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.

The level of oncogene H-Ras correlates with tumorigenicity and malignancy

Normal bovine adrenocortical cells and some human fibroblasts can be transformed by SV40T and H-Ras in a Ras-dependent manner. We recently reported that high levels of Ras derived from 5' LTR of retrovirrus can induce highly malignant and fast growing tumors, while lower levels of Ras derived from internal ribosome entry site(IRES) promotes slower tumor growth and loss of malignancy. Ras derived from CMV promoters resulted in much lower Ras levels and loss of tumor malignancy and growth. Further studies showed that the tumors formed in the presence of lower levels of Ras and dominant negative P53(P53DD) had fewer apoptotic cells and grew faster than the tumors formed from cells with same level Ras and SV40T. Our studies suggest that low levels of Ras are insufficient to inhibit apoptosis induced by pRb inactivation. In contrast, high levels of Ras not only allow normal cells to exit senescence and form tumors, but also protect against pRb inhibition-induced cell apoptosis.

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.

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.

Vascular endothelial growth factor,p53,and the H-ras oncogene in Egyptian patients with bladder cancer

AIM:To evaluate the relationship between vascular endothelial growth factor(VEGF),p53,and the H-ras oncogene and different clinicopathological parameters in Egyptian patients with Schistosoma-associated transitional cell carcinoma of the bladder.METHODS:The study included 50 patients with transitional cell carcinoma for whom radical cystectomy and urinary diversions were carried out.VEGF and p53 protein expressions were evaluated with an immunohistochemical staining method,and H-ras oncogene mutations were analyzed with a polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique.RESULTS:High grade tumors revealed higher p53 immunostaining than low grade tumors(P = 0.016).p53 and VEGF protein expressions,as well as H-ras oncogene mutations,had an insignificant impact on patient outcomes(P = 0.962,P = 0.791,and P = 967,respectively).Cancer extension to regional lymph nodes was associated with poor outcomes(P = 0.008).CONCLUSION:VEGF,p53 and the H-ras oncogene have no relation to patient survival and outcome in Schistosoma-associated transitional cell carcinoma.

Preliminary Study on c-Ha-ras Oncogene Mutations in Hydatidiform Mole Tissues

ve To study the presence of c-Ha-ras oncogene mutations in hydatidiform mole (HM) tissues and to further explore its relationship with mole's malignancy

1889例结直肠癌MSI、K-ras、N-ras和B-raf基因状态以及临床病理特征分析

目的通过对1889例结直肠癌微卫星不稳定(MSI)、K-ras、N-ras和B-raf基因检测,分析其基因状态与临床病理特征之间的关联性。方法收集东部战区总医院病理科结直肠癌患者手术切除大标本共计1889例,通过PCR荧光法联合毛细管电泳法对1889例结直肠癌进行MSI、K-ras、N-ras和B-raf检测,分析其基因状态与临床病理特征之间的关系,以及MSI与K-ras、N-ras和B-raf之间的关联性分析,并且对MSI自身位点的相关性分析。结果1889例结直肠癌患者中,微卫星高度不稳定(MSI-H)更易发生于右侧结肠(16.4%)、黏液腺癌(14.9%)、分化程度较低(18.7%)、年龄偏低(8.3%)、淋巴结未转移(9.1%)以及B-raf突变(5.2%)的患者中,K-ras突变更易发生于右侧结肠(50.7%)、黏液腺癌(50.7%)、分化程度较高(52.1%)、微卫星低度不稳定或稳定型(42.2%)的患者中,而B-raf突变更易发生在MSI-H(6.9%)的患者中,而N-ras基因则与临床病特征关联性不强。结论结直肠癌患者的MSI、K-ras和B-raf基因状态与临床病理特征存在着高度的关联性,因此通过分析其基因状态与临床病理特征之间的关系可以为临床靶向治疗以及预后提供更加可靠的依据。

Targeting the“undruggable”cancer driver genes:Ras,myc,and tp53

The term“undruggable”is to describe molecules that are not targetable or at least hard to target pharmacologically.Unfortunately,some targets with potent oncogenic activity fall into this category,and currently little is known about how to solve this problem,which largely hampered drug research on human cancers.Ras,as one of the most common oncogenes,was previously considered“undruggable”,but in recent years,a few small molecules like Sotorasib(AMG-510)have emerged and proved their targeted anti-cancer effects.Further,myc,as one of the most studied oncogenes,and tp53,being the most common tumor suppressor genes,are both considered“undruggable”.Many attempts have been made to target these“undruggable”targets,but little progress has been made yet.This article summarizes the current progress of direct and indirect targeting approaches for ras,myc,two oncogenes,and tp53,a tumor suppressor gene.These are potential therapeutic targets but are considered“undruggable”.We conclude with some emerging research approaches like proteolysis targeting chimeras(PROTACs),cancer vaccines,and artificial intelligence(AI)-based drug discovery,which might provide new cues for cancer intervention.Therefore,this review sets out to clarify the current status of targeted anti-cancer drug research,and the insights gained from this review may be of assistance to learn from experience and find new ideas in developing new chemicals that directly target such“undruggable”molecules.

三甲胺-N-氧化物协同RAS诱导动脉粥样硬化发展机制研究

目的 探究三甲胺-N-氧化物(TMAO)与肾素-血管紧张素系统(RAS)在人脐静脉内皮细胞(HUVECs)炎症中的关系及作用机制。方法 采用分子克隆技术,构建血管紧张素Ⅱ(AngⅡ)过表达和敲减的HUVECs;TMAO干预上述HUVECs后,检测细胞中炎症因子及细胞增殖、迁移、血管形成能力,并分析细胞中RAS重要组分的表达水平。结果 成功构建了AngⅡ过表达和敲减的HUVECs;AngⅡ过表达促进TMAO调节HUVECs炎症,抑制HUVECs增殖,抑制HUVECs迁移能力,抑制HUVECs血管形成,促进细胞中RAS组分AGT、ACE、AngⅡ、ATR的表达;敲减AngⅡ抑制TMAO调节HUVECs炎症,促进HUVECs增殖,促进HUVECs迁移能力,促进HUVECs细胞的血管形成,抑制细胞中RAS组分AGT、ACE、AngⅡ、ATR的表达。结论 TMAO能够协同RAS诱导HUVECs炎症。

树舌多糖GF对HepA瘤细胞N-ras基因表达的影响

目的探明树舌多糖GF对HepA瘤组织N-ras基因表达的影响。方法运用原位杂交法、SP免疫组化染色技术及多媒体彩色病理图文分析系统测定HepA瘤细包中N-rasmRNA量及N-Ras蛋白的表达量。结果树舌多糖组、猪苓多糖组中N-rasmRNA量及Ras蛋白的表达量均显著低于荷瘤组(P<0.01),树舌多糖组与猪苓多糖组无显著性差异。结论初步认为树舌多糖GF可通过降低N-rasmRNA量及Ras蛋白的表达,而抑制HepA瘤细胞的增殖。

重铬酸钾致N-ras基因DNA损伤作用研究

目的 研究重铬酸钾对N -ras基因的DNA损伤作用,探讨其致癌作用分子机制。方法 制备N -ras基因外显子1单链探针;重铬酸钾染毒细胞提取基因组DNA ,再经RDPCR扩增后与探针杂交显色。结果 重铬酸钾染毒剂量10 0 μmol L可检测到DNA损伤片段杂交条带,其损伤片段长于完整的N ras基因外显子1;更高剂量(10 0 0 μmol L)未能检测出DNA损伤作用。结论 重铬酸钾可能造成N- ras基因外显子1上游序列DNA损伤,且该损伤作用可能正是其致癌作用分子机制的关键所在。

大鼠肝癌湿热证、脾虚证与野生型p53 mRNA、N-ras表达相关性研究

目的探讨实验性肝癌湿热证、脾虚证与野生型p53 mRNA、N-ras表达的相关性,揭示中医证型分子水平的客观内涵。方法通过建立实验性大鼠肝癌湿热证和脾虚证模型,并设正常对照组,采用原位杂交和免疫组织化学法分别检测肝癌组织野生型p53 mRNA及N-ras蛋白表达。结果肝癌脾虚证组野生型p53 mRNA阳性表达率显著低于正常对照组(P<0.05),肝癌湿热证组与正常对照组比较,及肝癌脾虚证组与肝癌湿热证组比较差异无显著意义(P>0.05)。肝癌脾虚证组和肝癌湿热证组N-ras蛋白阳性表达水平比正常对照组均显著升高(P<0.05,P<0.01),肝癌脾虚证组与肝癌湿热证组比较差异无显著意义(P>0.05)。结论野生型p53 mRNA、N-ras异常表达与肝癌湿热证、脾虚证具有相关性,野生型p53 mRNA表达率显著降低是肝癌脾虚证的特征之一。

树舌多糖GF对HepA瘤细胞N-ras、C-myc基因表达的影响

目的为探明树舌多糖GF对HepA瘤组织N-ras、C-myc基因表达的影响。方法运用原位杂交法、SP免疫组化染色技术及多媒体彩色病理图文分析系统测定HepA瘤细胞中N-ras、C-myc mRNA及蛋白的表达量。结果树舌多糖组、猪苓多糖组中N-ras、C-myc mRNA及蛋白的表达量均显著低于荷瘤组(P<0.01),树舌多糖组与猪苓多糖组无显著性差异。结论树舌多糖GF可通过降低N-ras、C-myc mRNA及蛋白的表达,而抑制HepA瘤细胞的增殖。

益气养阴方及其拆方影响急性髓细胞白血病细胞Flt3和N-ras表达研究

目的研究益气养阴方及其拆方后的扶正方、祛邪方对Flt3和N-ras在人急性髓细胞白血病(acute myeloid leukemia,AML)表达的影响,探讨益气养阴方治疗白血病的作用机制。方法收集60例AML患者骨髓单个核细胞,将所提取每例患者的细胞混悬液分成4组:对照组不加药物,实验组分别加入益气养阴方、扶正方、祛邪方中药制剂。采用逆转录-聚合酶链反应(RT-PCR),免疫印迹法(Western bloting)观察益气养阴组、扶正组、祛邪组及对照组对Flt3、N-ras基因及FLT3蛋白表达的影响。结果 RT-PCR法检测:对照组、益气养阴组、扶正组、祛邪组Flt3基因表达率分别为(90.78±6.92)%、(38.18±4.50)%、(65.57±5.55)%、(61.35±6.39)%,N-ras基因表达率分别为(93.28±5.54)%、(34.38±6.69)%、(59.42±7.35)%、(65.28±7.64)%,益气养阴组、扶正组、祛邪组与对照组比较,差异均有统计学意义(P<0.05)。Westernbloting检测:对照组、益气养阴组、扶正组、祛邪组FLT3蛋白灰度值分别为0.8127±0.0284、0.4265±0.0353、0.5396±0.0274、0.5473±0.0282,对照组与益气养阴组、扶正组、祛邪组比较差异有统计学意义(P<0.01)。结论中药益气养阴方可抑制AML细胞的克隆性增殖,可降低Flt3和N-ras在AML细胞的表达水平,对AML治疗具有作用。

p53、p21^(ras)、nm23-H1基因在子宫内膜癌中表达与临床病理关系研究

目的 研究p53、p2 1ras、nm2 3 H1基因在子宫内膜癌中的表达与临床病理学关系。方法 应用免疫组织化学方法 (SABC法 )研究 18例正常增生期子宫内膜、12例子宫内膜非典型性增生过长、78例子宫内膜癌的p53、p2 1ras、nm2 3 H1基因的表达情况。结果 12例增生期子宫内膜中p53和p2 1ras基因表达阴性 ,3例子宫内膜非典型性增生过长中p53阳性 ;p53、p2 1ras、nm2 3基因在子宫内膜癌中表达率分别为 57 69% ,55 12 % ,71 79% ;p53的表达在不同的临床分期 ,病理学分级 ,有无淋巴结转移和是否绝经之间差异有显著性意义 (P <0 0 1) ;p2 1ras基因表达与病理学分级及有无淋巴结转移有关 (P <0 0 5) ,但与临床分期无关 ;nm2 3 H1的表达与病理学分级 ,临床分期 ,有无淋巴结转移有关。结论 p53、p2 1ras、nm2 3基因在子宫内膜癌中均高表达 ,p53的表达和肿瘤发生 ,发展有关 ,p2 1ras基因表达与肿瘤的进展有关 ,nm2 3的表达与淋巴结转移呈负相关。

正常子宫内膜、子宫内膜增殖症及子宫内膜癌ras、HER-2/neu癌基因表达的研究

为研究ｒａｓ、ＨＥＲ－２／ｎｅｕ癌基因的表达与子宫内膜癌发生、发展的关系，本文用免疫组化ＡＢＣ法检测了２３例正常子宫内膜、４４例子宫内膜增殖症及１０３例子宫内膜癌组织中ｒａｓ、ＨＥＲ－２／ｎｅｕ癌基因的表达情况。结果表明：在子宫内膜非典型增生中即存在ｒａｓ基因的过度表达，过度表达率为２０％，子宫内膜癌ｒａｓ的过度表达率为１８．４％，且ｒａｓ的过度表达与子宫内膜癌的病理分级、分期及患者的生存率无关，说明ｒａｓ癌基因的异常表达可能与癌形成的早期有关。子宫内膜癌中ＨＥＲ－２／ｎｅｕ的过度表达率为２７．２％，ＨＥＲ－２／ｎｅｕ的过度表达与子宫内膜癌的分期无关，与分级及患者的预后有关，存在ＨＥＲ－２／ｎｅｕ过度表达者的生存率低于无过度表达者，前者的５年生存率为５４．８％，后者为７９．６％，结果提示ＨＥＲ－２／ｎｅｕ的过度表达是判断子宫内膜癌预后的生物学指标。

人肝癌细胞N-ras基因RNA干涉有效靶点的确定

目的寻找人肝癌细胞N-ras基因的RNA干涉有效靶点。方法利用计算机网络资源及Oligo6.0、BlastResearch等生物学软件在N-ras基因编码区寻找RNAi有效靶点,并设计用于体内转录形成以U6启动子的siRNA发夹结构的DNA。结果成功筛选出RNA干涉的有效序列14个,并设计出siRNA发夹结构的DNA。结论这种对人肝癌细胞N-ras基因的RNAi有效靶点的筛选为进一步研究奠定了理论基础。其工作的开展将在RNAi治疗、肝癌N-ras基因功能研究、新药开发等方面发挥重要作用。

洛伐他汀对NB4细胞ras基因p21^(Ras)膜结合作用影响的体外试验

目的 :探讨洛伐他汀 (LOV)对人白血病NB4细胞的作用及其机制。方法 :以MTT比色法首先观察LOV对NB4细胞增殖的影响 ;利用逆转录 -聚合酶链反应半定量测定LOV作用于NB4细胞不同时间H -ras、K -ras、N -ras癌基因mRNA表达水平 ;同时采用流式细胞术测定NB4细胞p2 1Ras总蛋白、膜蛋白的表达。结果 :①LOV抑制NB4细胞增殖 ,IC5 0为 12 5 9μmol/L。②NB4细胞H、K、N -ras基因表达均为阳性。③LOV处理不同时间的NB4细胞H、K、N -ras基因转录水平无明显变化 ;p2 1Ras总蛋白水平也无变化 ,而细胞膜表面p2 1Ras蛋白水平随时间进行性下降。结论 :LOV抑制NB4细胞增殖。LOV靶向HMG -CoA还原酶 ,抑制p2 1Ras蛋白异戊二烯化、阻滞p2 1Ras蛋白与细胞膜结合 ;不影响ras癌基因以及p2 1Ras总蛋白的表达。

DDPH 对实验性心肌肥厚大鼠左心室 N-ras mRNA 及蛋白质表达的影响

目的：研究１－（２，６－二甲基苯氧基）－２－（３，４－二甲氧基苯乙氨基）－丙烷盐酸盐（ＤＤＰＨ）对心肌肥厚大鼠左室Ｎ－ｒａｓｍＲＮＡ及蛋白质表达的影响。方法：用部分狭窄腹主动脉方法造成大鼠心肌肥厚模型，从术后第４周开始，ｉｇ不同剂量的ＤＤＰＨ（２５和５０ｍｇ·ｋｇ－１·ｄ－１），持续８ｗｋ。用ＲＮＡ狭缝杂交及免疫组化的方法检测Ｎ－ｒａｓｍＲ－ＮＡ及蛋白质表达的变化。结果：术后１２ｗｋ，发现肥厚组心肌组织Ｎ－ｒａｓｍＲＮＡ表达是对照组的３．１倍，蛋白质表达是对照组的２．９倍，而二个给药组ｍＲＮＡ及蛋白质的表达明显低于肥厚组，但仍高于对照组。结论：ＤＤＰＨ对大鼠腹主动脉部分狭窄所致心肌肥厚时Ｎ－ｒａｓｍＲＮＡ及蛋白质表达的增加有一定的逆转作用。

PCR-SSCP-DNA序列分析法检测人肺癌中K-ras基因突变

应用聚合酶链式反应（ＰＣＲ）－单链构象多态性（ＳＳＣＰ）－ＤＮＡ序列分析法，对４０例肺癌样品中Ｋ－ｒａｓ基因突变进行检测。ＰＣＲ－ＳＳＣＰ银染分析，突变检出率为３０％（１２／４０），发现于肺腺癌的Ｋ－ｒａｓ基因突变率为４４％（１２／２７）；ＤＮＡ序列分析，１２例筛选突变阳性样品中的１１例检出了突变位点，９０％的突变发生于１２密码子，其突变谱以Ｇ→Ｔ颠换（占４０％）和Ｇ→Ａ转换（占６０％）为主。本研究结果显示ＳＳＣＰ银染分析对大量样品进行阳性筛选有很好的应用价值，ＰＣＲ－ＳＳＣＰ－ＤＮＡ序列分析法是检测基因突变的有效手段。