Musashi1基因调控结肠癌HCT116细胞恶性生物学行为的机制研究

背景与目的：Musashi1（Msi1）属于RNA结合蛋白家族中的一员,是RNA转录后表达的关键调控者,其是否参与肿瘤的发生、发展,以及具体分子机制仍不十分清楚。探讨沉默Msi1基因对结肠癌HCT116细胞恶性生物学行为的影响及可能的机制。方法：采用慢病毒载体构建稳定低表达Msi1的细胞株,细胞计数试剂盒（cell counting kit-8,CCK-8）实验检测细胞增殖能力,克隆形成实验检测细胞克隆形成能力,流式细胞术（flow cytometry,FCM）检测细胞周期的变化,裸鼠移植瘤模型观察沉默Msi1对裸鼠成瘤的影响。实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）和蛋白质印记法（Western blot）检测沉默Msi1基因后p27基因的mRNA表达和蛋白水平,双荧光素酶实验验证Msi1基因与目的基因p27 3’-非编码区（3’-untranslated region,3’-UTR）的相互作用。结果：沉默Msi1基因后,HCT116细胞的增殖能力显著下降,克隆集落数明显减少,G ₀ /G ₁ 期细胞增多,S期细胞明显减少,裸鼠移植瘤生长明显受到抑制。沉默Msi1基因后p27 mRNA表达未见明显变化,而p27蛋白水平明显上调,双荧光素酶实验证实Msi1基因能与p27 3’-UTR区域直接结合,抑制其翻译。结论：沉默Msi1基因通过靶向上调p27,导致结肠癌HCT116细胞G ₀ /G ₁ 期阻滞,从而抑制肿瘤细胞体内及体外增殖能力。     

Musashi 1在结肠癌中的表达及其与微血管密度及预后的关系

目的:探讨Musashi 1在结肠癌中的表达与肿瘤微血管密度(MVD)及预后的关系。方法:应用实时荧光定量PCR的方法检测36对结肠癌组织及其配对的正常结肠组织中Musashi 1 mRNA的表达情况。通过免疫组化法对96例结肠癌组织中Musashi 1表达情况和微血管密度(MVD)进行检测。结合随访术后生存情况,统计分析Musashi 1表达与结肠癌病理参数、预后及MVD之间的相关性。结果:实时荧光定量PCR法结果显示,在结肠癌组织中,Musashi 1 mRNA的相对表达水平显著高于正常结肠组织(P0.05)。免疫组化结果显示,96例结肠癌组织中,有61例(63.5%)高表达Musashi 1。Musashi1的表达与患者性别、肿瘤大小、分化程度无相关性(P0.05),而与肿瘤的浸润深度、Dukes分期、淋巴转移、TNM分期密切相关(P0.05)。Kaplan-Meier生存分析显示,Musashi 1高表达患者的5年生存率显著低于Musashi 1低表达者(23.0%vs60.0%;P0.01),且Musashi 1的表达与结肠癌血管密度相关(P0.01)。结论:结肠癌中Musashi 1的表达较正常癌旁组织高,具有统计学意义。结肠癌中Musashi 1的表达与肿瘤浸润深度、Dukes分期、淋巴转移、TNM分期和血管密度密切相关。Musashi 1蛋白可通过促进瘤内血管增生,从而促进结肠癌的生长和侵袭转移,进一步影响结肠癌患者的预后。     

艾芬地尔对脑缺血再灌注大鼠Musashi1表达的影响

目的探讨艾芬地尔对脑缺血再灌注不同时相大鼠脑组织的保护作用及对Musashi1表达的影响。方法将84只Wistar大鼠随机分为3组,假手术组、脑缺血再灌注组（模型组）、艾芬地尔组（干预组）各28只,采用线拴栓塞法建立大脑中动脉闭塞模型,分别于再灌注2、3、7、14d各处死7只大鼠,其中1只用于透射电镜观察神经细胞超微结构变化,6只用于尼氏染色观察神经细胞组织学变化,免疫组化研究Musashi1的表达规律。结果 （1）与假手术组比较,模型组有以下表现,电镜：神经元胞体及细胞器肿胀明显,胞浆密度降低;尼氏染色：神经元数目减少,胞体肿胀,尼氏小体减少或消失;免疫组化：Musashi1阳性细胞在再灌注2d时即出现轻微增多,3d时表达急剧上升,持续至14d仍有较高表达;（2）与模型组比较,干预组表现特点如下,电镜：神经元胞体及细胞器轻度肿胀;尼氏染色：尼氏小体较多;免疫组化：Musashi1阳性细胞较相同时点模型组表达增多,光密度值增高,表达规律同模型组。结论艾芬地尔能够减轻脑缺血再灌注造成的细胞损伤,具有促进神经再生因子Musashi1表达的作用,对于改善脑缺血再灌注损伤具有积极作用。     

Musashi1 antigen expression in human fetal germinal matrix development

Musashi1 is a highly conserved protein found in neural progenitor cells. We examined the expression dynamics of Musashi1 in conjunction with other representative neural progenitor antigenic determinants (Ki-67 and nestin) during 8 different stages of the developing human fetal germinal matrix. Our results indicate that Musashi1 is a useful marker for immature cells in periventricular areas inhabited by stem cells, progenitor cells, and differentiating cells.     

Musashi1基因表达对人结肠癌HCT116细胞的放射增敏作用及其机制

目的 研究沉默Musashi1对人结肠癌HCT116细胞放射敏感性的影响,为放射治疗提供新的增敏靶点。方法 用慢病毒载体建立Musashil（Msi1）低表达的稳转细胞株及阴性对照细胞株,将细胞分为沉默组、空白对照组和阴性对照组,通过克隆实验、流式细胞技术检测细胞凋亡及细胞周期,证实其放射敏感性。结果 成功构建了沉默Musashi1的HCT116细胞。沉默组的D₀、D_q、N、SF₂分别为1.55、0.88、1.76 Gy和0.43,空白对照组分别为2.17、1.51、2.01 Gy和0.64,阴性对照组分别为1.99、1.45、2.07 Gy和0.62。沉默组相对于空白对照组的放射增敏比为1.40,相对于阴性对照组的放射增敏比为1.28。8 Gy照射后24、48、72 h,沉默组凋亡比例始终高于阴性对照组和空白对照组（F=65.16,P<0.05）,而阴性对照组和空白对照组之间差异无统计学意义（P>0.05）。12 Gy照射后48 h,沉默组细胞周期中G₂/M期比例较空白对照组和阴性对照组显著下降（F=65.398,P<0.05）。结论 降低Musashi1的表达对人结肠癌HCT116细胞有放射增敏作用,可能通过促进其凋亡,解除G₂/M期阻滞而发挥放射增敏作用,有望成为新的增敏靶点。     

RNA结合蛋白Musashi1在结直肠癌中的表达及临床意义

Musashi1(Msi1)是一种进化保守的RNA结合蛋白,同时它是肠道、神经等多种组织中的干细胞标志物,对于维持自我更新与分化之间的平衡具有重要作用.近年来研究发现Msi1在多种肿瘤组织尤其是结直肠癌中高表达,参与调控细胞周期、增殖、凋亡等过程,逐渐成为多种癌变的关键调节剂,有望成为肿瘤治疗的新靶点.     

MicroRNA‐143/Musashi‐2/KRAS cascade contributes positively to carcinogenesis in human bladder cancer

It has been well established that microRNA (miR)‐143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR‐143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR‐143 negatively regulated the RNA‐binding protein Musashi‐2 (MSI2) in BC cell lines. MSI2 is an RNA‐binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR‐143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.     

Enteroendocrine,Musashi 1 and neurogenin 3 cells in the large intestine of Thai and Norwegian patients with irritable bowel syndrome

Objectives: The prevalence, gender distribution and clinical presentation of IBS differ between Asian and Western countries. This study aimed at studying and comparing enteroendocrine, Musashi 1 (Msi 1) and neurogenin 3 (neurog 3) cells in Thai and Norwegian IBS patients.

Material and methods: Thirty Thai and 61 Norwegian IBS patients as well as 20 Thai and 24 Norwegian controls were included. Biopsy samples were taken from each of the sigmoid colon and the rectum during a standard colonoscopy. The samples were immunostained for serotonin, peptide YY, oxyntomodulin, pancreatic polypeptide, somatostatin, Msi 1 and neurog 3. The densities of immunoreactive cells were determined with computerized image analysis.

Results: The densities of several enteroendocrine cell types were altered in both the colon and rectum of both Thai and Norwegian IBS patients. Some of these changes were similar in Thai and Norwegian IBS patients, while others differed.

Conclusions: The findings of abnormal densities of the enteroendocrine cells in Thai patients support the notion that enteroendocrine cells are involved in the pathophysiology of IBS. The present observations highlight that IBS differs in Asian and Western countries, and show that the changes in large-intestine enteroendocrine cells in Thai and Norwegian IBS patients might be caused by different mechanisms.