Nrip2通过非经典Wnt通路调控大肠癌干细胞的自我更新
发布时间：2019-07-05 20:14
【摘要】：目的：大肠癌是常见的消化道恶性肿瘤,占肿瘤相关死因的第二位。近年来尽管在提高患者生存率的化学疗法上有了很大的进展,但是大肠癌晚期患者的预后仍很差,总的死亡率在40%左右。阐明大肠癌分子机制是治疗大肠癌的基础和前提。研究提示大肠癌干细胞参与了大肠癌的发生、浸润、转移和药物抵抗。Wnt通路在大肠癌干细胞的自我更新中起了重要作用,然而Wnt通路作用于大肠癌干细胞的具体机制仍不明。本研究通过筛选和鉴定大肠癌干细胞中Wnt通路激活相关的蛋白,发现核受体相互作用蛋白2(Nuclear receptor-interacting protein2, Nrip2)在大肠癌干细胞中表达增加,并检测Nrip2在大肠癌干细胞自我更新中的作用,鉴定其关键作用靶蛋白,寻找Nrip2作用于大肠癌干细胞的潜在靶点,从而明确Nrip2对大肠癌生物学行为的影响。 方法：通过构建SW620克隆球cDNA逆转录病毒文库,筛选出Wnt通路激活相关的蛋白Nrip2表达增加。为探讨Nrip2在大肠癌中的表达情况,采用免疫组化和mRNA荧光原位杂交验证大肠癌病理组织切片中Nrip2蛋白的表达,并采用Real-time qPCR检测大肠癌细胞和临床样本富集的克隆球中Nrip2的表达情况。为评价Nrip2在大肠癌干细胞自我更新中的作用,构建稳定表达Nrip2的大肠癌细胞系,观察过表达Nrip2对大肠癌细胞克隆球成球能力、iPS和EMT相关因子的表达和体内致瘤能力的影响。为研究Nrip2对大肠癌干细胞自我更新的作用机制,质谱筛选Nrip2的靶蛋白Frizzled1(Fzd1),构建各种结构域突变体分析Nrip2与Fzdl蛋白的结合位点,以及Nrip2和下游靶分子的结合情况。通过免疫共沉淀(co-immunoprecipitation)和Western blot筛选和鉴定了Nrip2非经典Wnt通路的靶蛋白维甲酸相关孤儿受体β (Retinoic acid-related orphan receptor beta, Rorp),观察了过表达Rorp对大肠癌细胞克隆球形成能力、EMT相关因子的表达和体内致瘤能力的影响。通过基因芯片筛选出Rorp的下游靶基因HMG盒转录因子1(High Mobility Group-box transcription factor1, HBP1)。并通过PepstatinA处理过表达Nrip2的大肠癌细胞观察Nrip2的酶活性对其调控大肠癌干细胞自我更新的影响。 结果：构建SW620克隆球cDNA逆转录病毒文库,筛选出Wnt通路激活相关蛋白Nrip2表达增加。Nrip2表达于正常大肠组织和大肠癌组织细胞中,在大肠癌干细胞中表达明显升高。过表达Nrip2促进了大肠癌干细胞的自我更新。我们发现Nrip2能够与Fzdl结合,但不与β-catenin结合。免疫共沉淀筛选出Nrip2与Rorβ结合,其酶活性能够降解Rorβ,阻止Rorβ入核。Rorβ通过抑制Wnt通路活性抑制大肠癌干细胞的自我更新。基因芯片筛选发现Rorβ的下游靶基因是HBP1, HBP1通过竞争性结合TCF/LEF4抑制Wnt通路下游靶分子的转录。Nrip2的酶活性的灭活阻断了Nrip2对大肠癌干细胞的自我更新作用。 结论：首次发现了Nrip2和Rorp表达于正常大肠组织和大肠癌组织细胞中。Nrip2在大肠癌干细胞中表达增加,与Rorβ结合呈酶活性依赖型,通过降解Rorβ,抑制HBP1的转录,启动Wnt通路下游靶蛋白的表达,促进了大肠癌干细胞的自我更新,从而介导了一条新的非经典Wnt通路Nrip2/Rorβ/HBP1通路。Nrip2的酶活性是干预阻断大肠癌干细胞自我更新的潜在治疗靶点。
文内图片：
图片说明：富集大肠癌细胞克隆球大肠癌细胞系HT29SW620和原代大肠癌细胞P1在无血清克隆球培养基培养7-
[Abstract]:Objective: Colorectal cancer is a common malignant tumor of the digestive tract. In recent years, although much progress has been made in chemical therapy to improve the survival rate of patients, the prognosis of patients with advanced colorectal cancer is still poor and the total mortality is around 40%. The mechanism of molecular mechanism of colorectal cancer is the basis and premise of the treatment of large intestine cancer. The study suggests that colorectal cancer stem cells are involved in the occurrence, infiltration, metastasis and drug resistance of colorectal cancer. Wnt pathway plays an important role in the self-renewal of colorectal cancer stem cells. In this study, by screening and identifying the protein involved in Wnt pathway activation in colorectal cancer stem cells, the expression of nuclear receptor-interacting protein 2 (NIP2) in colorectal cancer stem cells was increased, and the role of NIP2 in the self-renewal of colorectal cancer stem cells was detected, and the key target protein was identified. The potential target of NIP2 on colorectal cancer stem cells was found, and the effect of NIP2 on the biological behavior of colorectal cancer was confirmed. Methods: Wnt pathway activation related protein Ntri2 expression was selected by constructing SW620 clone ball cDNA reverse transcription virus library. In order to study the expression of Ntri2 in colorectal cancer, the expression of the Ntri2 protein in the pathological tissue sections of the large intestine was verified by immunohistochemistry and mRNA fluorescence in situ hybridization, and the expression of the Ntri2 in the clone balls enriched by the colorectal cancer cells and the clinical samples was detected by Real-time qPCR. In order to evaluate the role of NIP2 in the self-renewal of colorectal cancer stem cells, a stable expression of NIP2-expressing colorectal cancer cell line was constructed, and the expression of the expression of the expression of NIP2 on the cloning of the colorectal cancer cells, the expression of iPS and EMT-related factors and the in vivo tumorigenicity were observed. In order to study the mechanism of Ntri2 on the self-renewal of colorectal cancer stem cells, the target protein Frizzled 1 (Fzd1) of Trip2 was screened by mass spectrometry, and the binding sites of the Ntri2 and Fzdl proteins were analyzed by various domain mutants, as well as the binding of the Ntri2 and the downstream target molecules. In this paper, the target protein, Retinoic acid-related receptor beta, Rorp, of the NIP2 non-classical Wnt pathway was screened and identified by co-immunopreservation and Western blot, and the expression of the EMT-related factors and the shadow of the in vivo tumorigenicity were observed. In response, the downstream target gene HMG-box transcription factor 1 (HBP1) of the Rorp was screened by the gene chip ). The human colorectal cancer cells overexpressing Ntri2 were treated with PepstatinA to observe the activity of NIP2 to regulate the self-renewal of colorectal cancer stem cells. In response to that result, a reverse transcription virus library of SW620 clone was construct, and the Wnt pathway activation related protein Ntri2 was screen. Up-to-date. Ntri2 is expressed in normal large intestine tissue and large intestine cancer cells, and is expressed in colorectal cancer stem cells. Significant increases. Overexpression of NIP2 promotes the self-adaptation of colorectal cancer stem cells. I'm updating. We've found that NIP2 can be combined with Fzdl, but not with I-cati. N-binding. The combination of NIP2 with Ror is screened by the immunoprecipitation, and the activity of the enzyme can degrade the Ror antigen and prevent the Ror. The inhibition of the activity of Wnt pathway in colorectal cancer stem cells by inhibiting Wnt pathway I update. The gene chip screen found that the downstream target gene of the Ror gene is HBP1 and HBP1 inhibits the downstream target molecule of the Wnt pathway by competitive binding of TCF/ LEF4 The inactivation of the enzyme activity of NIP2 blocks the self-improvement of the NIP2 to the colorectal cancer stem cells. Conclusion: Ntri2 and Rorp were found to be expressed in normal large intestine and colorectal cancer for the first time. In the tissue cells, the expression of NIP2 in the stem cells of the colorectal cancer is increased, and the expression of the target protein downstream of the Wnt pathway is inhibited by degrading the Ror antigen, inhibiting the transcription of the HBP1, starting the expression of the downstream target protein of the Wnt pathway, and promoting the stem cell of the colorectal cancer. Self-update to mediate a new non-classical Wnt pathway, Trip2/ Ror I/ H BP1 pathway. The enzyme activity of Trip2 is the potential for intervention to block the self-renewal of colorectal cancer stem cells
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
文内图片：
图片说明：富集大肠癌细胞克隆球大肠癌细胞系HT29SW620和原代大肠癌细胞P1在无血清克隆球培养基培养7-
[Abstract]:Objective: Colorectal cancer is a common malignant tumor of the digestive tract. In recent years, although much progress has been made in chemical therapy to improve the survival rate of patients, the prognosis of patients with advanced colorectal cancer is still poor and the total mortality is around 40%. The mechanism of molecular mechanism of colorectal cancer is the basis and premise of the treatment of large intestine cancer. The study suggests that colorectal cancer stem cells are involved in the occurrence, infiltration, metastasis and drug resistance of colorectal cancer. Wnt pathway plays an important role in the self-renewal of colorectal cancer stem cells. In this study, by screening and identifying the protein involved in Wnt pathway activation in colorectal cancer stem cells, the expression of nuclear receptor-interacting protein 2 (NIP2) in colorectal cancer stem cells was increased, and the role of NIP2 in the self-renewal of colorectal cancer stem cells was detected, and the key target protein was identified. The potential target of NIP2 on colorectal cancer stem cells was found, and the effect of NIP2 on the biological behavior of colorectal cancer was confirmed. Methods: Wnt pathway activation related protein Ntri2 expression was selected by constructing SW620 clone ball cDNA reverse transcription virus library. In order to study the expression of Ntri2 in colorectal cancer, the expression of the Ntri2 protein in the pathological tissue sections of the large intestine was verified by immunohistochemistry and mRNA fluorescence in situ hybridization, and the expression of the Ntri2 in the clone balls enriched by the colorectal cancer cells and the clinical samples was detected by Real-time qPCR. In order to evaluate the role of NIP2 in the self-renewal of colorectal cancer stem cells, a stable expression of NIP2-expressing colorectal cancer cell line was constructed, and the expression of the expression of the expression of NIP2 on the cloning of the colorectal cancer cells, the expression of iPS and EMT-related factors and the in vivo tumorigenicity were observed. In order to study the mechanism of Ntri2 on the self-renewal of colorectal cancer stem cells, the target protein Frizzled 1 (Fzd1) of Trip2 was screened by mass spectrometry, and the binding sites of the Ntri2 and Fzdl proteins were analyzed by various domain mutants, as well as the binding of the Ntri2 and the downstream target molecules. In this paper, the target protein, Retinoic acid-related receptor beta, Rorp, of the NIP2 non-classical Wnt pathway was screened and identified by co-immunopreservation and Western blot, and the expression of the EMT-related factors and the shadow of the in vivo tumorigenicity were observed. In response, the downstream target gene HMG-box transcription factor 1 (HBP1) of the Rorp was screened by the gene chip ). The human colorectal cancer cells overexpressing Ntri2 were treated with PepstatinA to observe the activity of NIP2 to regulate the self-renewal of colorectal cancer stem cells. In response to that result, a reverse transcription virus library of SW620 clone was construct, and the Wnt pathway activation related protein Ntri2 was screen. Up-to-date. Ntri2 is expressed in normal large intestine tissue and large intestine cancer cells, and is expressed in colorectal cancer stem cells. Significant increases. Overexpression of NIP2 promotes the self-adaptation of colorectal cancer stem cells. I'm updating. We've found that NIP2 can be combined with Fzdl, but not with I-cati. N-binding. The combination of NIP2 with Ror is screened by the immunoprecipitation, and the activity of the enzyme can degrade the Ror antigen and prevent the Ror. The inhibition of the activity of Wnt pathway in colorectal cancer stem cells by inhibiting Wnt pathway I update. The gene chip screen found that the downstream target gene of the Ror gene is HBP1 and HBP1 inhibits the downstream target molecule of the Wnt pathway by competitive binding of TCF/ LEF4 The inactivation of the enzyme activity of NIP2 blocks the self-improvement of the NIP2 to the colorectal cancer stem cells. Conclusion: Ntri2 and Rorp were found to be expressed in normal large intestine and colorectal cancer for the first time. In the tissue cells, the expression of NIP2 in the stem cells of the colorectal cancer is increased, and the expression of the target protein downstream of the Wnt pathway is inhibited by degrading the Ror antigen, inhibiting the transcription of the HBP1, starting the expression of the downstream target protein of the Wnt pathway, and promoting the stem cell of the colorectal cancer. Self-update to mediate a new non-classical Wnt pathway, Trip2/ Ror I/ H BP1 pathway. The enzyme activity of Trip2 is the potential for intervention to block the self-renewal of colorectal cancer stem cells
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
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