-ras 12(Val). Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with PacI and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad-k-ras 12(Val) was measured with the aid of GFP expression.
RESULTSThere were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack-CMV/k-ras 12 (Val) and pAdEasy-1 by method of CaCl₂. PCR test indicated each the recombinant adenovirus contained the insert of k-ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10¹² pfu/ml.
CONCLUSIONSThe method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad-k-ras 12(Val) paves a sound foundation for further study.
10.Effects of targeting inhibition of ERK1/2 signal transduction pathway on malignant biological behaviors of human high-metastatic large cell lung cancer cell line L9981.
Yin LI ; Qinghua ZHOU ; Zhilin SUN ; Zefang SUN ; Yanping WANG ; Yang QIN ; Wen ZHU ; Xiaohe CHEN
Chinese Journal of Lung Cancer 2005;8(6):504-509
BACKGROUNDIt has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.
METHODSThe expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods.
RESULTSThe ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P < 0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P < 0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P < 0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 (P > 0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 (P < 0.001).
CONCLUSIONSThe inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.