Tanshinone ⅡA improves Alzheimer’s disease via RNA nuclearenriched abundant transcript 1/microRNA-291a-3p/member RAS oncogene family Rab22a axis

BACKGROUND Alzheimer’s disease(AD)is a neurodegenerative condition characterized by oxidative stress and neuroinflammation.Tanshinone ⅡA(Tan-ⅡA),a bioactive compound isolated from Salvia miltiorrhiza plants,has shown potential neuroprotective effects;however,the mechanisms underlying such a function remain unclear.AIM To investigate potential Tan-ⅡA neuroprotective effects in AD and to elucidate their underlying mechanisms.METHODS Hematoxylin and eosin staining was utilized to analyze structural brain tissue morphology.To assess changes in oxidative stress and neuroinflammation,we performed enzyme-linked immunosorbent assay and western blotting.Additionally,the effect of Tan-ⅡA on AD cell models was evaluated in vitro using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Genetic changes related to the long non-coding RNA(lncRNA)nuclear-enriched abundant transcript 1(NEAT1)/microRNA(miRNA,miR)-291a-3p/member RAS oncogene family Rab22a axis were assessed through reverse transcription quantitative polymerase chain reaction.RESULTS In vivo,Tan-ⅡA treatment improved neuronal morphology and attenuated oxidative stress and neuroinflammation in the brain tissue of AD mice.In vitro experiments showed that Tan-ⅡA dose-dependently ameliorated the amyloid-beta 1-42-induced reduction of neural stem cell viability,apoptosis,oxidative stress,and neuroinflammation.In this process,the lncRNA NEAT1-a potential therapeutic target-is highly expressed in AD mice and downregulated via Tan-ⅡA treatment.Mechanistically,NEAT1 promotes the transcription and translation of Rab22a via miR-291a-3p,which activates nuclear factor kappa-B(NF-κB)signaling,leading to activation of the pro-apoptotic B-cell lymphoma 2-associated X protein and inhibition of the anti-apoptotic B-cell lymphoma 2 protein,which exacerbates AD.Tan-ⅡA intervention effectively blocked this process by inhibiting the NEAT1/miR-291a-3p/Rab22a axis and NF-κB signaling.CONCLUSION This study demonstrates that Tan-ⅡA exerts neuroprotective effects in AD by modulating the NEAT1/miR-291a-3p/Rab22a/NF-κB signaling pathway,serving as a foundation for the development of innovative approaches for AD therapy.

C-Ha-ras Oncogene in Oral Leukoplakia Tissues

The amplification and the G-T mutation at codon 12 of the C-Ha-ras oncogene in oral leukoplakia tissues were analyzed by using polymerase chain reaction (PCR) and molecular biologic technique. The results showed that these tissues had no amplification of Ha-ras oncogene. Only one case harbored G-T mutation in 11 oral leukoplakias, but this mutation was absent in 10 normal oral mucosal tissues. The possible role and significance of C-Ha-ras oncogene in oral precancerous lesions were also discussed.

Reduction of mitomycin C resistance in human bladder cancer T24 cells by knocking-down ras oncogene

Aim:Mitomycin C(MMC)is a commonly used as intravesical treatment for superficial bladder cancer.However,its role in combination with ras inhibition has not been investigated.The aim of this study was to explore the role of ras in MMC-induced apoptosis in T24 bladder cancer cells and to determine the efficacy of combination therapy in vitro.Methods:We measured the effects of various doses of MMC on apoptosis induction as well as on ras,ERK and Ki-67 protein expression by T24 cell line using immunocytochemistry,flow cytometry and Western blotting.We also tested the effect of siRNA on ras employed singly or in combination with MMC.Results:T24 cells expressed high level of ras protein.MMC treatment increased the level of ras and ERK protein expression after 24 h,and decreased these levels after 72 h.Ras siRNA(100 nmol/L)caused massive apoptosis associated with a marked decrease in ras expression in T24 cells.When combined with low doses of MMC,ras siRNA(50 nmol/L)sensitized T24 cells to apoptosis and decreased their expression of ras.The effect of combined therapy was higher than that of either compound used alone.Expression levels of ERK,a downstream target of ras,declined following combination therapy.Conclusion:Ras siRNA in combination with low dose MMC is a possible treatment strategy for patients with ras-positive bladder tumors.

EFFECTS OF LIMONENE,SALVIA MILTIORRHIZA AND TURMERIC DERIVATIVES ON H-RAS ONCOGENE EXPRESSION AND GAP JUNCTION INTERCELLULAR COMMUNICATION IN HUMAN SOLID TUMOR CELL LINES

Objective: To study gap junction intercellular communication (GJIC), H ras oncogene expression and ras oncogene product (P 21 ras protein) expression in four human solid tumor cell lines, W1-38,CACO 2,A549 and PaCa, and the effects of four compounds, Salvia miltiorrhiza derivative (SMD), d Limonene, Turmeric derivative I (TD I) and Turmeric derivative II (TD II), on them. Methods: The abilities of the four solid tumor cell lines to transfer dye to adjacent cells were examined by the scrape loading/dye transfer technique, and the H ras oncogene expression by Northern blotting and P 21 ras protein expression by Western blotting. Results: The results showed the loss of intercellular coupling in PaCa cells, slight GJIC in A549 and CACO 2 cells, and a good GJIC in W1-38 cells. The four compounds could improve the GJIC of PaCa to different extents. The amount of total and membrane associated P 21 ras in PaCa cells were decreased after treatment with SMD, d Limonene and TD I (2.5 μg/ml) for 48 h. Concomitantly, the growth of PaCa cells decreased in soft agar and had enhanced GJIC. The relative potency was found to be:d Limonene>SMD >TD I=TD II. There was no significant effect of the four compounds on H ras oncogene expression. Conclusion: It was suggested that there was an excellent correlation between loss of Lucifer Yellow dye transfer and ras gene mutation rate in the four solid tumor cell lines (ras gene mutation rate inversely correlated with average cell number coupled, r=0.98) i.e., the high ras gene mutation was closely correlated with loss of GJIC in these malignant human tumor cells; The antitumor effect of the monoterpene d Limonene and the phenol compound, SMD, might be related to inhibition of P 21 ras membrane association and enhancement of GJIC, whilst that of the others may be by a different mechanism; The inhibition of P 21 ras membrane association was directly related to the enhancement of gap junction intercellular com munication.

Correlated Flow Cytometric Analysis of H－ras p21 and DNA Ploidy in Acute Myelogenous Leukemia

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment,DNA diploidy occurred in 18 cases including 13 p21 negative ones,and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

Study on the Association Between Helicobacter Pylori Infection and the Pathogenesis of Gastric Cancer by Using Molecuar Biological Techniques

Recent epidemiological observations have indicated that the role of Helicobacter pylori (HP) infection in the pathogenesis of gastric cancer is an important but unresolved issue. We used the polymerase chain reaction, a sensitive and specific assay, to detect the HP infection in gastric biopsy specimens and examined the corrclations between HP infection and point mutation at 12 codon of H-ras oncogeneHras oncogene is higher in the group with HP infection than in those without HP infection. Furthermore, there is strong evidence that HP infection is associated with the increased expression of ras p21 protein , which suggested that HP infection increased the risk of ras oncogene activation. It is also noted that a significant relationship between infection with HP and the increase of DNA content and s% phase cells, indicated that rapid turnover of cells resulting from infection injury increases the risk of DNA damage.

MicroRNA-145 suppresses uveal melanoma angiogenesis and growth by targeting neuroblastoma RAS viral oncogene homolog and vascular endothelial growth factor

Background:Uveal melanoma(UM)is the most common primary intraocular malignancy in adults.It has been demonstrated that microRNA-145(miR-145)is correlated with the progression of various cancers by regulating the expression of multiple target genes,especially a number of genes that regulate angiogenesis and proliferation.However,the underlying mechanisms of miR-145 in tumor angiogenesis of UM are still not well illustrated.Thus,we aimed to explore the potential target genes or pathways regulated by miR-145 in UM and the effect of miR-145 on invasion and angiogenesis.Methods:Totally,24 choroid samples were collected in our study,including 12 UM samples and 12 normal uveal tissues.The expression of neuroblastoma RAS viral oncogene homolog(N-RAS),phosphorylated protein kinase B(p-AKT),and vascular endothelial growth factor(VEGF)in UM tissues and normal uveal tissues was analyzed using Western blotting analysis.Lentivirus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145.Transwell and endothelial cell tube formation assay were used to measure the effects of miR-145 on the invasion and angiogenesis of UM in vitro.The downstream target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase assay.BALB/c nude mice models were established to investigate the mechanisms of miR-145 on tumor growth and angiogenesis in vivo.Group data comparisons were performed using analysis of Student’s t test.A two-tailed P<0.05 was considered as statistically significant.Results:The results of Western blotting analysis indicated that the expressions of N-RAS(1.10±0.35 vs.0.41±0.36,t=3.997,P=0.012),p-AKT(1.16±0.22 vs.0.57±0.03,t=7.05,P=0.001),and VEGF(0.97±0.32 vs.0.45±0.21,t=3.314,P=0.008)inUMtumor tissues were significantly higher than those in normal uveal tissue.Luciferase assay demonstrated N-RAS and VEGF as downstream targets of miR-145.Moreover,tube formation assay revealed that miR-145-transfected human microvascular endothelial cell line formed shorter tube length(36.10±1.51mm vs.42.91±0.94 mm,t=6.603,P=0.003)and less branch points(350.00±19.97 vs.406.67±17.62,t=3.685,P=0.021)as compared with controls.In addition,the numbers of invaded MUM-2B and OCM-1 cells with miR-145 overexpression were significantly lower than the controls(35.7±3.3 vs.279.1±4.9,t=273.75,P<0.001 and 69.5±4.4 vs.95.6±4.7,t=21.27,P<0.001,respectively).In vivo,xenografts expressing miR-145 had smaller sizes(miR-145 vs.miR-scr,717.41±502.62mm3 vs.1694.80±904.33mm3,t=2.314,P=0.045)and lower weights(miR-145 vs.miR-scr,0.74±0.46 g vs.1.65±0.85 g,t=2.295,P=0.045).Conclusion:Our results indicated that miR-145 is an important tumor suppressor and the inhibitory strategies against N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future.

SMOOTH MUSCLE TUMORS OF THE GASTROINTESTINAL TRACT EXPRESSION OF RAS P21 ONCOGENE PRODUCT AND THE ASSOCIATION WITH CLINICOPATHOLOGY

Quantitative analysis of ras oncogene product P21 was performed on paraffin blocks from 55 smooth musele tumors of the gastrointestinal tract by immunofluorescence and flow cytometry.No positive evidence for P21 was found in 5 cases of normal smooth muscle tissues.

Oncogenic Ras/PI3K/Her2 share a common pathway in promoting cancer metastasis via inhibiting expression of p53-related ΔNp63α

Subject Code:H16With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Xiao Zhixiong(肖智雄)from the College of Life Science,Sichuan University demonstrates thatΔNp63αis a common inhibitory target in oncogenic PI3K/Ras/Her2-induced