Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis / 中国结合医学杂志

OBJECTIVE@#To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis.@*METHODS@#The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells.@*RESULTS@#The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01).@*CONCLUSION@#GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.

Effect and Mechanism of Yuxuebi Tablet Against Collagen-Induced Arthritis of Rats / 中国实验方剂学杂志

Objective:To explore the intervention effect of Yuxuebi tablet （YXB） on collagen-induced arthritis （CIA） in rats and its anti-inflammatory mechanism. Method:Following CIA modeling， the rats in the drug administration groups were separately treated with intragastric administration of YXB （0.1， 0.2， and 0.4 g·kg^-1） and methotrexate （MTX， 0.4 mg·kg^-1）， once a day. The incidence of CIA， mechanical pain threshold （MPT） and cold pain threshold （CPT） were evaluated once every three days. After continuous administration for 30 days， the peripheral blood of rats was collected for the determination of platelet （PLT） count and fibrinogen （FIB） content. The hematoxylin-eosin （HE） staining was conducted to analyze the pathological changes in joint tissues. The protein expression levels of interleukin （IL）-1<italic>β</italic>， IL-8， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） p65， phosphorylated NF-<italic>κ</italic>B （p-NF-<italic>κ</italic>B） p65， Ras， and Raf-1 in joint tissues of CIA rats were detected by immunohistochemistry （IHC） and Western blot. The rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS） were induced by tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>， 10 μg·L^-1） <italic>in vitro</italic> and then subjected to transwell migration/invasion assay， followed by the detection of protein expression levels of Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 in RA-FLS by Western blot. Result:Compared with the control group， the model group exhibited an increased incidence of CIA， significantly decreased MPT （<italic>P</italic><0.05，<italic>P</italic><0.01）， elevated CPT （<italic>P</italic><0.01） and PLT and FIB in the peripheral blood， worsened histopathological score of joints， enhanced RA-FLS migration and invasion， and up-regulated inflammatory factors （<italic>P</italic><0.01）. The comparison with the model group revealed that YXB at different doses obviously reduced the incidence of CIA， increased MPT， down-regulated CPT and PLT and FIB in the peripheral blood （<italic>P</italic><0.05，<italic>P</italic><0.01）， ameliorated the pathological changes like synovial hyperplasia and bone and cartilage destruction （<italic>P</italic><0.05，<italic>P</italic><0.01）， and inhibited RA-FLS migration and invasion. Besides， the low-， medium-， and high-dose YXB reversed the IL-1<italic>β</italic>， IL-8， Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 expression in joint tissues of CIA rats to different extents， as well as the protein expression of Ras， Raf-1 and p-NF-<italic>κ</italic>B p65 in RA-FLS （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:YXB reduces the incidence of CIA， ameliorates the clinical symptoms of RA and the pathological changes in joint tissues， and inhibits the formation of synovium， which may be attributed to its inhibition against Ras/Raf-1/NF-<italic>κ</italic>B signaling pathway.

Caracterización clínica y molecular de niños con síndrome de Noonan y otras RASopatías en Argentina / Clinical and molecular characterization of children with Noonan syndrome and other RASopathies in Argentina

Introducción. Las RASopatías son un conjunto de síndromes fenotípicamente superpuestos causados por mutaciones en genes implicados en la vía RAS/MAPK. La herencia es autosómica dominante, presentan características clínicas comunes, como baja talla, dismorfias craneofaciales, cardiopatia congénita, manifestaciones ectodérmicas y mayor riesgo de cáncer. El diagnóstico molecular es clave. Objetivo. Identificar mutaciones en los genes PTPN11, SOS1,RAF1, BRAFy HRAS,y comparar las principales características clínicas en pacientes con confirmación molecular. Población y métodos. Se estudiaron niños con diagnóstico clínico de RASopatía evaluados entre agosto de 2013 y febrero de 2017. Resultados. Se identificaron mutaciones en el 71 % (87/122) de los pacientes. El estudio molecular confirmó el diagnóstico en el 73 % de los pacientes con síndrome de Noonan. La mutación más prevalente fue c.922A>G (p.Asn308Asp) en el gen PTPN11. Se detectó una variante no descrita en RAF1, c.1467G>C (p.Leu489Phe). Se confirmó el sindrome cardiofaciocutáneo en el 67 % de los casos con mutaciones en el gen BRAF. El síndrome de Costello y el síndrome de Noonan con múltiples lentigos se confirmaron en todos los casos. Conclusión. La confirmación del diagnóstico clínico permitió un diagnóstico diferencial más preciso. Se determinó la prevalencia de las mutaciones en PTPN11 (el 58 %), SOS1 (el 10 %) y RAF1 (el 5 %) en niños con síndrome de Noonan, en PTPN11 (el 100 %) en el sindrome de Noonan con múltiples lentigos, en BRAF (el 67 %) en el síndrome cardiofaciocutáneo y en HRAS (el 100 %) en el sindrome de Costello.

Introduction. RASopathies are a set of syndromes with phenotypic overlapping features caused by gene mutations involved in the RAS/MAPK pathway. They are autosomal dominantly inherited and share common clinical characteristics, including short stature, craniofacial dysmorphisms, congenital heart disease, ectodermal manifestations, and a higher risk for cancer. A molecular diagnosis is a key factor. Objective. To identify PTPN11, SOS1, RAF1, BRAF, and HRAS mutations and compare the main clinical characteristics of patients with molecular confirmation. Population and methods. Children with a clinical diagnosis of RASopathy assessed between August 2013 and February 2017. Results. Mutations were identified in 71 % (87/122) of patients. The molecular test confirmed diagnosis in 73 % of patients with Noonan syndrome. The most prevalent mutation was c.922A>G (p.Asn308Asp) in the PTPN11 gene. A previously undescribed variant in RAF1 was detected: c.1467G>C (p.Leu489Phe). Cardiofaciocutaneous syndrome was confirmed in 67 % of cases with BRAF mutations. Costello syndrome and Noonan syndrome with multiple lentigines were confirmed in all cases. Conclusion. The confirmation of clinical diagnosis allowed for a more accurate differential diagnosis. The prevalence of PTPN11 (58 %), SOS1 (10 % ), and RAF1 mutations (5 %) in children with Noonan syndrome, of PTPN11 mutations (100 %) in those with Noonan syndrome with multiple lentigines, of BRAF mutations (67 %) in those with cardiofaciocutaneous syndrome, and of HRAS mutations (100 %) in those with Costello syndrome was determined.

LncRNA LINC00460 promotes the papillary thyroid cancer progression by regulating the LINC00460/miR-485-5p/Raf1 axis

BACKGROUND: Papillary thyroid cancer (PTC) is the most common malignancy of all thyroid cancers. LncRNA LINC00460 has been proved to play roles in the oncogenesis and progression of various tumors, including papillary thyroid cancer. However, the potential molecular mechanism of LINC00460 in PTC is poorly investigated. RESULTS: LINC00460 was upregulated in PTC tissues and cells. Raf1 was upregulated in PTC tissues, but miR-485-5p was down-regulated. High LINC00460 expression was associated with poor prognosis. LINC00460 knockdown suppressed proliferation, migration, invation and EMT of PTC cells. Bioinformatics prediction revealed that LINC00460 had binding sites with miR-485-5p, which was validated by luciferase reporter assay. In addition, miR-485-5p was confirmed to directly target Raf1 3'-UTR. Moreover, LINC00460 promoted PTC progression by sponging miR-485-5p to elevate the expression of Raf1. Knockdown of LINC00460 restrained tumor growth in vivo. CONCLUSION: LINC00460 induced proliferation, migration, invation and EMT of PTC cells by regulating the LINC00460/miR-485-5p/Raf1 axis, which indicated that LINC00460 may be a potential biomarker and therapeutic target for PTC.

The effects of microRNA-7 on proliferation and invasion of hepatocellular carcinoma HepG2 cells / 中华肿瘤杂志

Objective@#To investigate the effects of overexpression of microRNA-7 (miR-7) on the proliferation and invasion of HepG2 cells and the underlying mechanism in vitro.@*Methods@#The relative expression levels of miR-7 and Raf1 in hepatocellular carcinoma (HCC) tissues and adjacent normal tissues (ANT) were detected by quantitative real time-PCR (qRT-PCR). The relationship between the expression of miR-7 and the characteristics of HCC patients was analyzed. Cells were divided into blank control group, negative control (NC) group and miR-7 mimics transfected group, miR-7 mimics and NC were transfected into HepG2 cells by Lipofectamine™2000. The relative expression of miR-7 was detected by qRT-PCR. The proliferation ability of HepG2 cells was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The invasion of HepG2 cells was detected by Transwell assay. The target genes of miR-7 were predicted by TargetScan and the binding effect of miR-7 on the 3′UTR of Raf1 was verified by dual luciferase reporter assay.The expressions of Raf1 protein in hepatocellular carcinoma tissues, normal tissues and miR-7 mimics transfected HepG2 cells was detected by Western blot. The correlation of the levels of miR-7 and Raf1 mRNA was determined by Pearson correlation analysis.@*Results@#The relative expression level of miR-7 in HCC was 0.49±0.02, significantly lower than in ANT (1.21±0.05, P<0.01). The level of miR-7 was significantly correlated the tumor volume, metastasis and prognosis of HCC patients (P<0.05). The relative expression level of miR-7 in miR-7 mimics transfected HepG2 group was 12.67±0.40, significantly higher than that in blank group (P<0.01). Compared with the blank group, the A value and invasion ability of miR-7 mimics transfected group were significantly down-regulated at 48 hours and 72 hours after transfection (P<0.01). Compared with miR-7 NC group, the luciferase activity of wild-type Raf1 reporter gene in miR-7 mimics transfected group was significantly reduced (P<0.01). The relative expression of Raf1 protein in HCC was 3.15±0.10, significant higher than in ANT (0.53±0.03, P<0.01). The relative expression of Raf1 protein in miR-7 mimics transfected group was 0.24±0.01, significantly lower than in miR-7 NC group (0.98±0.02, P<0.01). Furthermore, an negative correlation was observed between the levels of miR-7 and Raf1 in HCC tissues (P<0.05).@*Conclusions@#The expression of miR-7 in HCC is significantly decreased and inversely correlated with poor survival of HCC patients. Overexpression of miR-7 can inhibit the proliferation and invasion ability of hepatocellular carcinoma cells HepG2 by downregulating Raf1 in vitro.

Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats / 中国比较医学杂志

Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.

DbcAMP inhibitted the proliferation of FTC-133 cell / 重庆医学

Objective To study the effect of dualdi butyryl cyclic AMP (dbcAMP)on the proliferation of FTC-133 cell line. Methods FTC-133 cells were normally cultured and divided into control group,dbcAMP treatment group (0.5,1.0,2.0 mmol/L). After FTC-133 cells were treated with dbcAMP (0.5,1.0,2.0 mmol/L)for 24 h or 48 h,the growth activity and growth curve was detected by MTT.Changes of the cell cycle were detected by flow cytometry.The mRNA and protein expression of Raf1 were measured by RT-qPCR and Western blotting.Results Compared with control group,the growth activity of FTC-133 cells was re-duced by different levels of dbcAMP in a dose-time dependence manner.The number of FTC-133 cells was decreased in the S phase and increased in the G2/M phase.The mRNA and protein expression of Raf1 of treatment group were both reduced compared with control group.Conclusion dbcAMP significantly reduced FTC-133 cells proliferation and promoted apoptosis,and which might be involoved by ERK MAPK signalling.

Effect of Zhengan Xifeng Decoction on Raf-1 mRNA and Protein Expression in Spontaneously Hypertensive Rats / 医药导报

Objective To study the effect of different doses of Zhengan xifeng decoction on Raf-1 mRNA and protein expression in the cardiovascular tissue of spontaneously hypertensive rats( SHR). Methods A total of 50 male SHR,24 weeks old,were randomly divided into the model,low dose,medium dose,high dose of Zhengan xifeng decoction and the compound apocynum groups,10 in each group. Ten homologous male rats( WKY)served as the normal control group. After gavaged for 5 weeks,western blotting and RT-PCR were used to detect the Raf-1 protein and mRNA expression in the cardiovascular tissue,respectively. Results Compared with the model control group,both Raf-1 protein and mRNA expressions significantly increased in all treatment groups( P〈0. 01 ). Conclusion The Zhengan xifeng decoction can stimulate cell proliferation and inhibit cell apoptosis by up-regulating the expression of Raf-1.

Molecular Mechanisms Underlying the Time-dependent Autophagy and Apoptosis Induced by Nutrient Depletion in Multiple Myeloma: a Pilot Study / 华中科技大学学报（医学）（英德文版）

This study explored the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in human multiple myeloma cell line RPMI8226 cells.RT-PCR and qRT-PCR were used to evaluate the transcriptional levels of Deptor,JNK1,JNK2,JNK3,Raf-1,p53,p21 and NFκB1 at 0,6,12,18,24 and 48 h after nutrient depletion in RPMI8226 cells.We found that transcriptional levels of Deptor were increased time-dependently at 0,6,12 and 18 h,and then decreased.Its alternation was consistent with autophagy.Transcriptional levels of Raf-1,JNK1,JNK2,p53 and p21 were increased time-dependently at 0,6,12,18,24 and 48 h accompanying with the increase of apoptosis.Transcriptional levels of NFκB 1 at 6,12,18,24 and 48 h were decreased as compared with 0 h.It was suggested that all the studied signaling molecules were involved in cellular response to nutrient depletion in RPMI8226 cells.Deptor contributed to autophagy in this process.Raf-1/JNK/p53/p21 pathway may be involved in apoptosis,and NFκB1 may play a possible role in inhibiting apoptosis.It remained to be studied whether Deptor was involved in both autophagy and apoptosis.

Effects of geldanamycin on cell proliferation and apoptosis of human ovarian cancer cell line SKOV3 and their mechanism / 肿瘤

Objective: To investigate the cytotoxic effects of geldanamycin on human ovarian cancer SKOV3 cells, and to explore the possible mechanisms of apoptosis and cell proliferation inhibition of SKOV3 cells induced by geldanamycin. Methods: MTT method was performed to evaluate the inhibitory effects of geldanamycin alone or in combination with cisplatin on proliferation of SKOV3 cells. Cell cycle and apoptosis rate of SKOV3 cells treated with geldanamycin were detected by flow cytometry. The expressions of Akt and Raf-1 proteins were examined by immunocytochemistry and flow cytometry. Results: The proliferation of SKOV3 cells was inhibited by different concentrations of geldanamycin in a time- and dose-dependent manners (P<0.05). Different concentrations of geldanamycin strengthened the inhibitory effect of cisplatin on proliferation of SKOV3 cells, and this inhibitory effect was strengthened with the increased concentration of geldanamycin within a certain concentration range. The apoptosis rate of SKOV3 cells was raised up gradually after treatment with 0-2000 nmol/L geldanamycin for 48 h, and the number of cells in G2/M phase was increased significantly (P<0.05). The results of immunocytochemistry and flow cytometry showed that the expressions of Akt and Raf-1 proteins in SKOV3 cells induced by geldanamycin were significantly down-regulated in a dose-dependent manner (P<0.05). Conclusion: Geldanamycin can inhibit the cell proliferation and induce the apoptosis of human ovarian cancer SKOV3 cells in vitro , which may be associated with downregulation of the expressions of Akt and Raf-1 proteins. Copyright© 2011 by TUMOR.

Expression of Raf-1 Kinase Inhibitory Protein in Extrahepatic Bile Duct Carcinoma

BACKGROUND: Raf-1 kinase inhibitory protein (RKIP) recently has been identified as a metastasis suppressor in a variety of human carcinomas. The prognostic significance of RKIP expression in extrahepatic bile duct (EBD) carcinoma has not been studied. The aims of the current study were to evaluate RKIP expression and to determine the prognostic significance of RKIP expression in EBD carcinoma. METHODS: Immunohistochemical staining for RKIP was performed for 131 cases of EBD carcinoma. The associations of RKIP expression with clinicopathologic parameters and patient outcomes were examined. Multivariate logistic regression analysis was used to identify independent predictive parameters for lymphovascular invasion and nodal and distant metastases. RESULTS: Loss of RKIP expression was observed in 55.0% (72/131) of cases. EBD carcinoma had significantly lower RKIP immunoreactivity than normal EBD (p < 0.001). Loss of RKIP expression was significantly associated with lymphatic invasion (p = 0.030) and nodal metastasis (p = 0.036), but it was not found to be a significant prognostic predictor for overall, disease-free or distant metastasis-free survival. In addition, loss of RKIP expression was an independent predictor for lymphatic invasion (p = 0.027). CONCLUSIONS: These results suggest that RKIP may play a role in the suppression of lymphatic invasion and nodal metastasis in EBD carcinoma.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.

Differential Physiological Effects of Raf-1 Kinase Pathways Linked to Protein Kinase C Activation Depending on the Stimulus in v-H-ras-transformed Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the molecular mechanism by which the Raf-1 kinase pathways that are linked to protein kinase C induce differential physiological effects, depending on the stimulus, by employing the pharmacological PKC activator PMA. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems and these cells were transiently transfected with the pMTH vector that encodes dominant-negative (DN) PKC-epsilon with using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. The Raf-1 kinase activity was measured by assessing the phosphorylation of recombinant MEK with using the immunoprecipitated Raf-1 proteins. The phosphorylated MEK protein bands were quantified by using Quantity One analysis software. RESULTS: The pharmacological PKC activator phorbol-12-myristate-13-acetate (PMA) and platelet-derived growth factor (PDGF) were able to induce the activation of Raf-1 kinase in the v-H-ras-transformed NIH3T3 fibroblasts. However, PMA was found to be much less sensitive PI3 kinase inhibitor or the chemical antioxidant than is PDGF. Especially, PMA mediated growth arrest while PDGF induced mitogenic signaling through the PKC-epsilon activation. Thus, the regulation of the Raf-1 cascade by both PDGF and PMA is likely to be intimately linked and they converge at the PKC level through different upstream pathways, as was shown by the inhibition of PDGF-induced Raf-1 kinase activation by the transient transfection with a dominant-negative mutant of PKC-epsilon. CONCLUSIONS: Taken together, these results imply that, depending on the stimulus, Raf-1 kinase leads to different physiological effects.

Effect of inhibiting c-raf-1 on radiosensitivity in human cervical carcinoma cell line HeLa / 重庆医科大学学报

Objective:To explore the effect of inhibiting c-raf-1 by antisense technology on radiosensitivity in human cervical carcinoma cell line HeLa.Methods:There were four groups in the study:control group,lipofectin group,sense oligodexynucleotides(S-ODN) group,antisense oligodexynucleotides(AS-ODN) group.The expression of c-raf-1 was detected by means of RT-PCR,Cellular response to irradiation was evaluated by the colony forming test,Apoptosis was observed by fluorescent staining.Bcl-2 protein expression was determined by flow cytometry.Results:AS-ODN group could significantly decreased the proliferation rate and increasing the apoptosis rate,significantly downregulating the expression of Bcl-2 of irradiated human cervical carcinoma cells,(vs lipofectin group or S-ODN group,P

Expression and Mutation Analysis of RKIP (Raf-1 Kinase Inhibitor Protein) in Human Gastric Cancer

PURPOSE: RKIP (Raf kinase inhibitor protein) is a novel candidate tumor suppressor, known to inhibit the MAPK signaling by interfering with the MEK phosphorylation by Raf-1. The aim of this study was to investigate the expression of RKIP and analyze the pattern of inactivation and mutation of the RKIP gene in human gastric cancer. METHODS: To explore if RKIP inactivation is implicated in gastric tumorigenesis, an expression analysis on the transcription and protein expression levels and a mutational analysis of RKIP were performed in 15 human gastric cancer cell lines and 92 primary carcinoma tissues. RESULTS: Abnormal reduction of the level of RKIP expression was frequently detected in the cancer cell lines and primary tumor tissues, at both the transcript and protein levels. Moreover, the expression level of RKIP in the tumor cells was inversely correlated with the level of Erk phosphorylation, indicating that RKIP plays a key role in the regulation of the Raf-MEK-Erk signaling pathway in human gastric cells. While the expression of the RKIP transcript was not re-activated in low expressor cells by treatment with the demethylating agent 5'Aza-dC, the genomic RKIP was detected at low levels in many cancer cell lines, suggesting that an abnormal reduction of level of RKIP expression in tumors might be caused by allelic deletion of the gene rather than transcriptional silencing due to aberrant DNA hypermethylation. A loss of heterozygosity study, using an intragenic polymorphic marker, revealed that approximately 21% of the gastric cancers harbored allelic loss of the RKIP gene. CONCLUSION: Collectively, this study has demonstrated that RKIP is a tumor suppressor, whose expression is frequently downregulated by allelic deletion in human gastric cancers. This study also suggests that an altered expression of RKIP might contribute to the development of gastric cancer via abnormal elevation of the Raf-Erk signaling pathway.

Expression of raf-1 in HepG2 strain stably transfected by HBx gene / 肿瘤研究与临床

Objective To investigate the effect on cell cycle of hepatitis B virus x protein and ap- proach the molecular mechanism of x protein's carcinogenesis. Methods Gene transfection mediated by the lipofectamine was used to introduce the eukaryotic expression vector of HBV x gene into human hepato- cellular carcinoma cell line HepG2(HepG2X cell).The selective medium containing G418 was used to select the cell clones which the X protein was expressed constantly.Flow eytometry was used to detect the cycle of the cells;raf-1 protein was detected by Western blot.Results X protein could be expressed on 21?10~3 loca- tion in the transfected cells,while there were no protein expressed in the control cells.The proliferation of X cells increased significantly according to MTS method(P

Expression of Raf-1 in colon carcinoma tissue and its clinical significance / 中国肿瘤生物治疗杂志

Objective: To investigate the relationship between the expression of Raf-1 and tumor angiogenesis,and to discuss its clinical significance.Methods: Tissue microarray technique was used to detect the expression of Raf-1 in 87 specimens of human colon carcinoma,their corresponding adjacent tissues,and incision margins.The patients were from the Department of Pathology of Xijing Hospital between 2005 and 2006.Microvessel density(MVD) was detected using immunohistochemistry with CD34 labeling.The correlation between Raf-1 expression and MVD with the tumor size,metastasis,and differentiation was analyzed.Results: The positive rates of Raf-1 in colon carcinoma tissues,adjacent tissues and incision margins were 86.47%,37.34% and 11.03%,respectively;there were significant difference among the 3 values(P

Mechanism of protein kinase C regulating CD44 gene expression in vascular endothelial cells / 中国免疫学杂志

Objective:To study the mechanism of protein kinase C regulating CD44 gene expression in vascular endothelial cells.Methods:Human umbilical vein endothelial cells (HUVECs)were taken as study model.The extract of Raf-1 kinase by immunoprecipitation was used for the Western blot analysis,and its activity was determined by enhanced chemiluminescene assay.CD44 gene expression was detected with RT-PCR,and phosphorylation was measurated by autoradiography.Results:CD44 phosphorylation in HUVECs was enhanced by 10ng/ml PMA treatment as compared with untreated cells, which reached the highest level at 30 minutes. Raf-1 kinase activity increased significantly after exposure to 10ng /ml PMA, and 0.05 ?mol/L Calphostinc could inhibit the role of Protein kinase C(PKC). CD44 gene expression level increased obviously after exposure to 10 ng /ml PMA (PKC activator) for only 1 minute(P

Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.

Inhibitory effects of c-raf-1 antisense oligodeoxynucleotides transfactionon proliferation of the human ovarian carcinoma SKOV_3 cell lines / 重庆医科大学学报

Objective: To investigate the effects and the mechanisms of c-raf-1 genes antisense oligodeoxynucleotides(ASODN) transfection in inhibiting the human ovarian carcinoma SKOV3 cell lines.Methods: There were 3 groups in our study: normal control group,c-raf-1 sense oligodeoxynucleotides(SODN) experimental group,and c-raf-1 antisense experimental group.at the different time points after liposome-mediated transfection,the cell proliferation,apoptosis,protein expressing level were observed by MTT assay,flow cytometry,fluorescent microscope and cloning test.Results: In the ASODN experimental group and SODN group,the OD-value were 0.272 and 1.307 respectively(P