MiR-23b在大鼠肝再生终止阶段的表达及作用机制研究
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摘要
大鼠70%肝切除(partial hepatectomy,PHx)后经1-2周时间基本恢复原来的体积和功能,这一过程被称为肝再生。肝再生进程大体分为三个阶段:启动阶段,增殖阶段,终止阶段。肝再生涉及的神奇现象一直受到人们的广泛关注:在启动阶段,肝细胞由平时的静转化为特殊时期的动,并有如此强大的再生能力;在增殖阶段,与其它一些组织、器官不同的是,肝脏的增殖并不只是由干细胞完成,而是由组成肝脏的多种细胞共同参与;在终止阶段,肝脏增殖的及时终止与肝脏肿瘤的无限增殖形成的鲜明对比。到目前为止,肝再生的相关研究主要集中在肝再生启动阶段和增值阶段,而肝再生终止阶段的分子机制仍不是特别清楚。MicroRNAs(miRNAs)是一类新发现的小RNAs,通过调控基因转录后水平的表达,参与生物生长和发育等许多生命过程的各个环节,包括器官发育、物质代谢、细胞分化、凋亡和癌变,是目前生命科学的研究热点和前沿领域。MiR-23b是一个参与调节多种信号通路的多功能miRNA,其作用涉及增殖、分化、凋亡、细胞粘附等各个方面,而且关于其功能及作用机制的研究仍在不断扩展。有趣的是,我们前期用芯片筛选肝再生后期差异表达的miRNA时发现miR-23b在肝再生终止阶段表达量明显降低。因此我们猜测miR-23b可能参与肝再生终止的调控,但目前miR-23b与肝脏的相关研究比较少,与肝再生相关的研究尚未见报道。鉴于上述分析,本课题利用经典的大鼠肝再生模型,研究miR-23b在大鼠肝再生终止阶段的作用并探讨其可能的分子机制,将有利于丰富对肝损伤和术后肝再生修复机制的认识和了解,并为更好的阐明肝再生终止的分子机理以及探寻肝脏肿瘤相关的治疗靶点提供实验依据。
第一部分MiR-23b在大鼠肝再生终止阶段的表达变化及功能探讨
MiR-23b参与多种信号通路的调节,其作用涉及增殖、分化、凋亡、细胞粘附等各个方面。最新关于miR-23b等研究证明了它还可以作为TGF-β1信号通路的辅助成分调节该通路的激活。而TGF-β1信号通路在肝再生终止阶段发挥重要的作用。我们前期用芯片筛选肝再生后期差异表达的miRNA时发现miR-23b在肝再生终止阶段表达量明显降低。综合以上因素我们猜测在肝再生后期miR-23b可能通过调节TGF-β1信号通路参与肝再生终止。本部分实验旨在验证miR-23b在肝再生终止阶段的表达变化并探讨其对肝再生终止的作用及可能的分子机制。
本部分研究包括动物实验和细胞实验两部分。通过动物实验,我们发现:在肝再生终止阶段,miR-23b的mRNA表达水平明显降低。结合实验中检测到肝再生终止阶段TGF-β1信号通路的持续激活状态(TGF-β1下游信号分子Smad3的mRNA和蛋白表达明显升高,磷酸化Smad3蛋白表达也呈现明显的升高趋势;细胞增殖活性明显降低,凋亡活性明显升高。),我们推测miR-23b可能通过激活TGF-β1信号通路参与调控肝再生终止。为了验证这一猜测,我们进一步作了体外的细胞试验来验证肝细胞内miR-23b对细胞生长及TGF-β1信号通路作用。在体外,通过培养正常大鼠肝细胞(BRL-3A),细胞转染miR-23b的模拟物(mimics)或抑制物(inhibitor)上调或下调miR-23b,进一步探讨miR-23b的功能,研究结果显示:①上调miR-23b促进肝细胞生长,下调miR-23b抑制肝细胞生长并抑制大鼠肝细胞细胞周期的进入,阻止细胞从G2期到M期转换;②Smad3是miR-23b的下游靶蛋白;③miR-23b通过转录抑制Smad3表达部分程度上抑制TGF-β1诱导的肝细胞凋亡;④TGF-β1是miR-23b的上有调节分子,转录抑制miR-23b的表达。
本部分实验我们发现在肝再生终止阶段miR-23b的表达量下调。TGF-β1是miR-23b的上游调节分子。在肝再生终止阶段,miR-23b的低表达降低了其对Smad3的转录抑制从而间接激活TGF-β1信号通路促进肝再生终止。
第二部分神经科粒素(neurogranin)在大鼠肝再生终止阶段的表达及功能探讨
第一部分实验中,我们用Target scan5.1软件预测miR-23b下游可能的靶基因时发现神经颗粒素是miR-23b下游潜在的靶基因之一。尽管经验证肝细胞内miR-23b的表达改变不能调节神经颗粒素的表达,但是我们前期用基因芯片筛选大鼠肝再生终止阶段差异表达的基因时发现神经颗粒素在肝再生终止阶段(部分肝切除后120h, 168h)明显升高。
神经颗粒素(neurogranin)是一种神经元特异性钙调蛋白结合蛋白,主要位于大脑等神经组织,近年来有研究发现其存在于胸腺及脾脏组织以及淋巴器官。神经颗粒素主要参与大脑等神经系统相关功能如学习、记忆、突出可塑性等的调节。最近有研究发现:神经颗粒素作为一种钙调蛋白结合蛋白,通过调节钙离子/钙调蛋白复合物的可用性调节细胞内钙离子浓度发挥促凋亡作用。
肝再生终止阶段的显著特征是肝细胞增殖活性降低,凋亡活性升高。作为一种促凋亡蛋白,神经颗粒素在肝再生终止阶段的表达上调与肝再生终止阶段凋亡活性的升高具有时效相关性。综合上述研究结果,我们猜测凋亡相关蛋白神经颗粒素可能参与调控肝再生终止。基于上述分析,本部分实验旨在研究神经颗粒素在大鼠肝再生终止阶段的表达变化并探讨及其在肝脏再生终止阶段的作用及分子机制。
本部分实验包括肝再生动物实验和离体细胞实验两个部分。通过动物实验,我们发现:①在肝再生终止阶段,神经颗粒素蛋白表达水平明显升高;②第一部分实验中检测到肝再生终止阶段细胞凋亡活性升高,线粒体凋亡通路处于激活状态;③已发现的对神经颗粒素的表达具有调节作用的分子有维甲酸、T3、NO、miR-23b等。但在肝细胞内对神经颗粒素具有调节作用的只有NO。NO具有一定的促凋亡作用,其调控的下游分子神经颗粒素也能发挥促凋亡效应。此外,在肝再生终止阶段NO浓度升高、神经颗粒素表达上调、线粒体凋亡通路的激活三者在时效上相关,因此我们猜测在肝再生终止阶段,NO浓度的升高很可能通过上调神经颗粒素表达参与线粒体凋亡通路的激活。为了验证这一推测,我们进一步通过离体细胞实验来说明神经颗粒素对肝细胞凋亡进程的调节作用。在体外,通过培养正常大鼠肝细胞(BRL-3A),细胞转染神经颗粒素干扰质粒下调神经颗粒素,进一步探讨神经颗粒素的功能。研究结果显示:①神经颗粒素干扰质粒能够在部分程度上抑制TGF-β1诱导的肝细胞凋亡进程。在这一过程中,肝细胞内钙离子浓度明显降低,线粒体膜电位明显降低;②在正常大鼠肝细胞内,外源性NO供体能够上调神经颗粒素的表达。
本部分实验我们发现在肝再生终止阶段神经颗粒素的表达量上调与miR-23b以及外源性甲状腺激素T3无关,NO是神经颗粒素的上有调节分子。NO通过诱导神经颗粒素表达上调,参与线粒体凋亡通路调控肝再生终止。
第三部分miR-23b在肝癌组织中的表达及其在早期肝癌术后预后中的作用研究
肝细胞癌(HCC)是人类最常见的致命性肿瘤之一,在全球肿瘤死亡原因中排列第五,中国大陆肝癌发病率占全球发病率的55%。根治性切除是HCC治疗的首选治疗方法,但是预后尚不理想,首要原因是肝内复发。寻找理想的肝癌术后预后价值指标,并据此选择合理的辅助治疗对改善预后有重要意义。
第一部分研究证明在肝再生终止阶段,miR-23b的低表达降低了其对Smad3的转录抑制从而间接激活TGF-β1信号通路促进肝再生终止。而TGF-β1信号通路在肝癌发生、发展中发挥着重要的作用,因此,我们猜测miR-23b可能在肝癌的发生、发展和预后等过程中发挥作用。已有研究表明miR-23b参与调节肝癌细胞增殖和侵袭等。因此,本部分实验结合临床病例和标本初步研究miR-23b在肝癌组织中的表达规律并初步探讨其对肝癌术后预后的影响,尤其是对早期肝癌预后的预测。
本部分实验包括两部分,①RT-PCR方法研究配对肝癌组织和癌旁组织中基因表达情况;②前瞻性观察2004.1-2004.6中连续入组的233例接受根治性切除患者癌组织中miR-23b的表达水平和肝癌术后预后的关系。RT-PCR结果发现miR-23b在肝癌组织中的表达比癌旁组织降低2倍左右。经调查分析发现miR-23b的表达水平与肿瘤患者血清AFP水平、肿瘤直径、肿瘤数目和TNM分期有关;多因素分析miR-23b是影响患者复发和生存的独立因素(hazard ratio HR,95% confidence interval, CI,复发:2.483,1.563-3.944,P<.001;生存:2.256,1.377-3.697,P<.001)。在BCLC A期患者中,miR-23b低表达患者复发率高于高表达患者,生存率则相反。
本部分实验我们发现肝癌组织中miR-23b的表达水平低于癌旁及正常组织。miR-23b的表达水平与肝癌患者术后预后有关,对早期肝癌患者预后有预测作用,miR-23b低表达患者术后可能会出现复发和转移,预后差。
总之,我们通过对大鼠肝再生终止阶段分子机制的一系列研究,揭示了miR-23b、神经颗粒素在肝再生终止阶段的可能作用,进一步完善了肝再生分子机制的研究。同时,我们通过对miR-23b表达水平与肝癌术后预后相关性研究证实miR-23b可以作为临床肝癌术后预后指标,不仅丰富了临床肝癌术后预后指标,同时为临床治疗肝癌提供了新的治疗靶点。
Following 70% partial hepatectomy (PHx), the remnant liver has a remarkable ability to regenerate to its original mass and function within 7-10 days after surgery in a process called liver regeneration (LR). LR after PHx is generally divided into three distinct phases: initiation, proliferation and termination. Despite multiple studies of LR, many aspects of this phenomenon remain unknown, including the proper temporal regulation of termination.
MicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown.
In a preliminary study using a miRNA microarray analysis, we found that miRNA 23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation.
In view of the above analysis, this study sought to elucidate if and how miR-23b was involved in the termination stage of LR, and this will provide basis to better illustrate the molecular mechanism of the termination liver regeneration after partial hepatectomy. The inquiry will also provide the basis for exploring therapeutic targets for liver cancer.
SectionⅠ: The expression of miR-23b and its role in the termination stage of liver regeneration after partial hepatectomy in rats
MicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown.
MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation. In a preliminary study using a miRNA microarray analysis, we found that miRNA23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. And a recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Considering the important role TGF-β1 plays during the termination stage of LR, the present work sought to elucidate if and how miR-23b was involved in the termination stage of LR. We also report evidences that miR-23b expression is remarkably diminished during the termination of LR, and miR-23b may contribute to the TGF-β1/Smad3 signalling pathway during the termination stage of LR.
In the present study we confirmed that miR-23b is down-regulated during the termination stage of LR. Our study also found that down-regulation of miR-23b inhibits cell proliferation, which may be attributed to G2/M arrest by flow cytometry investigation, as shown in figure 2. Therefore, down-regulation of miR-23b may contribute to the termination of LR, which is characterised by reduced proliferation and enhanced cellular apoptosis.
To further investigate the specific role of miR-23b during the termination stage of LR, it is very important to identify the target gene of miR-23b. A recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Thus, miR-23b plays a critical role in TGF-β1 /Smads signaling pathway. More importantly, it is widely known that TGF-β1 and activinA are natural terminators of LR. Therefore, we speculate miR-23b may take part in the termination stage of LR by targeting smads. Our study has confirmed that Smad3 but not Smad4 or Smad5 is a putative target gene of miR-23b in livers following PHx.
There is evidence that Smad3 plays a more significant role in the induction of TGF-β1-induced apoptosis than other Smads downstream of TGF-β1. Over-expression of Smad3 greatly potentiates the induction of apoptosis in response to TGF-β1, while expression of a dominant-negative mutant of Smad3 inhibits TGF-β1 induced apoptosis. Therefore, Smad3 is an important downstream molecular in TGF-β1 pathway. Interestingly, we have found that up-regulation of miR-23b could partially inhibit TGF-β1-induced apoptosis in BRL-3A cells by reducing the expression of Smad3. In addition, TGF-β1, Smad3, phosphorylated Smad3 and apoptotic activity increased and proliferative activity decreased when miR-23b was decreased from 72 to 168 h after PHx. Taken together, down-regulation of miR-23b may contribute to activation of the TGF-β1/Smad3 signalling pathway during the termination stage of LR.
In exploring the specific relationship between TGF-β1, miR-23b and Smad3, we considered TGF-β1 as a possible upstream regulator of miR-23b. Firstly, several reports have demonstrated that TGF-β1 can either positively or negatively regulate the expression of miRNAs including miR-192 and miR-24. More interestingly, even in the process of LR, miR-23b down-regulation occurs after 72h PHx, when TGF-β1 expression is at its highest levels. In addition, miR-23b can directly regulate the expression of Smad3 which is a downstream molecular in TGF-β1 signaling. Therefore, we presumed that miR-23b may act as a pitch point in the TGF-β1 signalling pathway. Then we confirmed that TGF-β1 can regulate the expression of miR-23b at the transcriptional level. Our present findings suggest at TGF-β1-miR-23b-smad3 signaling may act as a novel mechanism in the termination stage of LR.
In conclusion, miR-23b may contribute to TGF-β1/Smad3 signaling pathway during the termination stage of LR. Our findings reveal a miRNA-mediated regulation pattern during the termination stage of LR.
SectionⅡ: The expression of neurogranin and its role in the termination stage of liver regeneration after partial hepatectomy in rats
Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca2+/CaM complex and modulates the homeostasis of intracellular calcium in neurons. In recent years, studies have found that Ng exists in the thymus, spleen and other lymphatic organs.
In the first experimental part, we use Target scan5.1 to predict the potential target genes of miR - 23b have found that Ng is one of the potential target genes of miR-23b. but our study proved that miR - 23b is not the upstream regulator of neurogranin in the BRL-3A cells. Intereatingly, our prestudy using gene chip to indentify the differentially expressed gene during the termination stage (120,168h after partial hepatectomy) of liver regeneration after partial hepatectomy have found that the gene expression of Ng upregulated. In view of these above factors, we predicted that apoptosis related proteins Ng might be involved in the termination of liver regeneration after partial hepatectomy in rats. Based on the above analysis, this experimental part aimed at the investigation of the expression pattern of Ng during the termination stage of liver regeneration and the exploration of the related molecular mechanisms.
Our results showed that: the expression of Ng protein upregulated during the termination stage of liver regeneration which is characterized with a high apoptosis activity; the concentration of NO and T3 increased during the termination stage of liver regeneration; downregulation of Ng in BRL-3A cells will inhibits the growth of BRL-3A cells and partially inhibits TGF-β1 induced BRL-3A cell apoptosis through depressing the mitochondrial apoptosis pathway; exogenous NO donors SNP could upregulate the expression of Ng at the transcriptional level in BRL-3A cells, while there were no obvious change on the expression of Ng when BRL-3A cells were treated with miR-23b or T3.
This experimental part suggests that during the termination stage of liver regeneration, the upregulated expression of Ng attributed to the increased concentration of NO and contributed to the termination of liver regeneration by inducing mitochondrial apoptosis pathway.
SectionⅢ: The association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patients
Hepatocellular carcinoma (HCC) is the fifth most frequent human cancer worldwide, and, the second leading cause of cancer-related death in China. Major HCC risk factors include infection with hepatitis B (HBV) or C viruses (HCV) and cirrhosis associated with chronic inflammation. Hepatectomy is the first choice for HCC patients in China, but the outcome is poor due to high recurrence after surgery. Since most HCCs have dissemination within the liver before resection and are undetectable by imaging techniques, new and efficacious adjuvant therapy strategies are needed, along with new prognostic biomarkers for early detection of at-risk patients. Therefore, it is clinically meaningful that metastasis and prognosis related molecule and its functional mechanism were found. MicroRNAs (miRs) are small (21-25 nucleotide) nonprotein-coding RNAs that posttranscriptionally regulate gene expression. MiRNAs possess oncogenic or tumor suppressor activity in various tumors but little is known about miRNA expression pattern in hepatocellular carcinoma (HCC). The purpose of this part of study was to investigate the relationship between miR-23b expression and prognosis of HCC patients after curative resection.
We use quantitative reverse-transcriptase-polymerase-chain-reaction assays to investigate the expression of miR-23b in 115 patients with HCC who had undergone radical resection between 2004 and 2005. We assessed the association of miR-23b with tumor recurrence and patients’survival with HCC. The results showed that: Tumors had reduced levels of miR-23b expression, as compared with paired nontumorous tissues. In comparison with high miR-23b expression, low miR-23b expression in tumor tissue, which was correlated with encapsulation (P=.009) and TNM stage (P<.001) was associated with both tumor recurrence (median time to recurrence: 10.3±1.8 months vs 36.3±2.6 months, P<.001) and patients survival (1-, 3-, 5-year survival rates: 52%, 31%, 29% vs 84%, 63% 57%, P<.001). Multivariate analysis indicated that the expression level of miR-23b in tumor tissue was also an independent prognostic factor for both recurrence (hazard ratio [HR]: 2.483, 95% confidence interval [CI]: 1.563-3.944, P<.001) and survival (HR 2.256, 95%CI 1.377-3.697, P=.001).
Our analyses revealed that low expression of miR-23b was associated with multiple malignant characteristics of HCC. MiR-23b expression was low in a subgroup of patients with higher serum AFP level, >5cm in diameter and no complete capsule. In addition, patients with lower miR-23b expression were mostly in TNM stageⅢ, and these patients usually display microvascular invasion, and portal vein tumor thrombus. This suggests that miR-23b may be a tumor suppressor and that miR-23b low expression in hepatocytes may contribute to the development of a more aggressive phenotype of hepatocellular carcinoma. Furthermore, low expression of miR-23b might be associated with worse prognosis. Patients whose tumors had high miR-23b expression had a prolonged TTR and survival as compared with patients whose tumors had high miR-23b expression.
In short, this part of study evaluated for the first time the correlation between miR-23b expression, tumor recurrence, and survival in HCC patients. Our data strongly indicates that miR-23b expression in HCC is a potential biomarker for prognosis evaluation.
In conclusion, through the investigation of the termination stage of liver regeneration, we have found the potential role of miR-23b and neurogranin during the termination stage of liver regeneration in rat which will further improve the molecular mechanism of liver regeneration. Meantime, by analysing the association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patients, we have confirmed that miR-23b might be one of the prognostic indicators of HCC patients after curative resection which will provide the basis for exploring therapeutic targets for HCC.
第一部分MiR-23b在大鼠肝再生终止阶段的表达变化及功能探讨
MiR-23b参与多种信号通路的调节,其作用涉及增殖、分化、凋亡、细胞粘附等各个方面。最新关于miR-23b等研究证明了它还可以作为TGF-β1信号通路的辅助成分调节该通路的激活。而TGF-β1信号通路在肝再生终止阶段发挥重要的作用。我们前期用芯片筛选肝再生后期差异表达的miRNA时发现miR-23b在肝再生终止阶段表达量明显降低。综合以上因素我们猜测在肝再生后期miR-23b可能通过调节TGF-β1信号通路参与肝再生终止。本部分实验旨在验证miR-23b在肝再生终止阶段的表达变化并探讨其对肝再生终止的作用及可能的分子机制。
本部分研究包括动物实验和细胞实验两部分。通过动物实验,我们发现:在肝再生终止阶段,miR-23b的mRNA表达水平明显降低。结合实验中检测到肝再生终止阶段TGF-β1信号通路的持续激活状态(TGF-β1下游信号分子Smad3的mRNA和蛋白表达明显升高,磷酸化Smad3蛋白表达也呈现明显的升高趋势;细胞增殖活性明显降低,凋亡活性明显升高。),我们推测miR-23b可能通过激活TGF-β1信号通路参与调控肝再生终止。为了验证这一猜测,我们进一步作了体外的细胞试验来验证肝细胞内miR-23b对细胞生长及TGF-β1信号通路作用。在体外,通过培养正常大鼠肝细胞(BRL-3A),细胞转染miR-23b的模拟物(mimics)或抑制物(inhibitor)上调或下调miR-23b,进一步探讨miR-23b的功能,研究结果显示:①上调miR-23b促进肝细胞生长,下调miR-23b抑制肝细胞生长并抑制大鼠肝细胞细胞周期的进入,阻止细胞从G2期到M期转换;②Smad3是miR-23b的下游靶蛋白;③miR-23b通过转录抑制Smad3表达部分程度上抑制TGF-β1诱导的肝细胞凋亡;④TGF-β1是miR-23b的上有调节分子,转录抑制miR-23b的表达。
本部分实验我们发现在肝再生终止阶段miR-23b的表达量下调。TGF-β1是miR-23b的上游调节分子。在肝再生终止阶段,miR-23b的低表达降低了其对Smad3的转录抑制从而间接激活TGF-β1信号通路促进肝再生终止。
第二部分神经科粒素(neurogranin)在大鼠肝再生终止阶段的表达及功能探讨
第一部分实验中,我们用Target scan5.1软件预测miR-23b下游可能的靶基因时发现神经颗粒素是miR-23b下游潜在的靶基因之一。尽管经验证肝细胞内miR-23b的表达改变不能调节神经颗粒素的表达,但是我们前期用基因芯片筛选大鼠肝再生终止阶段差异表达的基因时发现神经颗粒素在肝再生终止阶段(部分肝切除后120h, 168h)明显升高。
神经颗粒素(neurogranin)是一种神经元特异性钙调蛋白结合蛋白,主要位于大脑等神经组织,近年来有研究发现其存在于胸腺及脾脏组织以及淋巴器官。神经颗粒素主要参与大脑等神经系统相关功能如学习、记忆、突出可塑性等的调节。最近有研究发现:神经颗粒素作为一种钙调蛋白结合蛋白,通过调节钙离子/钙调蛋白复合物的可用性调节细胞内钙离子浓度发挥促凋亡作用。
肝再生终止阶段的显著特征是肝细胞增殖活性降低,凋亡活性升高。作为一种促凋亡蛋白,神经颗粒素在肝再生终止阶段的表达上调与肝再生终止阶段凋亡活性的升高具有时效相关性。综合上述研究结果,我们猜测凋亡相关蛋白神经颗粒素可能参与调控肝再生终止。基于上述分析,本部分实验旨在研究神经颗粒素在大鼠肝再生终止阶段的表达变化并探讨及其在肝脏再生终止阶段的作用及分子机制。
本部分实验包括肝再生动物实验和离体细胞实验两个部分。通过动物实验,我们发现:①在肝再生终止阶段,神经颗粒素蛋白表达水平明显升高;②第一部分实验中检测到肝再生终止阶段细胞凋亡活性升高,线粒体凋亡通路处于激活状态;③已发现的对神经颗粒素的表达具有调节作用的分子有维甲酸、T3、NO、miR-23b等。但在肝细胞内对神经颗粒素具有调节作用的只有NO。NO具有一定的促凋亡作用,其调控的下游分子神经颗粒素也能发挥促凋亡效应。此外,在肝再生终止阶段NO浓度升高、神经颗粒素表达上调、线粒体凋亡通路的激活三者在时效上相关,因此我们猜测在肝再生终止阶段,NO浓度的升高很可能通过上调神经颗粒素表达参与线粒体凋亡通路的激活。为了验证这一推测,我们进一步通过离体细胞实验来说明神经颗粒素对肝细胞凋亡进程的调节作用。在体外,通过培养正常大鼠肝细胞(BRL-3A),细胞转染神经颗粒素干扰质粒下调神经颗粒素,进一步探讨神经颗粒素的功能。研究结果显示:①神经颗粒素干扰质粒能够在部分程度上抑制TGF-β1诱导的肝细胞凋亡进程。在这一过程中,肝细胞内钙离子浓度明显降低,线粒体膜电位明显降低;②在正常大鼠肝细胞内,外源性NO供体能够上调神经颗粒素的表达。
本部分实验我们发现在肝再生终止阶段神经颗粒素的表达量上调与miR-23b以及外源性甲状腺激素T3无关,NO是神经颗粒素的上有调节分子。NO通过诱导神经颗粒素表达上调,参与线粒体凋亡通路调控肝再生终止。
第三部分miR-23b在肝癌组织中的表达及其在早期肝癌术后预后中的作用研究
肝细胞癌(HCC)是人类最常见的致命性肿瘤之一,在全球肿瘤死亡原因中排列第五,中国大陆肝癌发病率占全球发病率的55%。根治性切除是HCC治疗的首选治疗方法,但是预后尚不理想,首要原因是肝内复发。寻找理想的肝癌术后预后价值指标,并据此选择合理的辅助治疗对改善预后有重要意义。
第一部分研究证明在肝再生终止阶段,miR-23b的低表达降低了其对Smad3的转录抑制从而间接激活TGF-β1信号通路促进肝再生终止。而TGF-β1信号通路在肝癌发生、发展中发挥着重要的作用,因此,我们猜测miR-23b可能在肝癌的发生、发展和预后等过程中发挥作用。已有研究表明miR-23b参与调节肝癌细胞增殖和侵袭等。因此,本部分实验结合临床病例和标本初步研究miR-23b在肝癌组织中的表达规律并初步探讨其对肝癌术后预后的影响,尤其是对早期肝癌预后的预测。
本部分实验包括两部分,①RT-PCR方法研究配对肝癌组织和癌旁组织中基因表达情况;②前瞻性观察2004.1-2004.6中连续入组的233例接受根治性切除患者癌组织中miR-23b的表达水平和肝癌术后预后的关系。RT-PCR结果发现miR-23b在肝癌组织中的表达比癌旁组织降低2倍左右。经调查分析发现miR-23b的表达水平与肿瘤患者血清AFP水平、肿瘤直径、肿瘤数目和TNM分期有关;多因素分析miR-23b是影响患者复发和生存的独立因素(hazard ratio HR,95% confidence interval, CI,复发:2.483,1.563-3.944,P<.001;生存:2.256,1.377-3.697,P<.001)。在BCLC A期患者中,miR-23b低表达患者复发率高于高表达患者,生存率则相反。
本部分实验我们发现肝癌组织中miR-23b的表达水平低于癌旁及正常组织。miR-23b的表达水平与肝癌患者术后预后有关,对早期肝癌患者预后有预测作用,miR-23b低表达患者术后可能会出现复发和转移,预后差。
总之,我们通过对大鼠肝再生终止阶段分子机制的一系列研究,揭示了miR-23b、神经颗粒素在肝再生终止阶段的可能作用,进一步完善了肝再生分子机制的研究。同时,我们通过对miR-23b表达水平与肝癌术后预后相关性研究证实miR-23b可以作为临床肝癌术后预后指标,不仅丰富了临床肝癌术后预后指标,同时为临床治疗肝癌提供了新的治疗靶点。
Following 70% partial hepatectomy (PHx), the remnant liver has a remarkable ability to regenerate to its original mass and function within 7-10 days after surgery in a process called liver regeneration (LR). LR after PHx is generally divided into three distinct phases: initiation, proliferation and termination. Despite multiple studies of LR, many aspects of this phenomenon remain unknown, including the proper temporal regulation of termination.
MicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown.
In a preliminary study using a miRNA microarray analysis, we found that miRNA 23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation.
In view of the above analysis, this study sought to elucidate if and how miR-23b was involved in the termination stage of LR, and this will provide basis to better illustrate the molecular mechanism of the termination liver regeneration after partial hepatectomy. The inquiry will also provide the basis for exploring therapeutic targets for liver cancer.
SectionⅠ: The expression of miR-23b and its role in the termination stage of liver regeneration after partial hepatectomy in rats
MicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown.
MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation. In a preliminary study using a miRNA microarray analysis, we found that miRNA23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. And a recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Considering the important role TGF-β1 plays during the termination stage of LR, the present work sought to elucidate if and how miR-23b was involved in the termination stage of LR. We also report evidences that miR-23b expression is remarkably diminished during the termination of LR, and miR-23b may contribute to the TGF-β1/Smad3 signalling pathway during the termination stage of LR.
In the present study we confirmed that miR-23b is down-regulated during the termination stage of LR. Our study also found that down-regulation of miR-23b inhibits cell proliferation, which may be attributed to G2/M arrest by flow cytometry investigation, as shown in figure 2. Therefore, down-regulation of miR-23b may contribute to the termination of LR, which is characterised by reduced proliferation and enhanced cellular apoptosis.
To further investigate the specific role of miR-23b during the termination stage of LR, it is very important to identify the target gene of miR-23b. A recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Thus, miR-23b plays a critical role in TGF-β1 /Smads signaling pathway. More importantly, it is widely known that TGF-β1 and activinA are natural terminators of LR. Therefore, we speculate miR-23b may take part in the termination stage of LR by targeting smads. Our study has confirmed that Smad3 but not Smad4 or Smad5 is a putative target gene of miR-23b in livers following PHx.
There is evidence that Smad3 plays a more significant role in the induction of TGF-β1-induced apoptosis than other Smads downstream of TGF-β1. Over-expression of Smad3 greatly potentiates the induction of apoptosis in response to TGF-β1, while expression of a dominant-negative mutant of Smad3 inhibits TGF-β1 induced apoptosis. Therefore, Smad3 is an important downstream molecular in TGF-β1 pathway. Interestingly, we have found that up-regulation of miR-23b could partially inhibit TGF-β1-induced apoptosis in BRL-3A cells by reducing the expression of Smad3. In addition, TGF-β1, Smad3, phosphorylated Smad3 and apoptotic activity increased and proliferative activity decreased when miR-23b was decreased from 72 to 168 h after PHx. Taken together, down-regulation of miR-23b may contribute to activation of the TGF-β1/Smad3 signalling pathway during the termination stage of LR.
In exploring the specific relationship between TGF-β1, miR-23b and Smad3, we considered TGF-β1 as a possible upstream regulator of miR-23b. Firstly, several reports have demonstrated that TGF-β1 can either positively or negatively regulate the expression of miRNAs including miR-192 and miR-24. More interestingly, even in the process of LR, miR-23b down-regulation occurs after 72h PHx, when TGF-β1 expression is at its highest levels. In addition, miR-23b can directly regulate the expression of Smad3 which is a downstream molecular in TGF-β1 signaling. Therefore, we presumed that miR-23b may act as a pitch point in the TGF-β1 signalling pathway. Then we confirmed that TGF-β1 can regulate the expression of miR-23b at the transcriptional level. Our present findings suggest at TGF-β1-miR-23b-smad3 signaling may act as a novel mechanism in the termination stage of LR.
In conclusion, miR-23b may contribute to TGF-β1/Smad3 signaling pathway during the termination stage of LR. Our findings reveal a miRNA-mediated regulation pattern during the termination stage of LR.
SectionⅡ: The expression of neurogranin and its role in the termination stage of liver regeneration after partial hepatectomy in rats
Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca2+/CaM complex and modulates the homeostasis of intracellular calcium in neurons. In recent years, studies have found that Ng exists in the thymus, spleen and other lymphatic organs.
In the first experimental part, we use Target scan5.1 to predict the potential target genes of miR - 23b have found that Ng is one of the potential target genes of miR-23b. but our study proved that miR - 23b is not the upstream regulator of neurogranin in the BRL-3A cells. Intereatingly, our prestudy using gene chip to indentify the differentially expressed gene during the termination stage (120,168h after partial hepatectomy) of liver regeneration after partial hepatectomy have found that the gene expression of Ng upregulated. In view of these above factors, we predicted that apoptosis related proteins Ng might be involved in the termination of liver regeneration after partial hepatectomy in rats. Based on the above analysis, this experimental part aimed at the investigation of the expression pattern of Ng during the termination stage of liver regeneration and the exploration of the related molecular mechanisms.
Our results showed that: the expression of Ng protein upregulated during the termination stage of liver regeneration which is characterized with a high apoptosis activity; the concentration of NO and T3 increased during the termination stage of liver regeneration; downregulation of Ng in BRL-3A cells will inhibits the growth of BRL-3A cells and partially inhibits TGF-β1 induced BRL-3A cell apoptosis through depressing the mitochondrial apoptosis pathway; exogenous NO donors SNP could upregulate the expression of Ng at the transcriptional level in BRL-3A cells, while there were no obvious change on the expression of Ng when BRL-3A cells were treated with miR-23b or T3.
This experimental part suggests that during the termination stage of liver regeneration, the upregulated expression of Ng attributed to the increased concentration of NO and contributed to the termination of liver regeneration by inducing mitochondrial apoptosis pathway.
SectionⅢ: The association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patients
Hepatocellular carcinoma (HCC) is the fifth most frequent human cancer worldwide, and, the second leading cause of cancer-related death in China. Major HCC risk factors include infection with hepatitis B (HBV) or C viruses (HCV) and cirrhosis associated with chronic inflammation. Hepatectomy is the first choice for HCC patients in China, but the outcome is poor due to high recurrence after surgery. Since most HCCs have dissemination within the liver before resection and are undetectable by imaging techniques, new and efficacious adjuvant therapy strategies are needed, along with new prognostic biomarkers for early detection of at-risk patients. Therefore, it is clinically meaningful that metastasis and prognosis related molecule and its functional mechanism were found. MicroRNAs (miRs) are small (21-25 nucleotide) nonprotein-coding RNAs that posttranscriptionally regulate gene expression. MiRNAs possess oncogenic or tumor suppressor activity in various tumors but little is known about miRNA expression pattern in hepatocellular carcinoma (HCC). The purpose of this part of study was to investigate the relationship between miR-23b expression and prognosis of HCC patients after curative resection.
We use quantitative reverse-transcriptase-polymerase-chain-reaction assays to investigate the expression of miR-23b in 115 patients with HCC who had undergone radical resection between 2004 and 2005. We assessed the association of miR-23b with tumor recurrence and patients’survival with HCC. The results showed that: Tumors had reduced levels of miR-23b expression, as compared with paired nontumorous tissues. In comparison with high miR-23b expression, low miR-23b expression in tumor tissue, which was correlated with encapsulation (P=.009) and TNM stage (P<.001) was associated with both tumor recurrence (median time to recurrence: 10.3±1.8 months vs 36.3±2.6 months, P<.001) and patients survival (1-, 3-, 5-year survival rates: 52%, 31%, 29% vs 84%, 63% 57%, P<.001). Multivariate analysis indicated that the expression level of miR-23b in tumor tissue was also an independent prognostic factor for both recurrence (hazard ratio [HR]: 2.483, 95% confidence interval [CI]: 1.563-3.944, P<.001) and survival (HR 2.256, 95%CI 1.377-3.697, P=.001).
Our analyses revealed that low expression of miR-23b was associated with multiple malignant characteristics of HCC. MiR-23b expression was low in a subgroup of patients with higher serum AFP level, >5cm in diameter and no complete capsule. In addition, patients with lower miR-23b expression were mostly in TNM stageⅢ, and these patients usually display microvascular invasion, and portal vein tumor thrombus. This suggests that miR-23b may be a tumor suppressor and that miR-23b low expression in hepatocytes may contribute to the development of a more aggressive phenotype of hepatocellular carcinoma. Furthermore, low expression of miR-23b might be associated with worse prognosis. Patients whose tumors had high miR-23b expression had a prolonged TTR and survival as compared with patients whose tumors had high miR-23b expression.
In short, this part of study evaluated for the first time the correlation between miR-23b expression, tumor recurrence, and survival in HCC patients. Our data strongly indicates that miR-23b expression in HCC is a potential biomarker for prognosis evaluation.
In conclusion, through the investigation of the termination stage of liver regeneration, we have found the potential role of miR-23b and neurogranin during the termination stage of liver regeneration in rat which will further improve the molecular mechanism of liver regeneration. Meantime, by analysing the association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patients, we have confirmed that miR-23b might be one of the prognostic indicators of HCC patients after curative resection which will provide the basis for exploring therapeutic targets for HCC.
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[5] Morte B., Martinez de A.C., Manzano J., Coloma A., Bernal J. Identification of a cis-acting element that interferes with thyroid hormone induction of the neurogranin (NRGN) gene. FEBS Lett 1999;464:179-183.
[6] Julie Vallortigara, Serge Alfos, Jacques Micheau, Paul Higueret, Valérie Enderlin. T3 administration in adult hypothyroid mice modulates expression of proteins involved in striatal synaptic plasticity and improves motor behavior[J]. Neurobiology of Disease 2008;31:378-385.
[7] Yang HM, Lee PH, Lim TM, Sheu FS. Neurogranin expression in stably transfected N2A cell line affects cytosolic calcium level by nitric oxide stimulation[J]. Brain Res Mol Brain. Res 2004;129:171-178.
[8] Sato T., Xiao D.M., Li H., Huang F.L., Huang K.P. Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein). J. Biol. Chem 1995;270:10314-10322.
[9] Junfang Wu et .al.Attenuation of Protein Kinase C and cAMP-dependent Protein Kinase Signal Transduction in the Neurogranin Knockout Mouse。J Biol Chem 2002; 22:277-282.
[10] S.T. Szabo et al. Glutamate receptors as targets of protein kinase C in the pathophysiology and treatment of animal models of Mania Neuropharmacology 2009;56 : 47–55
[11] Quintana, A.,et al.,. Calcium-dependent activation of T-lymphocytes. Pflugers Arch 2005;450;1–12.
[12] Han NL,et al. Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation[J]. Int JBiol Sci 2007;3:263-273.
[13] Jingang Gui et.al.,Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress。Int. J. Biol. Sci 2007;3:212-224
[14] Hildeman DA, Zhu Y, Mitchell TC, Kappler J, Marrack P. Molecular mechanisms of activated T cell death in vivo[J]. Curr Opin Immunol 2002;14:354-359.
[15] Devireddy LR, Green MR. Transcriptional Program of Apoptosis Induction following Interleukin 2 Deprivation: Identification of RC3, a Calcium/Calmodulin Binding Protein, as a Novel Proapoptotic Factor[J]. Molecular and Cellular Biology 2003;23:4532-4541.
[16]Gui J, et al.,. Characterization of transcriptional regulation of neurogranin by nitric xide and the role of neurogranin in SNP-induced cell death: implication of eurogranin in an increased neuronal susceptibility to oxidative stress[J]. Int J Biol Sci 2007;3:212-224.
[17] Timmins JM, etal.Calcium/ calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways. J Clin Invest 2009;119:292541.
[1] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132:2557-2576.
[2] Tang ZY. Hepatocellular carcinoma-Cause, treatment and metastasis. World J Gastroenterol 2001; 7:445-454.
[3] Portolani N, Coniglio A, Ghidoni S, Giovanelli M, Benetti A, Tiberio GA, et al. Early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications. Ann Surg 2006; 243:229-235.
[4] Bruix J, Boix L, Sala M, Llovet JM. Focus on hepatocellular carcinoma. Cancer Cell 2004; 5:215-219.
[5] Poon RT, Fan ST, Lo CM, Liu CL, Wong J. Intrahepatic recurrence after curative resection of hepatocellular carcinoma: long-term results of treatment and prognostic factors. Ann Surg 1999; 229:216-222.
[6] Yoon SK. Recent advances in tumor markers of human hepatocellular carcinoma. Intervirology 2008; 51:34-41.
[7]王杰,李杰,鲁凤民.MicroRNA在肿瘤方面的研究进展[J].周际遗传学杂志2007 ;(04):50-53.
[8] Visone R,Petrocca F,Croce CM.Micro-RNAs in gastrointestinal and liver disease[J].Gastroenterology 2008 ;135:1866-1869.
[9] CT Pager, KA Wehner, G Fuchs, P Sarnow. Chapter 5 MicroRNA-Mediated Gene Silencing. Prog Mol Biol Transl Sci 2009;90:187-210
[10] Lynn FC. Meta-regulation: microRNA regulation of glucose and lipid metabolism. Trends Endocrinol Metab 2009;20:452-459.
[11] Wang KC, Garmire LX, Young A, Nguyen P, Trinh A, Subramaniam S, Wang N, Shyy JY, Li YS, Chien S. Role of mircroRNA-23b in flow-regulation of Rb Phosphorylation and endothelial cell growth. Proc Natl Acad Sci 2010; 107:3234-3239.
[12] Kimura H, Kawasaki H, Taira K. Mouse microRNA-23b regulates expression of Hes1 gene in P19 cells. Nucleic Acids Symp Ser 2004;48:213-214.
[13] Liu W, Zabirnyk O, Wang H, Shiao YH, Nickerson ML, Khalil S, Anderson LM, Perantoni AO, Phang JM. MiR-23b targets praline oxidase, a novel tumor suppressor protein in renal cancer. Oncogene 2010;29 :4914-4924.
[14] Rogler CE, Levoci L, Ader T, Massimi A, Tchaikovskaya T, Norel R, Rogler LE. MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signalling and liver stem cell differentiation by targeting Smads. Hepatology 2009;50: 575-584.
[15] Salvi A, Sabelli C, Moncini S, Ventruin M, Arici B, Riva P, Portolani N, Giulini SM, De Petro G, Barlati S. MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells. FEBS J 2009; 276:2966-2982.
[16] Oe S, Lemmer ER, Conner EA, Factor VM, Leveen P, Larsson J, Karlsson S, Thorgeirsson SS. Intact Signaling by transforming growth factorβis not required for termination of liver regeneration in mice. Hepatology 2004;40:1098-1105.
[17] Zhong Z, Tsukada S, Rehman H, Parsons CJ, Theruvath TP, Rippe RA, Brenner DA, Lemasters JJ. Inhibition of transforming growth- beta/Smad signaling improves regeneration of small-for-size rat live grafts. Liver Transpl 2010;16:181-190.
[18] Dug Keun Lee, Seok Hee Park, Youngsuk Yi. The hepatitis B virus encoded oncoprotein pX amplifies TGF-βfamily signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis. Genes&Dev 2001;15:455-466.
[19] Diego F. Calvisi1, Valentina M. Factor, Roberto Loi. Activation ofβ-Catenin during Hepatocarcinogenesis in Transgenic Mouse Models Relationship to Phenotype and Tumor Grade. Cancer Research 2001; 61:2085-2090.
[20] Swayamjot Kaur, Fang Wang, Manickam Venkatraman. X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factorβ1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)*. Journal of Biological Chemistry 2005; 280:38599-38608.
[21] ZHEN XU, MIN XIONG SHEN, DONG ZHU MA. TGF?β1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells. Cell Research 2003;13:343-350.
[1] George A C, Carlo M C. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857–866.
[2] Chin D, Boyle G M, Porceddu S, et al. Head and neck cancer: Past, present and future. Expert Rev Anticancer Ther 2006; 6: 1111–1118.
[3] Deyrup A T. Epstein-Barr virus-associated epithelial and mesenchymal neoplasms. Hum Pathol 2008 ; 39: 473–483.
[4] Zhu J Y, Pfuhl T, Motsch N, et al. Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol 2009; 83: 3333–3341.
[5] Georgia S. Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell. RNA 2009; 15: 1443–1461.
[6] Filipowicz W, Bhattacharyya S N, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: Are the answers in sight?Nat Rev Genet 2008; 9: 102–114.
[7] Israel A, Sharan R, Ruppin E, et al. Increased microRNA activity in human cancers. PLoS One 2009; 4: 200-205.
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[1] Port JD, Sucharov C. Role of MicroRNAs in cardiovascular disease: therapeutic challenges and potentials. J Cardiovasc PHxarmacol 2010;56:444-453.
[2] CT Pager, KA Wehner, G Fuchs, P Sarnow. Chapter 5 MicroRNA-Mediated Gene Silencing. Prog Mol Biol Transl Sci 2009;90:187-210.
[3] Lynn FC. Meta-regulation: microRNA regulation of glucose and lipid metabolism. Trends Endocrinol Metab 2009;20:452-459.
[4] Chen XM. MicroRNA signatures in liver diseases. World J Gastroenterol 2009;15: 1665-1672.
[5] Wang KC, Garmire LX, Young A, Nguyen P, Trinh A, Subramaniam S, Wang N, Shyy JY, Li YS, Chien S. Role of mircroRNA-23b in flow-regulation of Rb Phosphorylation and endothelial cell growth. Proc Natl Acad Sci 2010;107:3234-3239.
[6] Rogler CE, Levoci L, Ader T, Massimi A, Tchaikovskaya T, Norel R, Rogler LE. MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signalling and liver stem cell differentiation by targeting Smads. Hepatology 2009;50:575-584.
[7] Oe S, Lemmer ER, Conner EA, Factor VM, Leveen P, Larsson J, Karlsson S, Thorgeirsson SS. Intact Signaling by transforming growth factorβis not required for termination of liver regeneration in mice. Hepatology 2004;40:1098-1105.
[1] Watson JB, Battenberg EF, Wong KK,Bloom FE, Sutcliffe JG. Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein[J]. J Neurosci Res 1990;26:397-408.
[2] A.A. Nielsen et al. Activation of the brain-specific neurogranin gene in murine T-cell lymphomas by proviral insertional mutagenesis Gene 2009;442 :55–62
[3] Bahler M, Rhoads A. Minireview:Calmodulin signaling via the IQ motif. FEBS Letters 2002;513:107-113.
[4] Huang K.P.,et al. Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3. Biochemistry 2000;39:7291-7299.
[5] Morte B., Martinez de A.C., Manzano J., Coloma A., Bernal J. Identification of a cis-acting element that interferes with thyroid hormone induction of the neurogranin (NRGN) gene. FEBS Lett 1999;464:179-183.
[6] Julie Vallortigara, Serge Alfos, Jacques Micheau, Paul Higueret, Valérie Enderlin. T3 administration in adult hypothyroid mice modulates expression of proteins involved in striatal synaptic plasticity and improves motor behavior[J]. Neurobiology of Disease 2008;31:378-385.
[7] Yang HM, Lee PH, Lim TM, Sheu FS. Neurogranin expression in stably transfected N2A cell line affects cytosolic calcium level by nitric oxide stimulation[J]. Brain Res Mol Brain. Res 2004;129:171-178.
[8] Sato T., Xiao D.M., Li H., Huang F.L., Huang K.P. Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein). J. Biol. Chem 1995;270:10314-10322.
[9] Junfang Wu et .al.Attenuation of Protein Kinase C and cAMP-dependent Protein Kinase Signal Transduction in the Neurogranin Knockout Mouse。J Biol Chem 2002; 22:277-282.
[10] S.T. Szabo et al. Glutamate receptors as targets of protein kinase C in the pathophysiology and treatment of animal models of Mania Neuropharmacology 2009;56 : 47–55
[11] Quintana, A.,et al.,. Calcium-dependent activation of T-lymphocytes. Pflugers Arch 2005;450;1–12.
[12] Han NL,et al. Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation[J]. Int JBiol Sci 2007;3:263-273.
[13] Jingang Gui et.al.,Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress。Int. J. Biol. Sci 2007;3:212-224
[14] Hildeman DA, Zhu Y, Mitchell TC, Kappler J, Marrack P. Molecular mechanisms of activated T cell death in vivo[J]. Curr Opin Immunol 2002;14:354-359.
[15] Devireddy LR, Green MR. Transcriptional Program of Apoptosis Induction following Interleukin 2 Deprivation: Identification of RC3, a Calcium/Calmodulin Binding Protein, as a Novel Proapoptotic Factor[J]. Molecular and Cellular Biology 2003;23:4532-4541.
[16]Gui J, et al.,. Characterization of transcriptional regulation of neurogranin by nitric xide and the role of neurogranin in SNP-induced cell death: implication of eurogranin in an increased neuronal susceptibility to oxidative stress[J]. Int J Biol Sci 2007;3:212-224.
[17] Timmins JM, etal.Calcium/ calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways. J Clin Invest 2009;119:292541.
[1] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132:2557-2576.
[2] Tang ZY. Hepatocellular carcinoma-Cause, treatment and metastasis. World J Gastroenterol 2001; 7:445-454.
[3] Portolani N, Coniglio A, Ghidoni S, Giovanelli M, Benetti A, Tiberio GA, et al. Early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications. Ann Surg 2006; 243:229-235.
[4] Bruix J, Boix L, Sala M, Llovet JM. Focus on hepatocellular carcinoma. Cancer Cell 2004; 5:215-219.
[5] Poon RT, Fan ST, Lo CM, Liu CL, Wong J. Intrahepatic recurrence after curative resection of hepatocellular carcinoma: long-term results of treatment and prognostic factors. Ann Surg 1999; 229:216-222.
[6] Yoon SK. Recent advances in tumor markers of human hepatocellular carcinoma. Intervirology 2008; 51:34-41.
[7]王杰,李杰,鲁凤民.MicroRNA在肿瘤方面的研究进展[J].周际遗传学杂志2007 ;(04):50-53.
[8] Visone R,Petrocca F,Croce CM.Micro-RNAs in gastrointestinal and liver disease[J].Gastroenterology 2008 ;135:1866-1869.
[9] CT Pager, KA Wehner, G Fuchs, P Sarnow. Chapter 5 MicroRNA-Mediated Gene Silencing. Prog Mol Biol Transl Sci 2009;90:187-210
[10] Lynn FC. Meta-regulation: microRNA regulation of glucose and lipid metabolism. Trends Endocrinol Metab 2009;20:452-459.
[11] Wang KC, Garmire LX, Young A, Nguyen P, Trinh A, Subramaniam S, Wang N, Shyy JY, Li YS, Chien S. Role of mircroRNA-23b in flow-regulation of Rb Phosphorylation and endothelial cell growth. Proc Natl Acad Sci 2010; 107:3234-3239.
[12] Kimura H, Kawasaki H, Taira K. Mouse microRNA-23b regulates expression of Hes1 gene in P19 cells. Nucleic Acids Symp Ser 2004;48:213-214.
[13] Liu W, Zabirnyk O, Wang H, Shiao YH, Nickerson ML, Khalil S, Anderson LM, Perantoni AO, Phang JM. MiR-23b targets praline oxidase, a novel tumor suppressor protein in renal cancer. Oncogene 2010;29 :4914-4924.
[14] Rogler CE, Levoci L, Ader T, Massimi A, Tchaikovskaya T, Norel R, Rogler LE. MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signalling and liver stem cell differentiation by targeting Smads. Hepatology 2009;50: 575-584.
[15] Salvi A, Sabelli C, Moncini S, Ventruin M, Arici B, Riva P, Portolani N, Giulini SM, De Petro G, Barlati S. MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells. FEBS J 2009; 276:2966-2982.
[16] Oe S, Lemmer ER, Conner EA, Factor VM, Leveen P, Larsson J, Karlsson S, Thorgeirsson SS. Intact Signaling by transforming growth factorβis not required for termination of liver regeneration in mice. Hepatology 2004;40:1098-1105.
[17] Zhong Z, Tsukada S, Rehman H, Parsons CJ, Theruvath TP, Rippe RA, Brenner DA, Lemasters JJ. Inhibition of transforming growth- beta/Smad signaling improves regeneration of small-for-size rat live grafts. Liver Transpl 2010;16:181-190.
[18] Dug Keun Lee, Seok Hee Park, Youngsuk Yi. The hepatitis B virus encoded oncoprotein pX amplifies TGF-βfamily signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis. Genes&Dev 2001;15:455-466.
[19] Diego F. Calvisi1, Valentina M. Factor, Roberto Loi. Activation ofβ-Catenin during Hepatocarcinogenesis in Transgenic Mouse Models Relationship to Phenotype and Tumor Grade. Cancer Research 2001; 61:2085-2090.
[20] Swayamjot Kaur, Fang Wang, Manickam Venkatraman. X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factorβ1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)*. Journal of Biological Chemistry 2005; 280:38599-38608.
[21] ZHEN XU, MIN XIONG SHEN, DONG ZHU MA. TGF?β1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells. Cell Research 2003;13:343-350.
[1] George A C, Carlo M C. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857–866.
[2] Chin D, Boyle G M, Porceddu S, et al. Head and neck cancer: Past, present and future. Expert Rev Anticancer Ther 2006; 6: 1111–1118.
[3] Deyrup A T. Epstein-Barr virus-associated epithelial and mesenchymal neoplasms. Hum Pathol 2008 ; 39: 473–483.
[4] Zhu J Y, Pfuhl T, Motsch N, et al. Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol 2009; 83: 3333–3341.
[5] Georgia S. Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell. RNA 2009; 15: 1443–1461.
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