沉默Musashi1基因对结肠癌细胞生物学特性及放疗敏感性的影响

发布时间：2018-04-30 02:14

  本文选题：结肠癌 + Musashi1　； 参考：《河北医科大学》2016年博士论文

【摘要】：结肠癌是全球范围内消化道最常见的恶性肿瘤之一,其发病率在我国仅次于胃癌和食道癌,位居第三,且近年来发病率持续升高,预后差。有关其发病机制及防治措施的研究是生物医学领域研究的热点之一。结肠癌的发生是在不同遗传背景下由致癌基因的激活和抑癌基因的失活共同作用的结果。转录后调控是基因表达的关键部分,这一过程主要受RNA结合蛋白(RNA-Binding Proteins,RBPs)的调节。业已证实,人体基因组中包含有800多种RBPs,其中的几种被发现控制着与癌变过程相关的基因网络。对RBPs深入的研究逐步揭示,它们与转录因子类似,具有抑癌和促癌作用。Musashi1(Msi1)作为一种进化保守的RNA结合蛋白家族成员,对于维持自我更新与分化之间的平衡具有重要作用。众多研究表明,Msi1可作为一种致癌基因,也是多种肿瘤干细胞和(或)祖细胞的重要标记物,能刺激肿瘤干、祖细胞的形成和发展。过度表达Msi1的肠上皮祖细胞可通过激活Wnt、Notch信号通路及调节p21这三个作用靶点,使细胞增殖旺盛,促进祖细胞的致瘤性,增加移植瘤的致瘤性。一般认为,肿瘤干细胞对放化疗抗拒,是肿瘤发生、发展和复发、转移的根源。Msi1可能通过促进β-链蛋白(β-catenin)的核内聚,增加结肠癌放疗的不敏感性,促进侵袭性肿瘤发展。此外Msi1位于多个信号通路的交汇点,是细胞基因表达的关键调节器,在细胞发育和内环境稳态中发挥重要作用。同时,Msi1也可作为多种癌变的关键调节剂,参与肿瘤的发生和发展。Msi1有望成为防治癌症的一个分子靶点。因此,确定Msi1在结肠癌中的作用及其机制具有重要的理论意义和潜在的应用价值。但目前有关Msi1对结肠癌细胞的作用及其机制尚不明确。本研究旨在探讨Msi1在结肠癌发生、发展中的作用及其机制,以及对放疗敏感性的影响。本课题研究将分四部分进行。1.首先在结肠癌患者的癌组织和结肠癌细胞系证实Msi1的高表达,通过RNA干扰技术构建沉默msi1稳转细胞系;2.探讨沉默msi1对结肠癌细胞生物学特性的影响;3.探讨沉默msi1对结肠癌细胞生长影响的分子机制;4.探讨沉默msi1对结肠癌细胞放疗敏感性的影响。第一部分结肠癌组织和细胞中musashi1蛋白表达及musashi1稳定低表达结肠癌细胞系构建目的:检测musashi1在结肠癌组织和结肠癌细胞系的表达水平;构建msi1稳定低表达结肠癌细胞系。方法:采用westernblot方法检测结肠癌患者癌组织、癌周正常组织、以及hct116、sw480和sw620三种结肠癌细胞系中msi1的蛋白表达,挑选msi1表达水平最高的结肠癌细胞系进行后续研究。应用rna干扰技术设计沉默msi1的基因序列及阴性对照序列,通过与质粒连接后感染工具细胞,将干扰质粒及阴性对照质粒植入慢病毒载体,随后感染结肠癌细胞;经过抗生素筛选获得稳定低表达细胞株,并运用real-timepcr及westernblot的方法从rna水平和蛋白水平检测空白组(blank)、阴性对照组(negativecontrol,nc)和敲低组(knockdown,kd)hct116结肠癌细胞基因沉默的效果。结果:通过westernblot方法检测,发现与正常肠粘膜相比,结肠癌组织及三种结肠癌细胞株中msi1蛋白均特异性高表达;在hct116、sw480、sw620三种结肠癌细胞株中,hct116细胞msi1蛋白的表达量最高,因此选取hct116细胞作为后续研究的目的细胞。本研究中,成功构建了带有干扰msi1基因序列gv248-msi1-shrna的慢病毒及阴性对照慢病毒gv248-nc-shrna,将干扰慢病毒及阴性对照慢病毒感染结肠癌hct116细胞,利用嘌呤霉素筛选出稳定的表达株。同时real-timepcr和westernblot结果显示,感染msi1干扰慢病毒载体后结肠癌hct116细胞msi1在rna水平和蛋白水平分别下降了73.85%和59.01%。小结:结肠癌组织及结肠癌细胞株中msi1蛋白均特异性高表达。利用结肠癌hct116细胞株可成功构建稳定低表达msi1细胞系。第二部分沉默musashi1对结肠癌hct116细胞生物学特性的影响目的:探讨沉默musashi1对结肠癌hct116细胞生物学特性的影响。方法:利用构建稳定低表达msi1的结肠癌hct116细胞,应用mts法检测沉默msi1后结肠癌细胞增殖情况,应用流式细胞仪检测细胞周期及凋亡的变化,transwell小室检测其体外侵袭能力,肿瘤球实验检测肿瘤细胞干性。并通过建立裸鼠移植瘤模型,观察沉默msi1对裸鼠成瘤的影响。结果:mts法及transwell小室检测结果显示,沉默msi1后,结肠癌hct116细胞体外增殖及侵袭能力显著下降。流式细胞检测结果显示,沉默msi1后肿瘤细胞的凋亡明显增加,g0/g1期细胞增多。肿瘤球实验结果显示,沉默msi1可明显降低肿瘤球形成的数量。结肠癌hct116细胞裸鼠皮下移植瘤结果显示,降低msi1表达可明显抑制移植瘤的生长。小结:沉默msi1能抑制结肠癌hct116细胞体外增殖能力和侵袭能力,导致细胞g0/g1期阻滞,诱导细胞凋亡。沉默msi1可明显降低结肠癌hct116细胞的干性,抑制移植瘤的生长。第三部分沉默musashi1对结肠癌hct116细胞生长抑制的分子机制目的:探讨沉默musashi1抑制结肠癌hct116细胞生长的分子机制。方法:利用real-timepcr和westernblot技术检测沉默msi1基因后细胞周期蛋白依赖性激酶抑制物p21(抑癌基因)mrna和蛋白的表达水平,并构建p213’-utr荧光素酶报告基因载体(p21wt)及其突变体(p21mu),将报告基因载体转染沉默msi1的细胞和对照组细胞株,计算msi1敲低组细胞与对照组细胞的荧光比值,并进行比较以判断p213’-utr区活性。结果:westernblot方法检测发现,与空白组及阴性对照组相比较,沉默msi1后结肠癌hct116细胞的p21蛋白表达明显上调;real-timepcr结果显示,3组细胞p21mrna水平的表达未见明显变化。进一步荧光素酶报告实验结果显示,与空白组、阴性对照组相比,敲低组p21wt的荧光素酶活性明显上升,msi1反应位点突变体p21mu的荧光素酶活性则没有明显变化。小结:结肠癌hct116细胞中,msi1能够与其靶基因p21mrna的3’-utr区特异性的结合,并抑制其翻译,下调p21蛋白表达,进而促进肿瘤细胞生长。沉默msi1后可通过上调p21蛋白的表达,发挥抑制肿瘤生长作用。第四部分沉默musashi1对结肠癌hct116细胞放疗敏感性的影响目的:探讨沉默musashi1对结肠癌hct116细胞放疗敏感性的影响。方法:采用慢病毒介导的Msi1干扰表达质粒感染HCT116细胞;对感染的HCT116细胞进行X线照射;运用克隆实验检测沉默Msi1基因对HCT116细胞放射敏感性的影响。通过MTS法检测照射后结肠癌细胞增殖情况,并计算其抑制率。运用流式细胞术检测照射后细胞周期及凋亡的变化。结果:克隆形成实验显示,经0~8Gy X线照射后,敲低组细胞存活曲线下降,D0、Dq、N和SF2值均明显低于空白组和阴性对照组,相对于空白组和阴性对照组放射增敏比SER分别为1.56和1.47。MTS试验结果显示,各时间段敲低组细胞的增殖活性均显著低于空白组和阴性对照组,而且随着时间和剂量的增加,各组抑制率逐渐增加。进一步流式细胞术显示,照射后各组细胞凋亡率明显增加,并呈现时间依赖性,随着时间延长,凋亡率逐渐增加。敲低组凋亡比例在各时间点增加的程度更加明显。并且照射后敲低组细胞G2/M期比例较空白组和阴性对照组显著下降。小结:抑制结肠癌HCT116细胞Msi1基因表达具有放疗增敏作用,其增敏机制可能是通过促进细胞凋亡,解除放疗后细胞G2/M期阻滞,从而增加结肠癌HCT116细胞的放疗敏感性。结论:1结肠癌组织及结肠癌细胞株中Msi1蛋白均特异性高表达,提示Msi1可能在结肠癌发生与发展中起重要作用;2利用结肠癌HCT116细胞可成功构建稳定低表达Msi1的细胞株;3沉默Msi1可抑制结肠癌HCT116细胞增殖和侵袭能力,导致细胞G0/G1期阻滞,诱导细胞凋亡,降低细胞干性,抑制移植瘤的生长;4沉默Msi1可上调结肠癌HCT116细胞Msi1的靶基因p21蛋白的表达,发挥抑制肿瘤生长的作用;5沉默Msi1可通过促进结肠癌HCT116细胞凋亡,解除放疗后细胞G2/M期阻滞,发挥放疗增敏作用。
[Abstract]:Colon cancer is one of the most common malignant tumors in the digestive tract around the world. Its incidence is third only second to gastric cancer and esophagus cancer in our country. In recent years, the incidence of cancer is increasing and the prognosis is poor. The research on its pathogenesis and prevention measures is one of the hot spots in the field of biomedical research. The occurrence of colon cancer is in different heredity In the background, the result of the activation of the oncogene and the inactivation of the tumor suppressor gene. Post transcriptional regulation is the key part of gene expression. This process is mainly regulated by the RNA binding protein (RNA-Binding Proteins, RBPs). It has been confirmed that there are more than 800 kinds of RBPs in the human genome, several of which have been found to be controlled and cancerous. RBPs in-depth studies have gradually revealed that they are similar to transcriptional factors..Musashi1 (Msi1), a member of the conserved RNA binding protein family, has an important role in maintaining the balance between self renewal and differentiation. Many studies have shown that Msi1 can be used as a carcinogenic basis. Because it is also an important marker for a variety of cancer stem and / or progenitor cells, it can stimulate the formation and development of tumor stem and progenitor cells. Overexpression of Msi1's upper intestinal progenitor cells can stimulate the proliferation of cells by activating Wnt, Notch signaling pathway and regulating the three targets of p21, which can promote the tumorigenicity of the progenitor cells and increase the tumorigenicity of the transplanted tumor. It is generally believed that tumor stem cells are resistant to radiotherapy and chemotherapy, which is tumor occurrence, development and recurrence. The origin of metastasis,.Msi1, may increase the nuclear cohesion of beta chain protein (beta -catenin), increase the insensitivity of colon cancer radiotherapy, and promote the development of invasive tumor. In addition, Msi1 is the key point of cell gene expression at the intersection of multiple signal pathways. The node plays an important role in cell development and internal environment homeostasis. At the same time, Msi1 can also be a key regulator of multiple carcinogenesis..Msi1 is expected to be a molecular target for the prevention and control of cancer. Therefore, the role of Msi1 in colon cancer and its mechanism have important theoretical significance and potential application price. However, the effect of Msi1 on colon cancer cells and its mechanism are not yet clear. The purpose of this study is to explore the role and mechanism of Msi1 in the development of colon cancer and its effect on the sensitivity of radiotherapy. The study will be divided into four parts for.1., first of all, in cancer tissue and colon cancer cell lines in colon cancer patients to confirm the high table of Msi1 To construct silenced msi1 stable cell lines by RNA interference technique; 2. to investigate the effect of silent msi1 on the biological characteristics of colon cancer cells; 3. to explore the molecular mechanism of the effect of silent msi1 on the growth of colon cancer cells; 4. to explore the effect of silent msi1 on the sensitivity of colon cancer cells. Part one colon cancer tissue and cell musashi1 protein table Construction of musashi1 stable and low expression colon cancer cell lines: to detect the expression level of musashi1 in colon and colon cancer cell lines and to construct msi1 stable low expression colon cancer cell lines. Methods: Westernblot method was used to detect cancer tissue of colon cancer patients, cancer Zhou Zhengchang tissue, and HCT116, SW480 and SW620 three types of colon cancer The protein expression of msi1 in the cell line and the follow-up study of the colon cancer cell lines with the highest msi1 expression level were selected. The RNA interference technique was used to design the gene sequence and negative control sequence of the silent msi1. The plasmid and negative control particles were implanted into the lentivirus vector and then infected with the colon cancer cells after the infection of the plasmid with the plasmid. A stable low expression cell line was obtained by antibiotic screening, and the real-timepcr and Westernblot methods were used to detect the blank group (blank) from RNA level and protein level, and the effect of silence on the HCT116 colon cancer cell based on the negative control group (negativecontrol, NC) and the knockout group (knockdown, KD). Results: detection by Westernblot method was found. Msi1 protein was highly expressed in colon and three colon cancer cell lines in normal intestinal mucosa; the expression of msi1 protein in HCT116 cells was the highest in the three colon cancer cell lines of HCT116, SW480, and SW620. Therefore, HCT116 cells were selected as the purpose of the follow-up study. In this study, the msi1 gene was successfully constructed. The lentivirus and negative control lentivirus gv248-nc-shrna of sequence gv248-msi1-shrna will interfere with the HCT116 cells of colonic cancer infected by lentivirus and negative control lentivirus, and use purinamycin to screen out the stable expression strain. Meanwhile, real-timepcr and Westernblot results show that the HCT116 cell msi1 of colon cancer is in RNA after the infection of msi1 dry virus carrier. The level and protein level decreased by 73.85% and 59.01%., respectively: msi1 protein was highly expressed in colon and colon cancer cell lines. A stable low expression msi1 cell line was successfully constructed using HCT116 cell line of colon cancer. The effect of second partially silent musashi1 on the cell biological characteristics of colon cancer HCT116: To explore the silent Mu The effect of sashi1 on the biological characteristics of colon cancer HCT116 cells. Methods: using the construction of HCT116 cells with stable low expression of msi1, the proliferation of colon cancer cells after silent msi1 was detected by MTS method. The changes of cell cycle and apoptosis were detected by flow cytometry. The invasiveness of the cells in vitro was detected by the Transwell compartment and the tumor ball test was detected. The effect of silent msi1 on tumor formation in nude mice was observed through the establishment of a nude mouse model. Results: the results of MTS and Transwell cell detection showed that after silent msi1, the proliferation and invasion ability of HCT116 cells in colon cancer decreased significantly in vitro. Flow cytometry results showed that the apoptosis of tumor cells after silent msi1 increased obviously. The results of tumor ball test showed that silencing of msi1 could significantly reduce the number of tumor formation. The result of subcutaneous transplantation of HCT116 cells in HCT116 cells of colon cancer showed that the decrease of msi1 expression could obviously inhibit the growth of the transplanted tumor. The silence msi1 could inhibit the proliferation and invasion ability of HCT116 cells in colon cancer and lead to cell G 0/g1 phase arrest, inducing cell apoptosis. Silence msi1 can obviously reduce the stem of colon cancer HCT116 cells and inhibit the growth of xenografts. Third the molecular mechanism of silent musashi1 on the growth inhibition of colon cancer HCT116 cells: To explore the molecular mechanism of silence musashi1 to inhibit the growth of HCT116 cells in colon cancer. Methods: using real-timepcr and West Ernblot technique detected the expression level of mRNA and protein of cell cyclin dependent kinase inhibitor p21 (tumor suppressor gene) after silencing the msi1 gene, and constructed P213 '-utr luciferase reporter gene vector (p21wt) and its mutant (p21mu), and transfected the reporter gene vector into the cells of the silent msi1 and the control group, and calculated the cells of the msi1 knockout group. The fluorescence ratio of the cells in the control group was compared to determine the activity of P213 '-utr region. Results: the results of Westernblot method detection showed that the expression of p21 protein in HCT116 cells of colon cancer after silent msi1 was up to be up obviously compared with the blank group and negative control group. The results of real-timepcr showed that the expression of p21mrna level in the 3 groups did not change obviously. The results of further luciferase reporter assay showed that the luciferase activity of p21wt in the knockout group was significantly higher than that in the blank group and the negative control group, and the luciferase activity of the msi1 reaction site mutant p21mu was not significantly changed. Msi1, in colon cancer HCT116 cells, was enough to bind to the 3 '-utr region specific to the target gene p21mrna. And inhibit its translation, down regulate the expression of p21 protein, and then promote the growth of tumor cells. After silent msi1, the expression of p21 protein can be used to inhibit the growth of tumor. The effect of the fourth part of silence musashi1 on the radiotherapy sensitivity of colon cancer HCT116 cells: To explore the effect of silent musashi1 on the radiotherapy sensitivity of colon cancer HCT116 cells Methods: HCT116 cells were infected with lentivirus mediated Msi1 interference expression plasmid, the infected HCT116 cells were irradiated by X-ray, and the effect of silent Msi1 gene on the radiosensitivity of HCT116 cells was detected by cloning experiment. The proliferation of colon cancer cells after irradiation was detected by MTS method and the inhibition rate was calculated. Flow cytometry was used to detect the cell proliferation. Changes in cell cycle and apoptosis after irradiation. Results: the clone formation experiment showed that after 0~8Gy X-ray irradiation, the survival curve of the cells in the knock low group decreased, and the values of D0, Dq, N and SF2 were significantly lower than those in the blank group and the negative control group. The radiosensitivity ratio of the blank group and the negative control group was 1.56 and the 1.47.MTS test results showed, respectively, at each time period. The proliferation activity of the cells in the knockout group was significantly lower than that in the blank group and the negative control group, and the inhibition rate of each group increased gradually with the increase of time and dose. Further flow cytometry showed that the apoptosis rate of each group increased obviously after irradiation, and the apoptosis rate gradually increased with time. The proportion of the G2/M phase in the lower group after irradiation was significantly lower than that in the blank group and the negative control group. A summary: the inhibition of Msi1 gene expression in colon cancer HCT116 cells has the effect of radiation sensitization, and its sensitizing mechanism may be by promoting cell withering and relieving G2/M block after radiotherapy, thus increasing the growth of cells. Conclusion: the Msi1 protein in 1 colon and colon cancer cell lines is highly expressed, suggesting that Msi1 may play an important role in the development and development of colon cancer; 2 the cell lines with low expression of Msi1 can be successfully constructed by using colon cancer HCT116 cells, and 3 silenced Msi1 can inhibit the HCT116 fine of colon cancer. Cell proliferation and invasion can induce cell G0/G1 phase block, induce cell apoptosis, reduce cell stem and inhibit the growth of transplanted tumor. 4 silent Msi1 can up regulate the expression of p21 protein of Msi1 target gene of colon cancer HCT116 cells and play the role of inhibiting tumor growth; 5 silent Msi1 can promote apoptosis of colon cancer HCT116 cells and remove the fine after radiotherapy. Cell G2/M phase block, and play the sensitizing effect of radiotherapy.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.35

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