MiR-7 affects the progression of abdominal aortic aneurysms by up-regulating the expression of ERK and MMP-9 proteins / 中华胸心血管外科杂志

Objective:To explore the effect of miR-7 on the formation of abdominal aortic aneurysm by up-regulating the expression of ERK and MMP-9 proteins.Methods:Download the miRNAs expression profile chip data from the GEO database, the differentially expressed miRNAs were screened out by GEO2R and the correlation between target genes and ERK genes was analyzed; twenty 4-week male C57BL/6J mice with SPF grade were selected and randomly divided into a model group (only 0.6% BAPN solution was given, n=10) and miR-7 group (fed with 0.6% BAPN solution+ injected with 1×10 9 PFU/ml lentivirus containing miR-7 over expression gene via tail vein, n=10), after 7 weeks of continuous feeding, the mice were anesthetized intraperitoneally with chloral hydrate. After the abdominal cavity and thorax were dissected, the abdominal aorta was separated from the left ventricle perfusion with normal saline under physiological pressure and pathological sections were prepared. Masson staining and α-SMA staining were used to evaluate the lesion degree of abdominal aortic vessels in each group; the protein expression levels of p-ERK1/2 and MMP-9 in the diseased abdominal aorta of each group were detected by WB. Results:By screening differential genes, we found that miR-7 was highly expressed in patients with abdominal aortic aneurysm, and further analysis revealed that miR-7 was positively correlated with ERK1/2; Masson staining showed that the tumor of abdominal aortic aneurysm in miR -7 group was significantly larger than that in the model group, and the difference was statistically significant ( P<0.05), the results of α-SMA histochemical staining showed that the number of α-SMA positive VSMC in miR-7 group was significantly lower than that in the model group, and there was almost no-SMA staining in some VSMC; WB results showed that the highest expressions of P-ERK1/2 and MMP-9 proteins in the abdominal aorta of miR-7 group were significantly higher than that of the model group, with statistically significant differences ( P<0.05). Conclusion:MiR-7 accelerated the formation of abdominal aortic aneurysms by up-regulating the expression of ERK and MMP-9 proteins.

Construction of let-7d expression vector and its inhibitory effect on HMGA2 and ras expression in human ovarian cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins.</p><p><b>METHODS</b>The pre-let-7d sequence was synthesized and inserted into pcDNA6.2GW/EmGFPmiR and transfected into ovarian cancer IGROV1 cells to cause pre-let-7d overexpression. Real-time quantitative RT-PCR was employed to examine the expression levels of let-7d miRNA and HMGA2 mRNA, and Western blotting was performed to detect the expressions of HMGA2 and ras protein in the transfected cells. The effect of pcDNA6.2GW-let-7d transfection on IGROV1 cell proliferation was determined using MTT assay and the cell apoptosis rate was measured using flow cytometry.</p><p><b>RESULTS</b>The eukaryotic expression vector containing the target gene let-7d was successfully constructed and transfected into IGROV1 cells. The transfected cells showed a marked reduction of HMGA2 expression but a less obvious down-regulation of ras expression. Transfection with pcDNA6.2GW-let-7d to suppress the expression of HMGA2 caused alterations of the phenotype of IGROV1 cells shown by a reduced proliferative activity and increased cell apoptosis.</p><p><b>CONCLUSION</b>Let-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.</p>

High resolution melting analysis for the rapid and sensitive detection of KRAS codon 12 and 13 mutations in colorectal cancer / 中华检验医学杂志

Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients.Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2,notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS.HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues.The results were compared with the direct sequencing data.Results After the PCR amplification,the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability.HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA.All 60 samples identified the KRAS mutation by HRM and sequencing.17 samples were positive(28.3%) by HRM for KRAS exon 2 mutations,and 15 samples were confirmed the presence of codon 12 or 13 mutations(25.0%) and the other 2 samples were wild type by sequencing.The 60 samples detected by HRM were given 100% sensitivity with 96% specificity.Conclusions HRM is a sensitive intube methodology to screen for mutations in clinical samples.HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.

siRNA in silencing the expression of DNA-dependent protein kinase and its effect on radiosensitivity of lung adenocarcinoma cells / 中华放射肿瘤学杂志

Objective To discuss the role of DNA-dependent protein kinase catalytic subunit (DNA-DPKCS) in human lung adenocarcinoma cell line (A549) by using small interfering RNA (siRNA) to specifically knockdown DNA-DPKCS expression and its effects on cell proliferation, cell cycle and radio-sensitivity. Methods The DNA-DPKCS-siRNA expression vector was constructed and transfected into A549 cell line. The transformed clones were randomly selected and isolated. The cell cycle distribution and apop-tesis were analyzed by flowcytometry analysis. Cell survival was detected by using clonogenic formation as-say. Results With specific inhibition of DNA-DPKCS expression, stable transfected cell line 549pRNA-DNA-DPKCS was constructed by RNA interference technique. The 549pRNA-C and 549pSUPER cell lines were the control cell lines tansfected with control and blank plasmids, respectively. Compared with A549 cells, the expression levels of DNA-DPKCS mRNA (0.110: 1. 000), protein (0. 870: 2.967) and activity of DNA-DPKCS (0.004: 0.266) in 549pRNA-DNA-DPKCS cells were significantly lower (F = 80.55 ,P < 0.01;F=63.96, P<0.01;F=51.62,P<0.01, respectively). The analysis of SF_2(0.25:0.76), D_0 (1.42:1.62) and D_q (0.06: 1. 00) showed significant difference between 549pRNA-DNA-DPKCS and A549 cells (F = 996.86, P < 0.01 ; F = 17.41, P < 0.05 ; F = 68.92, P < 0.01). The number of 549pRNA-DNA-DPKCS cells in S (24.5%: 35.5%) and G_2 (10.7%: 11.0%) phases was significantly decreased (F = 4.83, P<0.05 and F=32.04, P <0.01, respectively). Conclusions In A549 cells, inhibit of DNA-DPKCS gene expression can enhance the radiosensitivity and affect cell cycle distribution.

Establishment and its biological characteristics of patient-derived lung cancer xenograft models / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>With the ongoing need to improve therapy for lung cancer, there has been an increasing interest in the development of reliable preclinical models to test novel therapeutics. The aim of this study is to establish a patient-derived lung cancer xenograft model in mice and to observe the biological characteristics of xenografts.</p><p><b>METHODS</b>Surgically resected tumor specimens from patients with lung cancer were implanted in the subcutaneous layer of the NOD/ SCID mice. Cancer specimens of percutaneous lung biopsy by CT fluoroscopy were implanted into the subrenal capsule of nude mouse. The subcutaneous carcinoma was surgically removed when it grew to approximately 1.0 cm in diameter, and then re-transplanted into new nude mice. The growth process of transplanted tumor was observed. Expression of CEA, cytokeratin, and Ki67 were detected by immunohistochemistry. Mutations in the exons 18-21 of EGFR and exons 12,59 of K-ras of primary and xenograft tumors were examined. The cell cycle of xenograft tumor cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>Eleven cases were conducted for NOD/SCID mice and nude mice modelling. The patient-derived lung cancer xenografts have been established successfully, and the tumor could be passed to new nude mice, including No 2 model (adenocasinoma), No. 3 model (small cell lung cancer), and No.5 model (squamous cell cancer). High homogeneity was found between xenograft tumors and human lung cancer in histopathology, immunohistochemical phenotype, and EGFR, K-ras mutation status. The S-phase fraction of xenograft cell cycle was prolonged, which indicated that the xenografts remains highly proliferated.</p><p><b>CONCLUSION</b>The xenotransplantation models established for patient-derived lung cancer in immune deficient mice. The success rate is 27%. This model system displayed the biological characteristics of human lung cancer, suggesting that it may provide a stable, reliable, and useful animal model in human lung cancer research.</p>

VEGF-D expression in lung adenocarcinoma with or without acquired resistance to gefitinib and normal lung tissue / 中国肺癌杂志

<p><b>BACKGROUND</b>The epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) is playing an important role in the treatment of non-small cell lung cancer.The acquired resistance of EGFR-TKI has become a hot spot.This study aims to investigate VEGF-D expression level in lung adenocarcinoma with or without acquired resistance to gefitinib and in normal lung,so as to explore the role of VEGF-D in resistance of gefitinib.</p><p><b>METHODS</b>Lung adenocarcinoma,normal lung tissues adjacent to the carcinoma and adenocarcinoma with acquired resistance to gefitinib including some metastastic lymph nodes were obtained during operation.SYBR Green real-time PCR with beta actin gene as the endogenous control was performed to examine VEGF-D gene expression semi-quantitatively.After positive expression of VEGF-D was determined,the expression ratio and level in each group was analyzed qualitatively and quantitatively.</p><p><b>RESULTS</b>Exact significance was computed to compare VEGF-D expression ratios in groups of lung adenocarcinoma with acquired resistance to gefitinib(1/6,16.7%),lung adenocarcinoma(7/14,50.0%) and normal lung(16/16,100.0%) and the difference was significant(P=0.000 091 7).VEGF-D relative expression level in the seven lung adenocarcinoma cases was significantly lower than its corresponding normal lung tissue by analysis of nonparametric test(P < 0.01).</p><p><b>CONCLUSIONS</b>VEGF-D has a high-level expression in normal lung tissues,while has a decreased expression in lung adenocarcinoma with or without acquired resistance to gefitinib,suggesting that VEGF-D functions in lung adenocarcinoma with or without acquired resistance to gefitinib in a way,which is different from in normal lung.</p>

Cloning of human preferentially expressed antigen of melanoma gene and its expression in E.coli / 医学研究生学报

Objective: To clone and express the preferentially expressed antigen of melanoma (PRAME) gene in E.coli. Methods: The cDNA encoding human PRAME gene was extracted from human AML cell line HL-60 and amplified by RT-PCR, then the PRAME gene was inserted into plasmid PGEM-T-easy. After sequenced, it was cloned into the prokaryotic expression vector pGEX-4T-2 to construct a clone with high level expression and the recombinant plasmid was cloned in E.coli. Results:The protein product reached the highest level at 5 h after IPTG induction. Conclusion: Since PRAME is only expressed at low levels in a few normal tissues and encodes an antigen recognized by autologous cytotoxic T lymphocytes, while expressed at high levels in patients with leukemia, it might be a new targeting candidate for tumor immunotherapy.

Induction of specific cytotoxic T lymphocytes using hepatoma antigenic peptide mixed with HSP70 in vitro / 中国免疫学杂志

Objective:To explore the possibility of inducing cell mediated immune response with HSP70 antigenic peptide complex in vitro.Methods:HSP70 peptide complex was reconstituted in vitro.Granulocyte/macrophage colony stimulating factors and interleukin 4 were used to cultivate DC from peripheral blood of HLA A2 positive healthy donors.HSP70,HSP70 peptide complex or peptide was used to activate the DC individually,which will initiate homogenize T lymphocyte to form cytotoxic T lymphocyte(CTL).The cytotxicity of the CTL was detected by MTT assay.Results:It was found that peptide specific CD8 + CTL responses were readily elicited by HSP70 peptide complex or peptide.The CTL response primed by HSP70 peptide complex was more potene than peptide alone.Conclusion:The results suggest that HSP70 peptide complex is immunogenic and HSP70 can lead to great efficient CTL response,antigenic peptides and HSP70 complex may be used as peptide vaccines for cancer immunotherapy.

Construction of recombinant adenovirus vector carrying human miR-133a and its expression in vitro / 中国病理生理杂志

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.

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells / 中国病理生理杂志

AIM: To investigate the inhibitory effect of vector-based RNA interference(RNAi) on the expression of melanoma associated antigen A3(MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells.METHODS: A vector for transcribing specific small hairpin RNA(shRNA) targeting MAGEA3 gene was constructed,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000.The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively.The cell apoptosis was studied by DNA fragmentation,electron microscopy,TUNEL assay,and annexin V/PI staining.RESULTS: The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells.After the shRNA expression vector was transfected into the MEL-ED1 cells,the expression of MAGEA3 gene was inhibited significantly(by 90%).DNA fragmentation,electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ?1.98%,significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group(both P

Expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in hepatocellular cell lines transfected by PRL-2 gene / 中国病理生理杂志

AIM: To investigate the possible mechanism of PRL-2 in invasive metastasis of tumors.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the transfectants were selected by growth in the medium supplemented with G418.Zymographic analysis of metalloproteinases(MMPs) activity was performed,RT-PCR was used to determine the mRNA levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2,the protein levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2 were analyzed by Western blotting.The effects of the special inhibitor of PRL-2 on transfected cells were also observed.RESULTS: The stable cell line selected by G418 was identified by RT-PCR and Western blotting.More abundance of MMP-2,MMP-9 and its activated type were secreted by the CL-1-PRL-2 cells than untransfected cells and transfected vector cells(P

Microsurgical treatment and survivin expression for intramedullary gliomas / 中华显微外科杂志

Objective To detect the expression of survivin protein in intramedullary gliomas and evaluated the clinical significance of the survivin expression. Method Seventeen cases of intramedullary gliomas were removed by using microsurgical technique.It composed of 8 cases of ependymomas and 9 ones of astrocytomas.The patients were followed up by MRI scanning periodically.The survivin protein expression of the intramedullary gliomas were examined by immunohistochemical stain (SP). Results Total resection of the tumor was obtained in 7 cases of ependymomas and subtotal resection was undertaken in the other one case.For 9 cases of astrocytomas,total resection of the tumor achieved in 3 cases,subtoal resection in 5 cases and partial resection in one case.Survivin expression was detected in 2 samples of ependymomas and 6 ones of astrocytomas.2 astrocytoma cases were moderate positive staining,who suffering from intracranial and vertebral subarachnoid dissemination of the tumor. Conclusion The result of microsurgical treatment for intramedullary ependymomas is satisfactory.The survivin expression in astrocytoma samples is significantly higher than that in ependymoma.Moderate positive staining may correlated with subarachnoid dissemination of the tumor.

Experimental gene therapy study on rat ovarian cancer with naked human wild type p53 plasmid delivered by echo contrast agent mediated by ultrasound / 中华超声影像学杂志

Objective To determine whether ultrasound mediated microbubble destruction could increase naked human wild type p53 plasmid delivery to ovarian cancer in rats by intratumoral injection. Methods Forty rats with ovarian cancer successfully induced by chemical intoxicant were divided into four groups.The wild type p53 gene was cloned into a high expressing efficiency eukaryotic plasmid pcDNA 3.1+ and the mixture of naked human wild type p53 plasmid was carefully injected directly into the center of ovarian neoplasms with or without echo contrast agent, and it was exposed to ultrasound for twenty minutes once a week.Two months after p53 gene transfection, semiquantitative RT-PCR was used to detect wild type p53 mRNA expression and western blot was to evaluate p53 protein expression level. Results Recombinated eukaryotic expression plasmid DNA encoding pcDNA 3.1+ /p53 was successfully constructed and confirmed by sequence analyzing and endonuclease cutting. The ovarian cancer model of rat was obtained by exposure to intoxicant. Expression of human wild type p53 mRNA was significantly higher in rats transfected with wild type p53 plasmid by means of ultrasound and contrast agent than others(P