RNA interference against hypoxia inducible factor-1α inhibited growth of cervical cancer by limiting vascularization

Objective: Cervical cancer has become a major public health problem. The development of effective, systemictherapies for cervical cancer is highly desired. We show here that hypoxia inducible factor-1α (HIF-1α) was indicated as anattractive therapeutic molecular target for cervical cancer. Methods: Firstly, we observed the expressional level of HIF-1α incervical cancer and Hela and Siha cell lines. Secondly, by constructuring HIF-1α shRNA targeting human HIF-1α mRNA commonsequence and transfecting it with plasmid to cervical cell, we detected the changes of HIF-1α and its downstream geneslevels VEGF. Then we injected selected stably transfected cell line into athymic nude mice to estimate its' antitumor effects.Results: We observed that HIF-1α inhibition was related to down-regulated VEGF resulting in prevention of angiogenesis,then leading to slower-growing tumors. Conclusion: The underlying concept of transfecting a HIF-1α shRNA expression vectorto block the HIF-1α holds promise as the clinical potential of gene therapy for cervical cancer.     

Study on inhibition of growth of ACHN cells via shRNA expression vector-mediated cyclinE1 gene silencing

OBJECTIVE To explore the inhibition of ACHN cells via shRNA expression vector mediated cyclinE1 gene silencing. METHODS The shRNA targeting at cyclinE1 gene was designed and synthesized. By ligation, the fragment was inserted into pGenesil-1-U6 to construct the recombinant plasmid pGenesil-1- U6-cyclinE1. The identified recombinant plasmid was introduced into ACHN cells with lipofectamine 2000. The inhibition of cyclinE1 mRNA and protein expression were analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion and speed of cell migration were detected by transwell chamber invasive models and cell scratch method. RESULTS The inhibition of expression of cyclinE1 in ACHN cells mediated by recombinant vector （0.0933 ± 0.05） was significantly lower than that in the group of transfected with empty vector （0.8827 ± 0.04） and the control group （0.9021±0.03） （P 〈 0.05）. Flow cytometry showed that recombinant cells were blocked in the G1 phase and the apoptotic ratio was increased significantly （11.15 ± 4.00）% （P 〈 0.05）. The curves of cell growth indicated that the proliferation of cell transfected with recombinant plasmid was inhibited significantly compared with that in control group （P 〈 0.05）. The results of transwell and cell scratch suggested that the abilities of invasion and migration of the cells transfected with recombinant plasmid were decreased conspicuously （P 〈 0.05）. CONCLUSION The expression of cyclinE1 could be inhibited successfully by RNA interference induced by shRNA expression vector. This consequently inhibits the cell growth and induces apoptosis. Our study provided a preliminary result in searching of RNA interference （RNAi） therapy for renal cell carcinoma.     

TIME- AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.     

Expression of a mutant hTERT in human bladder carcinoma cell line T24 and its clinical significance

Objective: To construct a mutant pEGFP- hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods:PCR amplification was performed by using primers based on the known gene sequence of hTERT. PCR production was cloned into plasmid pGEMT-T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sa/I sites of the pEGFP-C1 vector. After transfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and senescence correlated [3-galactosidase staining. Results: Identification of pEGFP-hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated ~-galactosidase in transfected cells gradually increased with extended cultured time and cell growth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.     

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR HUMAN CCL21 AND CHARACTERIZATION OF ITS CHEMOTACTIC ACTIVITY

Objective： To obtain recombinant human CCL21 with biological activity from eukary0tic expression system for further use in cancer gene therapy. Methods： A fragment of human CCL21 gene was obtained from pSK-hCCL21 plasmid digested by Xho I and BamH I, inserted into the responding sites of eukaryotic expression vector pVAX1, and then transfected into COS-7 cells by electroporation method. The expression of hCCL21 protein was detected by western blotting analysis. The in vitro chemotaxis assay was used to test the chemotactic function of the expression product to lymphocytes. Results： Human CCL21 protein was expressed by transfected COS-7 cells with recombinant plasmid containing hCCL21 gene, and was verified by western blotting. The in vitro chemotaxis assay demonstrated that human CCL21 protein had a potent chemotactic function to lymphocytes. Conclusion： Human CCL21 was successfully and transiently expressed in eukaryotic cells, which lays some foundation for the study of CCL21 gene therapy in murine tumor models.     

ISOLATION OF HUMAN TRANSCRIPTS EXPRESSED IN 16HBE CELLS RELATED TO CHLOROPHYLLIN ANTITRANSFORMING ACTIVITY AGAINST ANTI-BPDE BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

Objective: To analyze the differentially expressed eDNA sequences related to ehlorophyllin (CHL) mediated inhibition of malignant transformation of human bronchial epithelial cell line (16HBE). Methods: 16HBE cells treated with chlorophyllin and anti-BPDE were conducted as tester, 16HBE cells treated only with anti-BPDE were conducted as driver,and eDNA representational difference analysis (eDNA RDA) was used to compare the differential gene expression between the two kinds of cells. The eDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA was sequenced and compared with database in GenBank by BLASTN. Results: Among the 5 cloned eDNA sequences, three were novel and were registered in dbEST database, two showed sequence homology to alpha-enolase and a newly found gene ribosomal protein S18/S6-1ike. Conclusion: These 5 eDNA sequences might play important roles in antitransforming effect of chlorophyllin.     

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.     

TSLC1基因真核表达载体的构建及在HepG2细胞中的表达

Objective： To construct the eukaryotic expression vector for human TSLC1 gene, and to express TSLC1 in HepG2 cells for investigating its effect on HepG2 cell growth. Methods： Full length of TSLC1 cDNA was amplified from RNA of normal human liver by RT-PCR, and cloned into pCI-neo expression vector. The recombinant plasmid pCI-TSLC1 was identified with restriction enzyme and sequenced, and then was stably transfected into HepG2 cells with lipofectamine 2000. The positive clones were examined by western-blotting and immunofluorescence, cell growth was analyzed with MTT assay. Results： The eukaryotic expression vector pCI-TSLC1 was successfully constructed and the stable cell line highly expressing TSLC1 protein was obtained. The growth of TSLCl-transfected cells was significantly suppressed in vitro. Conclusion： The HepG2 stable cell line could highly express TSLC1 protein, which provided a basis for further exploring the roles of TSLC1 in hepatocellular carcinoma.     

转染AP-2α基因对结肠癌SW620细胞增殖及凋亡的影响(英文)

Objective:We investigated the effects of exogenous AP-2α gene on SW620 cell cycle, apoptosis and proliferation. Methods:The Plasmid pcDNA3.1(+)-AP-2α was transfected into colorectal carcinoma SW620 cells line by liposome mediation for transient expression. After AP-2α transfected SW620 cells, the exogenous AP-2α mRNA and protein express were determined by the method of Real-time PCR and Western blot. In order to elucidate the effect of expression of exogenous AP-2α gene on the colorectal cancer cell SW620, ...     

Preparation of Superparamagnetic Dextran-coated Iron Oxide Nanoparticles used as a Novel Gene Carrier into Human Bladder Cancer Cells

Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion:The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.     

Expression of nitroreductase gene NOR<Subscript>1</Subscript> in E.Coli and the preparation of antiserum

Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyelonai antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transeription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases.The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunobiot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsuifate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography.The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.     

婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.     

肿瘤坏死因子相关的凋亡诱导配体对胰腺癌细胞抗肿瘤作用的研究

Objective To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL).Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry.Procaspase-8 and procaspase-3 were determined by Western blot. Results The stable overexpression of TRAIL was detected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore,co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P ＜ 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and themechanisms involved in this effect may be proapoptosis and bystander effect.     

Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

Background: The mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer (CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells, using a synthetic lethal short hairpin RNA (shRNA) screening approach. Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183 (RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF183-knockdown cell lines were generated by infection of lentivi-ruses that express RNF183 shRNA, and small interference RNA (siRNA) was used to knock down RNF183 transiently. Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the protein abundance. MTT assay, colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation. Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8 (IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo. Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.     

A traditional Chinese medicine Huaier triggers G1 cell cycle arrest and apoptosis through cyclins-CDKs-CKIs machinery in MOLT4 cells

Objective： The purpose of the study was to study the effect of Huaier, a traditional Chinese medicine, on the cell cycle adjustment in MOLT4 cells in vitro. Methods： We used MTT assay to test cell viability, flow cytometry to detect cell cycle and apoptosis and western blot to examine the expression of cell-cycle and apoptotic proteins in MOLT4 cells induced by Huaier. Results： Huaier could reduce the viability of MOLT4 cell by inducing G1 arrest and apoptosis. The induction of apoptosis after treatment with Huaier for 24 h was demonstrated in a dose- and time-dependent manner by flow cytometry analysis. G1 arrest induced by Huaier was modulated through the increased expression of Cdki proteins （p21^cip/waf1 and p27^kip1） with a simultaneous decrease in Cdk2, Cdk4, Cdk6, cyclin D1 and cyclin E expression. Huaier also induced Bax and Bcl-2 expression and activation of Caspase-3. Conclusion： It is firstly demonstrated that Huaier can inhibit proliferation of MOLT4 cells via G1 arrest and apoptosis. These results suggest that Huaier is a cell-cycle anti-cancer drug.     

61. STUDY ON MUTAGENICITY OF PENICILLIUM DIGITATUM

Penicillium digitatum (P. digitatum) is a pathogenic fungus mildewed fruits and its process products. There were some events of poisoning on the clinic because fruits and its process products mildewed by the fungi are eaten. A lot of reports were about the study on acute poisoning on this hand. But there were few reports about the study on genetoxicity, and therefor we determined the mutagenicity of P. digitatum with different methods to provide scientific basis for prevent the effect of human genetoxicity. METHODS: ① The preparation of extract from P. digitatum: Preponderant fungus separated from mildewed fruits was incubated in the Czapak's medium for two weeks, and then it was extracted with CHCL2 and evaporated. It was dissolved by dimethylsulfoxide (DMSO) for use. ② Bacterial reverse mutation assay: The assay was without S9 mix. E. coli ND-160 strain was used in the assay. The assay set up negative control, positive control and test group, The test group contained four concentrations (3.125 mg/plate, 6.25 mg/plate, 12.5 mg/plate and 25 mg/plate). ③ Micronucleus assay of polychromatic erythrocytes (PCE) in mice bone marrow. The assay was individed into negative control group (0.9% NaCl, 20 ul/mice), positive control group (cyclophosphamide, CP, 30 mg/kg B.W.) and test group (extract, 250 mg/kg B.W.). ④ Unscheduled DNA synthesis (UDS) assay in primary cells of lung and liver of rat. Fresh cells separated from lung and liver were incubated. There were three groups in the test. They were negative control group SO, 1%(v/v)], positive control group (10-7 mol/L 3-MC for lung cell; 10-7 mol/L HN2. HCL for liver cell). The radioactivity of cells that were treated was detected. The result showed unscheduled incorporation index, and the index represented the level of UDS. ⑤ Mutation assay in E. coli K12 infA gene. Using E. coli K12 strain as mutation target, partial infA gene was amplified by PCR, and the overlapping fragments were cloned into PGEN-T vector. Seqences were determined and then analyzed by computer. RESULTS: ①The extract remarkblely induced the reverse mutation of E. coli ND-160 strain and there was a linear dosage-effect relation. ②The extract significantly increased the micronucleus rate of mouse bone marrow (P<0.01). ③The extract could induced UDS in the primary cells of the lung and liver of rat, and UDS was particularly obvious in the primary cell of lung (P<0.01). ④Five point mutations in nucleotide of infA gene were induced by the extract, and one of them led to amino acid variation (Lys-Val). CONCLUSION: P. digitatum could cause gene mutation and eating mildewed fruits and its process products should be avoided.     

肝部细胞中乙肝病毒启动子介导的特异性表达

Objective： To investigate four expression vectors carrying enhanced green fluorescent protein （EGFP） genes, which under the control of different HBV promoters, were detected and compared as to their expressions in hepatocytes and nonhepatic cell lines. Methods： Four HBV promoters （including enhancers） were amplified by PCR, subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were analyzed and identified by restriction enzymes and sequencing. After transfection into human hepatoma cell lines and non-liver cells, transfection efficiency was measured by EGFP expression through fluorescence microscopy and analyzed with fluorescence activated cell sorter （FACS）. Results： All target fragments were separately obtained and successfully cloned into the expression vector. The transfection results showed that in hepatoma cells, the expression of EGFP was more obvious than it was in non-hepatic cells. Among the four promoters, S gene promoter presented the strongest transfection efficiency. Conclusion： Our findings indicate that HBV promoters could lead specific expression in hepatocytes, different promoters had different outcomes, which might be a potential for the gene therapy of liver diseases.