Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.     

Effects of thyroid hormone on the myosin heavy chain mRNA expression in cardiac myocytes induced by angiotensin Ⅱ

AIM: To study the effect of thyroid hormone on the expressional change of myosin heavy chain(MHC) gene in cardiomyocyte induced by angiotensinⅡ(AngⅡ) and its potential mechanism. METHODS: Cardiac myocyte was cultured according to the method of Simpson. 10^-8 mol/L T₃ and 10^-7 mol/L AngⅡ were added to the culture medium, respectively or synchronously. After 48 h, the expression of α and β-MHC mRNA in myocytes were detected by RT-PCR. The protein kinase C activation were detected by PepTag non-radioactive PKC assay. The incorporation of -Leucine and [³H]-thymine to test the protein and DNA synthesis in myocytes were also performed. RESULTS: AngⅡalone increased the incorporation of [³H]-Leucine of myocytes while it had no effect on the incorporation of [³H]-thy mine. The expression of β-MHC mRNA was increased and the expression of α-MHC mRNA was decreased significantly at the condition of AngⅡ. The enhanced PKC activation was induced by AngⅡalso. When AngⅡand T₃ were added to the culture medium synchronously, though the incorporation of [³H]-leucine and [³H]-thymine were not changed compared with AngⅡ treated alone. The α-MHC mRNA expression was increased and the β-MHC mRNA expression was decreased significantly. The PKC activation of the myocytes also was decreased. CONCLUSIONS: T₃ inhibited the expressional change of myosin heavy chain gene in cardiac myocytes induced by AngⅡ. The effect of T₃ on the change of PKC activation in cardiac myocytes may be one of its mechanisms.     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Effect of captopril and betaloc on the changes in expression of MHC of left ventricle in hypertensive rats

AIM:To investigate myosin heavy chain(MHC)gene expression and the effects of captopril and betaloc at an early stage of hypertension. METHODS:Model of hypertension was made by partly narrowing two bilateral renal arteries(2K2C). The rats were divided into four groups at random. (1) control group; (2)2K2C group;(3)captopril group;(4)betaloc group.Levels of α-MHC and β-MHC mRNA were determined by dot-blot. RESULTS:α-MHC mRNA expression were gradual1y reduced in 2K2C group, while β-MHC mRNA expression were increased, and the marked changes were observed at 72h postoperation. Captopril could inhibit the changes in MHC gene expression; but betaloc could not. CONCLUSION: The expression of MHC gene has changed at an early stage of renal hypertensive rat, and renin-angiotensin system may play an important role in this change.     

Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas

AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients.     

Cholesterol depletion up-regulates expression of prohibitin in human prostate carcinoma PC-3 cells

AIM: To study how cholesterol delpetion affects prohibitin expression and the development of prostate cancer. METHODS: Human prostate carcinoma PC-3 cells were cultured in normal medium (NM) and cholesterol delpetion medium (CDM) for 48 h.The mRNA expression of prohibitin was detected by real-time PCR. Prohibitin promoter was cloned and inserted into PGL3-Basic to reconstruct plasmid. The plasmid was transiently transfected into PC-3 cells. The cells were cultured in the medium with different concentrations of cholesterol for 48 h and luciferase expression was detected.RESULTS: The results of real-time PCR showed that the mRNA level of prohibitin increased about 19 times in PC-3 cells in the presence of CDM. After transfected with prohibitin promoter plasmid for 48 h, PC-3 cells cultured in CDM had higher luciferase expression than the cells cultured in NM or in CDM with cholesterol. CONCLUSION: Cholesterol depletion up-regulates prohibitin expression in PC-3 cells, which may be one of the self-prophylactic and regulatory mechanisms to protect PC-3 cells from apoptosis caused by cholesterol insufficiency.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.     

Effects of inhibiting myosin light chain kinase on endothelin-1 induced proliferation and apoptosis of pulmonary arterial smooth muscle cells in rats

AIM: To investigate the effect of inhibiting myosin light chain kinase(MLCK) on endothelin-1(ET-1) induced proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs). METHODS: Rat PASMCs were cultured and stimulated with ET-1. The cells were randomly divided into control group, ET-1 group and ET-1+MLCK inhibitor group(ET-1+M). Western blotting, MTT assay, [³H]-TdR incorporation and flow cytometry were employed to test the expression of myosin light chain(MLC) and MLCK, cell proliferation, cell cycle and apoptotic rate of PASMCs,respectively. The phosphorylation of MLC was determined by glycerol-PAGE coupled with Western blotting. RESULTS: Compared with control group, the protein expression of MLCK and MLC phosphorylation significantly enhanced after ET-1 stimulation. ET-1 markedly induced the proliferation and decreased the percentage of apoptotic rate in the PASMCs. However, pretreatment with ML-7, a MLCK inhibitor, significantly reversed the above effects induced by ET-1. CONCLUSION: MLCK inhibitor effectively inhibits the ET-1-induced proliferation and the cell cycle progression.     

Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

AIM:To explore the recombined human proinsulin gene containing glucose reaction element (GLRE) expression in transfected CBRH7919 cells. METHODS:The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis. RESULTS:38 h after transfection, at the glucose levels of 0-25 mmol/L, the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L, respectively (P<0.05). One month after transfection, under above glucose levels, insulin values were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L (P<0.05). CBRH7919 cells including PLXSN-(GLRE)3-BP-1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were (2.10±0.23)U/L and (2.05±0.17)U/L, respectively. PCR products of transfected cells showed target band, but control cells did not. CONCLUSIONS:Recombined proinsulin gene was transfected successfully in CBRH7919 cells. The cells combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.     

Relationship between STEAP expression and hydrogen peroxide in human prostate tissues

AIM:To observe the STEAP expression and its function in human prostate tissues.METHODS:The expressions of STEAP mRNA and protein were detected by RT-PCR and Western blotting. H₂O₂ and SOD levels were detected by molybdic acid reduction and xanthine oxidation methods respectively.RESULTS:STEAP mRNA and protein were highly expressed in prostate cancer tissues. H₂O₂ and SOD levels in prostate cancer were obviously higher than those in normal prostate tissue.CONCLUSION:The function of STEAP gene is possibly to induce intracellular H₂O₂ and promote cell growth.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

转化CaMV基因芸薹属蔬菜植株抗病性鉴定及其遗传分析

 以转化CaMV 弱株系Bari1 基因&#1048578; 的大白菜、菜薹、紫菜薹和花椰菜植株为试材, 研究了CaMV 弱株系Bari&#1048577;1 基因&#1048578; 所提供的遗传工程交叉保护及其遗传规律。结果表明,在转化CaMV 弱株系Bari&#1048577;1 基因&#1048578; 的大白菜、菜薹、紫菜薹和花椰菜植株中, 48. 08%对CaMV强株系CABB&#1048577;BJI 具有较强的抗性, 51. 92%表现为敏感。抗性基因( CaMV Bari&#1048577;1 基因) 在自交代( S1) 中大多数( 76%) 表现为典型的孟德尔单基因显性遗传, 部分株系的抗性出现了15 1, 1 1, 1 3 和1 95 的非孟德尔遗传现象。     

Effect of curcumin on expression of p21 and CD44V6 in human breast cancer xenografts

AIM:To study the effect of curcumin on the expression of p21 and CD44V₆ in breast carcinoma in nude mice.METHODS:Nude mice were xenografted with human breast cancer cell line MCF-7 and randomly divided into 2 groups (n=4 in each group): control group and curcumin group. In latent period,the percentage of tumor development was observed. Tumors were measured and the surface areas were calculated. RT-PCR was performed to detect the expression level of cyclin D1,p21 and CD44V₆ mRNA. RESULTS:The tumor surface areas in the curcumin group were significantly lower than those in control group. In curcumin treatment group,the expression of p21 was up-regulated while cyclin D1 was nearly not changed. The expression of CD44V₆ was significantly down-regulated in curcumin group.CONCLUSION:Curcumin inhibits the expression of CD44V₆ and up-regulates the expression of p21 in nude mice bearing human breast cancer cell line MCF-7.     

Regulation of estrogen on the expression of KLK6 mRNA and protein in human breast cancer cell line MCF-7

AIM:To investigate the effects of estrogen and tamoxifen on the expression of KLK6 mRNA and protein (hK6) in human breast cancer cell line MCF-7. METHODS:MCF-7 cells were incubated with 17-βE2 and tamoxifen at different concentrations for 72 hours, respectively. The expression levels of kallikrein 6 (KLK6) mRNA and protein were evaluated by fluorescence quantitative RT-PCR and flow cytometry, respectively. RESULTS:Compared with ethanol control, KLK6 mRNA expression levels were significantly decreased when 17-βE2 was added at concentrations of 10^-10 and 10^-8 mol/L (P<0.01). No statistical change was observed when 17-βE2 was at 10^-12 mol/L (P>0.05). Flow cytometry showed the same results. The average fluorescence intensity (AFI) that represents the level of hK6 was decreased after incubated with 17-βE2 (P<0.01). After incubation with tamoxifen, the levels of KLK6 mRNA and hK6 were increased (P<0.01). CONCLUSION:Estrogen down-regulates the expression levels of KLK6 mRNA and protein (hK6), while tamoxifen has an opposite effect.     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.