CCL18 downregulates the expression of miR98 in breast cancer cells via N-Ras/c-myc/lin28 pathway / 西安交通大学学报(医学版)

Objective To explore whether CCL18 is involved in regulating the expression of miRNAs in breast cancer.Methods The expression profile of miRNAs in the breast cancer cell following CCL18 treatment was determined by miRNAs microarray analysis.Then we performed QRT-PCR and Luciferase Reporter Assay to validate the results from the miRNAs microassay.We used transient transfection to change the expression of miR98 and c-myc in breast cancer cells.We then used QRT-PCR and Western blot to analyze the mechanism by which CCL18 downregulates the expression of miR98 in breast cancer cells.Results miRNAs microarray analysis showed that cells treated with CCL18 differentially expressed 20 miRNAs genes compared with those in the control group. Our QRT-PCR and Luciferase Reporter Assay confirmed the result.The mRNA and protein expressions of C-myc and lin28 were increased after CCL18 stimulation in breast cancer cells.Transfection with c-myc siRNAs rescued the increase of lin28 and loss of miR98 expression caused by CCL18 stimulation.Our results also showed that CCL18 could upregulate the expression of N-Ras at post-transcription level.Conclusion CCL18 downregulates the expression of miR98 via N-Ras/c-myc/lin28 pathway.The downregulated miR98 increases the expression of N-Ras after transfection,which further activates c-myc/lin28 pathway and forms a positive feedback loop.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

Effect of N-Ras by RNA interference on radiosensitivity of hepatoma carcinoma cell MHCC97-H line / 中华放射医学与防护杂志

Objective To investigate the radiosensitivity of silencing N-Ras by RNA interference in hepatoma carcinoma cell MHCC-97.Methods N-Ras RNA interference (RNAi) vector was constructed by using pcDNA 6.2-GW/EmGFP-mir plamid.The RNAi effect was detected by RT-PCR,Western bolt,immunohistochemisty and MTT method.Survival curve for each cell line were obtained by measuring the clone forming abilities of irradiated cell populations.Results After silencing the N-Ras by RNAi,The expression level of N-Ras mRNA,N-Ras protein,immunohistochemisty were decreased 96.9% ±0.159%(t=40.377,P＜0.05),89.8%±0.012% (t=31.595,P＜0.05),90%,respectively,and The survival of hepatoma carcinoma cell MHCC-97 line were inhibited 21.9% (F = 4.63,P < 0.05).Which have significant difference in statistics.The SER of hepatoma carcinoma cell MHCC-97 line after interference was 1.15.Conclusions RNAi targeting silence N-Ras may increase the radiosensitivity of hepatoma carcinoma cell MHCC-97 line.

Mutation of the N-ras Gene in a Patient Suffering from the Blast Phase of Chronic Myelogenous Leukemia

The blast phase in chronic myelogenous leukemia (CML) is associated with mutation of several genes. It is well known that p53 gene mutation plays a key role in the myeloid or lymphoid blast phase of CML. But for the case of the N-ras gene, the association between N-ras mutations and the blast phase of CML is not yet known. We report here on a case of detecting N-ras point mutation without p53 mutation in a 64 year-old man who suffered from the lymphoblastic blast phase of CML.

Fatal outcome of a young woman with papillary thyroid carcinoma and graves' disease: possible implication of "cross-signalling" mechanism / Desfecho Fatal de uma Paciente com Carcinoma Papilífero de Tiróide e Doença de Graves: possível Implicação do Mecanismo de Sinalização Cruzada

A 29 yrs-old patient was referred to our hospital due to generalized convulsions. She had hyperthyroidism treated with methimazole. Her MRI showed 4 metastatic lesions in the brain. She had a goiter with a "cold" nodule and a palpable ipsilateral lymph node. The FNAB disclosed a papillary thyroid carcinoma. Under 5 mg of MMI treatment, she had a subclinical hyperthyroidism and TRAb were 47.8 percent (n.v. < 10 percent). The CT scan also showed lung metastasis. She underwent a total thyroidectomy with a modified neck dissection and she received an accumulated radioiodine dose of 700 mCi during the following two years. She died from the consequences of multiple metastatic lesions. Studies were performed in DNA extracted from paraffin-embedded tissue from the tumor, the metastatic lymph node and the non-tumoral thyroid. The genetic analysis of tumoral DNA revealed point mutations in two different genes: the wild type CAA at codon 61 of N-RAS mutated to CAT, replacing glycine by histidine (G61H) and the normal GCC sequence at codon 623 of the TSHR gene was replaced by TCC, changing the alanine by serine (A623S). In the non-tumoral tissue no mutations were found. In vitro studies showed a constitutive activation of the TSHR. It is very probable that this activating mutation of the TSHR is unable to reach the end point of the PKA cascade in the tumoral tissue. One possibility that could explain this is the presence of a cross-signaling mechanism generating a deviation of the TSH receptor cascade to the more proliferative one involving the MAPKinase, giving perhaps a more aggressive behavior of this papillary thyroid cancer.

Paciente de 29 anos foi encaminhada ao Hospital de Clínicas por causa de convulsões generalizadas. Apresentava hipertiroidismo tratado com metimazol (MMI). A ressonância magnética mostrava quatro lesões metastáticas cerebrais. Possuía bócio com nódulo frio e linfonodo palpável ipsilateral. Usando 5 mg de MMI, a paciente apresentava hipertiroidismo subclínico e TRAb = 47,8 por cento (normal < 10 por cento). A tomografia computadorizada também mostrava metástases pulmonares. A paciente foi submetida a tiroidectomia total com dissecção cervical modificada e recebeu dose acumulada de radioiodo de 700 mCi durante o período de dois anos. Foi analisado o DNA extraído de tecido emblocado em parafina do tumor, do linfonodo metastático e de tecido tiroidiano não-tumoral. Foram encontradas mutações pontuais em dois genes: uma substituição do genótipo selvagem CAA no códon 61 de /N-RAS/ por CAT, substituindo a glicina pela histidina (G61H) e uma substituição da seqüência normal GCC no códon 623 do gene TSHR por TCC, trocando a alanina pela serina (A623S). Não foram encontradas mutações no tecido não-tumoral. Estudos in vitro mostraram ativação constitutiva de TSHR. Já que esta mutação ativadora de TSHR foi incapaz de atingir o final da cascata PKA no tecido tumoral, sugere-se que um mecanismo de cross-signaling possa explicar o desvio da cascata do receptor de TSH para outra mais proliferativa, envolvendo MAPKinase e levando ao comportamento mais agressivo deste câncer papilífero.

Effect of BPDE on Expression of N-Ras Gene in Human Bronchial Epithelial Cells / 环境与健康杂志

Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE

Secondary structure of target RNA impacts on RNA interference efficiency / 基础医学与临床

Objective To study the effects of the RNA secondary structure on the RNA interference efficacy and to optimize the RNAi target sequence selection.Methods N-Ras mRNA in K562 human chronic erythro-leukemia cell line was selected as the target for RNAi experiments.Four siRNAs were designed aiming at secondary structure region and none-secondary structure region separately.The N-Ras expression change and the cell growth was tested by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR),light microscopy,MTT,and flow cytometry.Results Knock-down efficiency was recorded in all of the 4 experiment groups,but the degree varied a lot.The siRNAs targeting none-secondary structure region presented higher silencing efficiency than those targeting secondary structure region.Conclusion The secondary structure of mRNA closely relate to the RNA interference efficiency,which should be considered in the processing of the target sequence selection.

Significance of N-ras Oncogene Expression in Acult Myelogenous Leukemia / 中国医师杂志

ObjectiveTo investigate the effect of N-ras oncogene expression in acute myelogenous leukemia(AML).Methods N-ras oncongene in 10 cases of AML were determined using reverse transcriptase-polymerase chain reaction(RT-PCR).Results 3/10 cases were the positive expression,the positive rates were 30%;2/3 patients were treated to complete remmision(CR).Their N-ras oncogene change negative,1/3 patients was no remmision(NR),his N-ras oncogene change negative,1/3 patient was no remmision(NR),his N-ras oncogene keeps positive and dead after 4 months.Conclusions Determination of the N-ras oncogene expression in AML has worthy for diagnose and prognosis.

Construction of Mutant N-ras/61 Gene Recombinant Vaccinia Virus / 中国肿瘤生物治疗杂志

Objective: To construct the recombinant vaccinia virus of mutant N-ras/61 gene and enhance the immunogenecity of mutant N-ras／61 protein produced by the recombinant vaccinia virus. Methods: N-ras／61 gene was inserted into P1108 and transfected into CV-1 cell infected with vaccinia virus by Cell FECTIN. PCR and Western blot were used to identify the recombinants. Results: We get recombinant vaccinia virus rV-N-ras/61 by PCR and tk- selecting. The expression of N-ras gene was detected by Western blot. Conclusion: This study is a test for studing effective vaccine of mutant N-ras/61 gene. The efficacy in vivo of the N-ras／61 vaccine in immunotherapy is under investgation.

Analysis of mutations of ras and p53 gene in multiple myeloma / 대한내과학회지

BACKGROUND: Multiple myeloma is a malignant prolif eration of plasma cells producing monoclonal immunog lobulins. The pathogenesis of this disease is still unkno wn. Karyotypic complexity and stepwise disease progre ssion in multiple myeloma suggest that the development of multiple myeloma is a multistep process in genetic events, such as oncogene activation or tumor suppressor gene inactivation. Alterations of ras oncogene or p53 tumor sup pressor gene are involved in various type of human cancers. The aim of this study was to determine the frequency of ras and p53 gene mutations in multiple myeloma, and to analyze its association with clinical parameter and clinical outcome. METHODS: Mutations of N-, K-ras exon 1 & 2 and p53 exon 5-8 were observed in 33 patients with multiple myeloma. Genomic DNA was isolated from mononuclear cells separated from bone marrow samples. Extracted DNAs were screened for mutations by single-strand con formation polymorphism analysis of PCR products (PCR SSCP). DNA fragments displaying an altered electrophore tic mobility were further studied by direct sequencing to confirm and characterize the nature of the mutations. RESULTS: No mutation was found at N-, K-ras exon 1, K-ras exon 2 or p53 exon 5-8. Only one patient has N-ras exon 2 mutation(1/33 patients, 3%). By direct sequencing of PCR products, I confirmed and detected a CAA-->AAA transversion(glutamine-->lysine). The patient was a 61-year-old male in progressive state. M protein was IgG/kappa type. Bone marrow aspirate revealed a 67% plasma cell infiltration. CONCLUSION: Although the number of patients is small, these data revealed low frequency of N-ras, K-ras and p53 gene mutation in multiple myeloma. Ras and p53 gene mutations may have limited role in pathogenesis of mulitple myeloma and may associate with tumor progres sion rather than initiation.

EFFECTS OF EICOSAPENTAENOIC ACID IN COMBINATION WITH RETINOIC ACID ON N-RAS EXPRESSION IN HL-60 CELLS / 营养学报

Objective:To study the effects of eicosapentaenoic acid (EPA) and retinoic acid (RA) on proliferation and differentiation of HL 60 cells and the corresponding mechanisms. Methods:Flow cytometry was used to assay cell proliferation function, NBT reduction experiments to evaluate cell differentiation, and Western blot to analyse pRB expression. Results:The proliferation index (PI) in all experimental groups were all lower than that of the control , and EPA in combination with RA was the lowest one. NBT reduction experiments showed that the OD value of cells treated by EPA and RA was about 5.9 times of the control, but correspondingly by RA was 2.6 times. Moreover, p21 N ras expression of HL 60 cells were all decreased by treatment with EPA or/and RA. Conclusion:The synergistic effects of EPA and RA on proliferation and differentiation of HL 60 cells may be partly due to the change of p21 N ras expression.l proliferation function,NBT reduc