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

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。

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。

骨肉瘤N-ras、c-myc基因异常及其蛋白产物的表达

目的：探讨癌基因Ｎ－ｒａｓ、ｃ－ｍｙｃ及其蛋白表达与骨肉瘤的关系。方法：Ｓｏｕｔｈｅｒｎｂｌｏｔ和免疫组织化学（ＬＳＡＢ法）同步检测９例人骨肉瘤Ｎ－ｒａｓ、ｃ－ｍｙｃ癌基因和其ｐ２１ｒａｓ、ｃ－ｍｙｃ蛋白产物表达。结果：Ｎ－ｒａｓ基因有１例为缺失改变，检出率１１％（１／９），ｃ－ｍｙｃ基因有３例为扩增改变，检出率３３％（３／９）。ｐ２１ｒａｓ、ｃ－ｍｙｃ基因蛋白表达阳性率分别为７８％（７例）、８９％（８例）。ｃ－ｍｙｃ基因蛋白在骨肉瘤的表达强度为最高。在９例骨肉瘤中，两种原癌基因蛋白共同表达６例，占７０％。结论：癌基因蛋白过度表达在骨肉瘤是一个常见现象，并从癌基因和其蛋白水平上提示癌基因Ｎ－ｒａｓ、ｃ－ｍｙｃ和ｐ２１ｒａｓ。

滋养细胞肿瘤N-ras基因原位分子杂交研究

目的 :为探讨滋养细胞肿瘤发病原因。方法 :采用核酸原位分子杂交技术 ,用地高辛标记的N ras基因探针与 40例滋养细胞肿瘤患者的刮宫标本及子宫切除标本进行原位杂交。用图像分析仪分别测各组阳性信号的灰度值作定量分析。结果 :随着恶性程度的增加 ,N ras基因阳性信号的表达呈增高趋势 ,恶性葡萄胎和绒毛膜癌明显高于良性葡萄胎和正常绒毛 ,有极显著性差异 (P <0 .0 1 )。结论 :N ras基因与滋养细胞的增殖、分化、恶变有关 ,由于N ras基因的过度表达而致恶变。

癌基因p21^(pan-ras)和p62^(c-myc)在皮肤T细胞淋巴瘤中的表达及其意义

目的：研究癌基因在皮肤Ｔ细胞淋巴瘤中的作用。方法：采用免疫组织化学技术检测了３２例蕈样肉芽肿／Ｓéｚａｒｙ综合征（ＭＦ／ＳＳ）皮肤组织中癌基因ｒａｓ和ｍｙｃ蛋白产物ｐ２１和ｐ６２的表达。结果：３２例ＭＦ／ＳＳ中有１２例ｐ２１阳性，有１７例ｐ６２阳性，有１０例同时示ｐ２１和ｐ６２阳性。其中有皮肤外播散的进展期ＭＦ／ＳＳ比局限于皮肤的早期ＭＦ／ＳＳ增高明显。结论：ｐ２１ｐａｎｒａｓ和ｐ６２ｃｍｙｃ的表达，与ＭＦ／ＳＳ细胞增殖转化相关。ｒａｓ和ｍｙｃ癌基因可能在ＭＦ／ＳＳ的多阶段发病过程中起协同作用。

5-氟尿嘧啶及其与甲酰四氢叶酸合并用药对7402肝癌细胞株N-ras癌基因表达的影响

为了探讨5－氟尿嘧啶（5-Fu）和5-Fu合并甲酰四氢叶酸（FH4）用药对7402肝癌细胞N-ras癌基因表达的影响。采用了异硫氰酸胍法从加有5-FU和5-FU与FH4。合用的7402肝癌细胞中分别提取总RNA，应用缺口平移法，用[α-(32)P]-dATP，dCTP标记N-ras癌基因DNA探针，与各实验组提取的7402肝癌细胞RNA进行斑点杂交。结果显示：单独使用5-FU，杂交斑点明显变小。5-Fu与FH4合并使用，杂交斑点完全消失。表明5-Fu可以明显抑制7402肝癌细胞N-ras癌基因的表达。5-Fu与FH4联合使用，其抑制效果更为明显。实验提示N-rasRNA降低并不是细胞毒作用而是药物对细胞DNA合成抑制的结果。

多发性骨髓瘤患者中N-ras基因突变的检测及其意义

目的 了解多发性骨髓瘤 (multiplemyeloma ,MM)患者中N ras基因突变率及临床意义。方法 采用聚合酶链反应 单链构象多态性银染法检测MM患者的N ras基因突变 ,并结合临床分析。结果 14例MM患者中 ,4例在N ras第 6 1密码子 ,1例在第 12、13密码子处发生突变 ,突变率为 35 .7%。突变者中 4例已死亡 ,其中 2例检测突变时已处于疾病终末期。结论 N ras基因在MM中有较高的突变率 ,对其的检测有助于病程进展和预后的分析。

原位杂交法检测树舌多糖GF对HepA瘤细胞N-ras、C-myc基因mRNA表达的影响

目的:探明树舌多糖GF对HepA瘤细胞癌基因N-ras、C-myc mRNA水平的影响。方法:应用地高辛标记探针原位杂交技术,检测树舌多糖GF对HepA瘤细胞中癌基因N-ras、C-myc mRNA丰度的影响。结果:树舌多糖组、猪苓多糖组中mRNA表达量均显著低于模型组(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可通过调节N-ras、C-myc基因mRNA的表达,而抑制HepA瘤细胞的增殖。

寡脱氧核苷酸抑制N-ras癌基因表达及肝癌细胞增殖的实验研究

目的 了解三螺旋形成寡核苷酸（ＴＦＯ）、反义寡核苷酸（ＯＤＮａｓ）抑制肝癌细胞生长的作用。方法 以Ｎ－ｒａｓ第二转录起始位点及翻译起始区１－６密码为靶点，合成１２聚硫代磷酸ＴＦＯ（ＴＦＯ１２）、１９聚硫代磷酸ＯＤＮａｓ（ＯＤＮａｓ１９）、同时合成１９聚无关序列硫代磷酸寡核苷酸对照（ＯＤＮｃｏｎ）。在人肝癌Ｂｅｌ７４０２细胞观察了充代磷酸寡核苷酸对Ｎ－ｒａｓ表达ｐ２１蛋白的抑制及对细胞增殖的影响。

PCR-SSCP在恶性血液病N-ras癌基因点突变检测中的应用

目的：检测血液系统肿瘤中Ｎ－ｒａｓ癌基因点突变的活性。方法：采用ＰＣＲＳＳＣＰ技术分析了２８例恶性血液病Ｎｒａｓ基因的突变活性，包括：ＡＮＬＬ６例，ＡＬＬ１２例，ＨＤ２例，ＮＨＬ３例，ＭＤＳ５例。结果：２８例中ＰＣＲＳＳＣＰ检测与正常对照，发生阳性者４例（１４．６％），其中ＡＬＬ１例（８．３％）、ＡＮＬＬ１例（１６．６％）、ＭＤＳ２例（４０％）。４例中有２例临床缓解后持续阳性，１例为ＡＬＬ患者，于骨髓移植后１０４ｄ复发死亡。１例ＭＤＳ患者转化为急性白血病，另２例临床缓解后３个月检测转阴，其中１例为ＡＮＬＬ，１例为ＭＤＳ。结论：ＰＣＲＳＳＣＰ方法可作为一种癌基因点突变检测手段常规应用于临床，但值得注意的是，ＰＣＲＳＳＣＰ方法只能提供分析区域是否存在突变点。

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.

急性髓细胞白血病亚型N-ras癌基因突变分析

为全面了解Ｎ－ｒａｓ癌基因突变与急性髓细胞白血病（ＡＭＬ）亚型的关系，从新鲜骨髓和库存骨髓片提取ＤＮＡ，应用银染聚合酶链反应－单链构型多态性（ＰＣＲ－ＳＳＣＰ）技术对９８例ＡＭＬ患者的Ｎ－ｒａｓ癌基因第１和第２外显子的点突变进行了研究。结果显示：共有２９例检测到突变，其中第１和第２外显子突变分别为１８例和１４例，两外显子同时突变３例；突变率为２９．５９％。除Ｍ［７］未检测外，其它各亚型均发现突变；而以Ｍ４ａ为多见，Ｍ［３ａ］为少见。因此，Ｎ－ｒａｓ癌基因突变是ＡＭＬ常见的肿瘤标志。

胰腺癌组织中p21ras蛋白表达与微血管密度的关系及其临床意义

目的检测胰腺癌组织中p21ras蛋白的表达及微血管密度(MVD)计数,探讨两者的关系及其临床意义。方法选择病理证实为胰腺导管腺癌的48例患者的石蜡切块标本,应用EnVision显色系统免疫组织化学方法进行p21ras蛋白检测和MVD计数。结果p21ras蛋白表达率胰腺癌组织为60·41%,癌旁组织为37·5%(P<0·05)。MVD计数p21ras表达阳性组为22·207±5·815,p21ras阴性组为18·053±5·502(t=3·597,P<0·01)。结论胰腺癌组织中p21ras的表达与MVD计数相关,提示ras基因突变可能促进肿瘤微血管的形成。

结直肠癌APC、K-ras、p53基因突变检测

目的:探讨结直肠癌中APC、K-ras、p53基因突变模式。方法:应用酚/氯仿法提取48例结直肠癌组织及其相应正常黏膜组织的DNA,用聚合酶链反应(PCR)、单链构象多态性分析(SSCP)和DNA测序等方法检测APC基因第15外显子突变密集区(mutation cluster region,MCR)区段、K-ras和p53基因的突变。结果:APC、K-ras和p53基因的突变率分别为37.5%(18/48)、43.8%(21/48)和35.4%(17/48)。48例结直肠癌组织中,有42例发生APC、K-ras或p53基因突变,突变率高达87.5%(42/48),其中仅有APC、K-ras或p53 1种基因发生突变的发生率分别为16.7%(8/48)、25.0%(12/48)和20.8%(10/48)。单独1种基因发生突变的总发生率为62.5%(30/48)。APC和p53,APC和K-ras或p53和K-ras 2种基因均有突变的发生率分别为6.3%(3/48)、10.4%(5/48)和4.2%(2/48)。APC、K-ras和p53 3种基因均发生突变的发生率为4.2%(2/48)。2种和3种基因均发生突变的总发生率为25%(12/48)。结论:结直肠癌的发生、发展并不完全遵循由正常结直肠黏膜上皮细胞向腺瘤和侵袭性癌转化的过程中,及依次发生“APC→K-ras→p53→DCC”突变累积这一经典的结直肠癌发生发展模式,可能存在其他结直肠癌发病机制。