Effect of the combination of miR-34a and miR-let7 on the biological properties of pancreatic cancer cells / 中华胰腺病杂志

Objective To investigate the influence on biological characteristics in human pancreatic cancer cells after beding transfected by two anti-carcinoma miRNAs at the same time.Methods Pancreatic cancer cells PANC1,SW1990 and normal pancreatic cells AR42J were transfected by miR-34a and(or) miR-let7 by liposome.Cells transfected with negative control miRNA (miR-NC) and untransfected were as controls.The expression of miR-34a and miR-let7 were detected by real-time fluorescent quantitative RT-PCR.The cell proliferation was detected by MTT test and the migration and invasion were evaluated by transwell assay.The apoptosis rate was measured by flow cytometric analysis.Results After being transfected with miRNAs,the expression of miR-34a and miR-let7 in double transfection group (miR-34a and miR-let7 were transfected at the same time),miR-34a transfection group,miR-let7 transfection group was significantly up-regulated than those in miRNA-NC transfection group and untransfected group in PANC1 cells,SW1990 cells and AR42J cells,repectively.The difference which was statistically significant (P ＜0.05)indicating that cells were successfully transfected.The cell proliferation in double transfection group of PANC1 cells and SW1990 cells were (0.665 ± 0.01,0.6375 ± 0.03),which were significantly inhibited compared with (0.974 ± 0.03,0.971 ±0.05) in miR-NC group and (0.8875 ±0.05,0.8625 ±0.06) in miR-let7 group.The difference was statistically significant(P ＜ 0.05).The cell proliferation activity in double transfection group was lower than those in miR-34a group (0.795 ±0.06,0.7925 ±0.06),but did not have statistically significant difference.There was no significant change in AR42J cells.Cell invasion assay showed that the number of PANC1 cells permeating substrate membrane in miR34a group (103.7 ± 3.28) and miR-let7 group (100.7 ± 1.76) were significantly fewer than miR-NC group (231.3 ±2.6) and untransfected group (153.7 ±2.6).The number of cells permeating substrate membrane in double transfection group(61.67 ± 3.18)was fewer than miR-34a group and miR-let7 group,respectively.The difference was statistically significant (P ＜ 0.01).The migration test had consistent results with invasion test.The changes of invasion and migration in SW1990 cells were similar to those in PANC1 cells.The apoptosis rate of PANC1 cells in miR-34a group,miR-let7 group,double transfection group,miR-NC group and untransfected group was (16.66 ± 1.27) ％,(15.46 ± 0.33) ％,(23.35 ± 1.80) ％,(9.33 ± 0.31) ％ and (8.83 ± 0.36) ％ respectively.Single transfection group had higher apoptosis rate than miR-NC group and untransfected group (P ＜0.05).Double transfection group had a significantly higher apoptosis rate than miR-let7 group (P ＜ 0.05),while there was no significant difference between double transfection group and miR-34a group.Conclusions The cell proliferation,invasion and migration in double miRNAs transfected pancreatic cancer cells were significantly down-regulated compared with those in single miRNA transfected cells,while apoptosis rate in double miRNAs transfection group was higher than single miRNA transfection group.Thus,the combination of two anti-cancer miRNAs may exert a more significant synergistic antitumor effect.

Construction of adenovirus carrying dual-target shRNA for Oct-4 and Survivin and its inhibitory effect on human hepatocellular carcinoma cells / 生物工程学报

The transcriptional factor Oct-4 and Survivin are the key regulatory factors in cancer cell proliferation and mitosis. A dual cancer-specific shRNA adenovirus vector, Ad5-Dual-shRNA, targeting Oct-4 and Survivin genes was constructed by molecular cloning and recombination. After cells were infected with virus, hepatocellular carcinoma cell line EHBH-H1 was used for detecting the expression of Oct-4 and Survivin proteins by Western blotting. The viral cytotoxic effect on cancer cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay in vitro, and the inhibition effect on tumor xenografts was observed in nude mice. The results showed that the expression of Oct-4 and Survivin in cancer cell line EHBH-H1 could be silenced markedly by Ad5-Dual-shRNA. In MTT and animal experiments, Ad5-Dual-shRNA also represented much stronger anti-tumor effect on tumor growth than Ad5-Surv-shRNA and Ad5-Oct4-shRNA. From this research we can draw a conclusion that the cancer-specific adenovirus vector expressing dual-shRNA targeting Oct-4 and Survivin genes may provide us a more effective, specific and convenient gene therapy method.

Construction and identification of recombinant adenovirus containing heat shock protein70 gene driven by carcinoembryonic antigen promoter / 中华胰腺病杂志

Objective To construct the recombinant adenoviros containing heat shock protein70 (Hsp70) gene driven by carcinoembryonic antigen (CEA) promoter. Methods Hsp70 gene and CEA promoter were amplified by RT-PCR and PCR, and then subcloned into the shuttle vector pDC316 to construct the recombinant vector PDC316-pCEA-Hsp70. The recombinant vector was co-transfected with adenoviral backbone plasmid into HEK293 cells to generate the recombinant adenovirus Ad5-pCEA-Hsp70. The recombinant adenovirus was purified by CsCl banding and titrated by 50％ tissue culture infective dose (TCID50) assay. After transfection of the recombinant adenovirus into human pancreatic cell lines SW1990 and BxPC3, the expression of mRNA and protein level of Hsp70 were determined by RT-PCR and ELISA,respectively. Results Digestion and DNA sequencing certified that the Hsp70 gene and CEA promoter was successfully inserted into pDC316 plasmid. Virus acquired through co-transfection with backbone plasmid was confirmed to be constructed successfully by PCR amplification. The particles finally expressed was 2.2 ×1011vp/ml, and the titer was 1.5 x 1010 PFU/ml. BxPC3 cancer cells with positive CEA expression showed increased expression of Hsp70 mRNA and protein after infected by recombinant adenovirus; while SW1990 cancer cells with negative CEA expression showed no change of expression of Hsp70 mRNA and protein after infected by recombinant adenovirus. Conclusions The recombinant adenovirus Ad5-pCEA-Hsp70 which can express Hsp70 gene in CEA positive cancer cells is constructed successfully.

Antitumor effect on gastric cancer in vitro of selective replicative-competent adenovirus CNHK300-mIFN-γ / 肿瘤研究与临床

Objective To compare the expression of mIFN-γ, replicative activities and anti-tumor activities of CNHK300-mIFN-γand Ad-mIFN-γin normal and gastric cancer cells. Methods The replicative activities of viruses in cells were measured by viral replication assay. CPE assay was used to detect the antitumor effect of viruses. The expression level of mIFN-γ in cancer cells was detected by ELISA. Results The infection of CNHK300-mIFN-γled to an obvious expression of mIFN-γin gastric cancer cells. The vector system CNHK300-mIFN-γpossessed more replicated potential than Ad-mIFN-γ, and could specifically kill most of BGC-823 cells at MOI value of 0.1, which was much better than that by the traditional adenoviral vector. Conclusion CNHK300-mIFN-γcan selectively replicate and effectively express mIFN-γ in tumor cells, and specifically kill gastric cancer cells, suggesting a splendid future as a new anticancer agent.

Construction and expression in vitro of an RU486 inducible vector carrying DsRed protein / 生物工程学报

The regulation of a target gene expression is very important in gene therapy. However, constitutive or inappropriate expression of the genes with traditional expression system may interfere with the effect of the gene therapy, even may lead to lethal side effect. We constructed an RU486 inducible eukaryotic vector carrying DsRed protein and evaluated its regulatable effect in vitro. The single vector named PDC-RURED was constructed with molecular biological methods, which contained DsRed gene, promoter and RU486-inducible system. To minimize any potential interference, we spaced the two transcriptional elements with a 1.6 kb insulator. The vector was identified by different enzyme restrictions, sequencing analysis and PCR assay. We demonstrated the regulatable expression of this vector after transfection in HEK293 cells by fluorescence microscopy and flow cytometry. In the absence of RU486, no significant DsRed protein activation was observed, whereas in the presence of RU486 up to 40 fold activation of the DsRed protein was observed in cultured cells. The data show that the novel eukaryotic expression plasmid vector can be used to regulate the expression level of genes of interest in appropriate time under the control of RU486. This inducible expression vector provides a powerful tool for the research of gene regulation and gene therapy.

Relationship of cyclin D1 and P16 with the biological characters of lung cancer / 中国组织工程研究

BACKGROUND:Cyclin D1 and P16 promote cellular proliferation and inhibit cellular division by regulating the activity of CDK4. There existes to some extent amplification or overexpression of cyclin D1 gene together with P16 gene the inactivation of P16 gene in the many tumors.OBJECTIVE: To probe into the relationship of the amplification of cyclin D1 and the deletion of P16 and lung cancer.DESIGN: Controlled trial of case samples.SETTING: Department of Biochemistry, the 81 Hospital of Chinese PLA.PARTICIPANTS: Tissue samples of 40 cases of lung cancer were obtained from surgical specimens of tumor tissues and normal lung tissues at the 81 hospital of Chinese PLA between June 1998 and June 1999. Informed consent was obtained from the patients.METHODS: Method of multiplex polymerase chain reaction (PCR) was adopted. Cyclin D1 gene and P16 gene were amplified in the same tube,using extracted genome DNA as template. 10 μL PCR products were separated on a 15 g/L agarose gel.MAIN OUTCOME MEASURES: ① Amplificative result of cyclin D1and P16 in the pathological tissue of the patients with lung cancer. ② Relationship of cyclin D1 and P16 and the biological character of lung cancer.RESULTS: ① The amplified positive rates of cyclin D1 gene and P16gene in lung cancer were 55.0%(22/40) and 65%(26/40) respectively.②Relationship of cyclin D1 and P16 and biological characters of lung cancer :The amplified positive rate of both of them is closely related to the metastasis of lymph node and the staging of clinical pathology. With the incidence and development of metastasis, amplification positive rate of cyclin D1 was increased while the positive rate of P16 decreased.CONCLUSION: The high amplification of Cyclin D1 and the high deletion of P16 gene is closely related to the high activity of proliferation of lung cancer. To detect the changes of these two genes would help us to judge the metastasis, malignant extent and patients' prognosis.

Analysis on point mutation of the CDKN2/p16 gene in lung cancer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the status of CDKN2/p16 gene point mutation in lung cancer.</p><p><b>METHODS</b>Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing were used to detect the point mutation of CDKN2/p16 gene exon 2 in 89 cases of lung cancer.</p><p><b>RESULTS</b>In 69 cases of the lung cancer without deletion of CDKN2/p16 gene exon 2, 16 cases were found to have suspicious abnormality of CDKN2/p16 gene exon 2 by PCR-SSCP, and in these 16 cases, 9 were found to harbor point mutations of CDKN2/p16 gene exon 2 by automated sequencing analysis.</p><p><b>CONCLUSION</b>The point mutation is one of the mechanisms for CDKN2/p16 gene inactivation, but it is not the chief mechanism. The inactivation of CDKN2/p16 gene aroused by point mutation plays a role to some extent in the genesis and progression of lung cancer.</p>

Therapeutic effect of replication-deficient adenovirus carrying p16 gene on gastric cancer xenografts in nude mice / 中国肿瘤生物治疗杂志

Objective:To construct a replication-deficient adenovirus carrying p16 gene and to investigate its anti-tumor activity on human gastric cancer xenografts in nude mice.Methods:p16 cDNA was amplified by PCR and was inserted into the plasmid pSuCMV,the latter was then used to recombine the replication-deficient adenovirus AdCMV-p16 in 293 cells.Human SGC-7901 gastric cancer xenograft models were established in nude mice and were divided into 3 groups:AdCMV-p16,Ad-LacZ,and control groups.Mice in AdCMV-p16 group received intratumoral injections of 2?108 pfu/100 l AdCMV-p16(injected every other day for 5 times).Mice in control group received the same volume of virus preserving solution.The tumor volumes were measured at predefined time points.The anti-tumor effect of AdCMV-p16 was observed by p16 immunochemical study and TUNEL detection of cell apoptosis.Results:The replication-deficient adenovirus expressing p16 gene evidently inhibited the growth of human gastric cancer xenografts in nude mice(P0.05),only with a inhibition rate of 4.26%.The pathological examination showed that apoptoses were the main changes in AdCMV-p16 group,and p16 gene was found in the cancer cells.Conclusion:The replication-deficient adenovirus harboring p16 gene can recover the expression of p16 in gastric cancer cells and subsequently inhibit the growth of human gastric cancer.

EXPRESSION OF THE p16 AND Rb GENES mRNA AND PROTEIN LEVELS IN LUNG CANCER / 解放军医学杂志

The authors used immunohistochemistry and in situ hybridization to observe the expression of the p16 and Rb mRNA and protein levels in 89 cases of lung cancer. The results showed that the loss of p16 gene expression took place mainly in non small cell lung cancer, and loss of Rb gene expression mainly in small cell lung cancer. The expression of these genes on protein level fundamentally corresponded with mRNA level. It is suggested that the expression of these two genes are related to the histological type of lung cancer, and the p16 and Rb genes can be considered as one of molecular biological targets for gene classification diagnosis. There may exist a mechanism at gene translation level which leads to the inactivation of the p16 and Rb genes.

EFFECT OF PROTEIN DEFICIENCY ON MACROPHAGES OF MICE / 营养学报

We have observed and studied the immune response, ultrastructure and phagocytosis of peritoneal macrophages (M?) of mice in protein deficiency by means of indirect fluorescent antibody test (IFAT), immunoenzymatic staining technique (IEST),fluorescence isothiocyanate antibody (FITC-Ab) quantitative assay, M? phagocytosis test, scanning electron microscopy (SEM) and im-munoelectron microscopy (IEM).The results showed that the body weight of mice was continuously declined after fed protein deficient diet. In the same time fluorescence reaction and enzyme stain on the M? surface was retarded. The amount of FITC-Ab on the M? membrane was decreased. The villi on the M? surface were shortened, the positive rates and positive degree of cells were lowered,the reaction of cell membrane and nuclear membrane was retarded in SEM and IEM.The phagocytic function of M? was inhibited.The results showed that in protein deficiency, the immune reaction, structure and function of peritoneal M? of mice were markedly affected.