MicroRNA-381靶向调控LRH-1对结肠癌细胞增殖和侵袭影响及机制的研究
发布时间：2019-05-24 03:15
【摘要】：结肠癌是一种常见的恶性肿瘤,是在内、外因素作用下发生的多基因表达失调的一种个体化疾病,严重威胁着人类生命健康。虽然大量研究已经证实,饮食和遗传因素等在结肠癌的发生和发展中起着重要作用,但结肠癌恶性转化及肿瘤细胞增殖、侵袭的关键因素还有待进一步研究确定。微小RNA(micro RNAs,mi RNA)是一类长度约19-22核苷酸的RNA,通过与靶基因m RNA的3’-UTR非编码区互补匹配来调控基因转录后表达。这种相互作用导致翻译阻遏,多数情况下降低m RNA的表达水平。研究表明,多种mi RNA在人肿瘤中表达下调或上调,包括结肠癌,这为肿瘤发生提供了新的分子基础。Tang等首先报道了mi R-381在神经胶质瘤细胞中的作用。结果表明,mi R-381可促进神经胶质瘤细胞在体外和体内的增殖,此作用与减弱抑制MEK/ERK和AKT信号有关。然而,Rothschild等发现mi R-381在肺腺癌中的表达显著下调,低mi R-381表达水平与预后不良相关。此外,mi R-381通过上调Cdc2的活性来增强5-FU在肾癌细胞中的化疗敏感性。mi R-381表达的变化也与MDR1基因的表达和多种药物耐药性的发展呈负相关。这些结果表明,mi R-381在癌细胞中的作用因肿瘤的不同而不同,因此,有必要研究其在结肠癌中的的生物学功能,为深入理解结肠癌的发生和发展开辟新的研究路径。目的:在本研究中,我们首先检测mi R-381在结肠癌组织和癌旁组织中的表达及与临床病理特征的关系,再进一步探讨mi R-381对结肠癌细胞生长、增殖和侵袭的影响,然后预测并验证mi R-381的靶基因,阐明mi R-381在结肠癌发生发展中的作用,为深入理解结肠癌发病的潜在分子机制和开发新的有效分子治疗靶点提供实验依据。方法:通过RT-PCR方法,观察mi R-381在不同临床分期的结肠癌组织及相应癌旁组织中的表达及与临床病理特征的关系;通过应用RNA干扰技术沉默mi R-381表达,观察其对结肠癌细胞株生长、增殖及侵袭的影响,以及体内成瘤能力的影响;通过应用mi RNA靶基因预测软件Target Scan、mi Randa和mi RWalk分析mi R-381的潜在靶基因,发现mi R-381能够靶向结合LRH-1 3’UTR的ACAUAUA序列,并通过双荧光素酶报告基因分析系统验证mi R-381是否与之特异结合;再以人结肠癌细胞株SW480和HCT116为研究对象,应用mi R-381沉默和mi R-381过表达技术,观察其对结肠癌细胞株LRH-1表达的影响,进一步验证mi R-381的靶点;通过应用RNA干扰技术沉默LRH-1表达,观察其对mi R-381抑癌作用的影响。结果:本课题三部分的具体结果内容如下:第一部分mi R-381在结肠癌组织和癌旁组织中的表达情况本部分实验主要利用荧光定量RT-PCR方法观察mi R-381在临床切除的结肠癌标本和癌旁组织中的表达情况。结果如下:1 mi R-381在结肠癌组织中表达情况RT-PCR结果表明mi R-381在结肠癌组织中的相对表达水平(0.50±0.08)显著下调,是癌旁组织(1.00±0.13)的0.5倍,两者差异具有统计学意义。2 mi R-381与临床病理特征的关系mi R-381在20例III期和5例IV期结肠癌中的相对表达(0.28±0.06)水平明显低于3例I期和7例II期结肠癌(1.00±0.12)组织,P0.001。并且mi R-381的表达还与肿瘤浸润深度、组织学分化程度、远处转移等因素有关(P0.05),而与患者性别、年龄因素无关,与淋巴结转移数量无关。第二部分mi R-381对结肠癌细胞生长、增殖和侵袭能力的影响本部分实验主要利用基因转染、MTT比色法、Brd U法、Transwell小室侵袭实验和体内成瘤的方法检测沉默mi R-381后结肠癌细胞的生长、增殖和侵袭能力。结果如下:1抑制mi R-381对细胞在体外生长、增殖和侵袭的影响为明确mi R-381下调在结肠癌中的作用,转染了其反义核苷酸至SW480和HCT116细胞,然后分析细胞的生长、增殖和侵袭能力。MTT比色法和Brd U掺入实验显示,抑制mi R-381显著促进细胞的生长和增殖能力。此外,这两种细胞的浸润能力也明显增强。结果表明,下调mi R-381能促进体外培养的结肠癌细胞生长、增殖和侵袭。2抑制mi R-381对肿瘤在裸鼠体内生长的影响皮下注射经筛选并鉴定稳定过表达mi R-381反义核苷酸或阴性对照的SW480细胞至裸鼠前肢皮肤。结果,抑制mi R-381明显促进小鼠体内癌细胞的生长,肿瘤的大小和重量显著增加。第三部分mi R-381通过靶向LRH-1调控结肠癌细胞的生长、增殖和侵袭本部分研究通过应用mi RNA靶基因预测软件Target Scan、mi Randa和mi RWalk分析mi R-381的潜在靶基因,发现mi R-381能够靶向结合LRH-13’UTR的ACAUAUA序列,并通过双荧光素酶报告基因分析系统验证mi R-381是否与之特异结合;再以人结肠癌细胞株SW480和HCT116为研究对象,应用mi R-381沉默和mi R-381过表达技术以及Western blot实验,检测mi R-381对靶基因LRH-1的调控作用,及沉默LRH-1表达后结肠癌细胞的生长、增殖和侵袭能力。结果如下:1 mi R-381在结肠癌细胞中的靶向调控基因通过3个生物信息学软件,我们预测并鉴定了mi R-381的调控靶标并揭示其潜在分子机制。编码肝受体类似物-1(LRH-1)的基因3’UTR区域存在一个潜在的mi R-381结合位点。Western blot实验表明,抑制mi R-381导致SW480和HCT116细胞中LRH-1的蛋白水平增加。另外,过表达mi R-381模拟物降低了这两种细胞中LRH-1的表达。因此,成功克隆了LRH-1基因的3’-UTR非编码序列,为研究mi R-381是否能与LRH-1基因的3’-UTR非编码序列特异结合,我们进一步构建了突变型3’-UTR非编码序列,分别插入荧光素酶报告基因载体。实验结果表明,抑制了mi R-381表达的结肠癌细胞株增强了携带野生型3’-UTR的荧光素酶活性,但不能增强携带突变型3’-UTR的荧光素酶活性。2沉默LRH-1对mi R-381抑癌作用的影响在SW480细胞中转染了LRH-1 si RNA后,内源性LRH-1基因表达沉默。LRH-1的内源性缺失抑制了mi R-381反义核苷酸的促进细胞生长、增殖和侵袭的作用,提示LRH-1对mi R-381在细胞生长、增殖和侵袭方面有非常重要的作用。结论:综合上述三部分内容,本研究得出如下结论:1 mi R-381在人结肠癌组织中显著下调,mi R-381的表达降低与结肠癌临床分期有关。2体外和体内研究进一步表明,抑制mi R-381促进结肠癌细胞的生长、增殖和侵袭,同时促进裸鼠体内肿瘤的生长。3 LRH-1是mi R-381的靶基因,mi R-381的下调在结肠癌的发生发展过程中起着重要的作用。4 mi R-381是结肠癌发生发展过程中一个重要的生物标志物,具有靶向LRH-1治疗癌症的潜能。
[Abstract]:Colon cancer is a common malignant tumor, which is an individual disease of multiple gene expression disorders that occur under the effects of internal and external factors, which is a serious threat to the health of human life. Although a large number of studies have confirmed that diet and genetic factors play an important role in the occurrence and development of colon cancer, the key factors for malignant transformation of colon cancer and the proliferation and invasion of tumor cells are still to be further studied and determined. MicroRNAs (micro RNAs, mi RNA) are a class of RNA with a length of about 19-22 nucleotides and regulate the post-transcriptional expression by complementary matching with the 3 '-UTR non-coding region of the target gene m RNA. This interaction results in translation repression, in most cases the level of expression of m-RNA. The study shows that the expression of multiple mi-RNA in human tumors is down-regulated or up-regulated, including colon cancer, which provides a new molecular basis for tumorigenesis. Tang et al. first reported the role of mi R-381 in glioma cells. The results show that mi R-381 can promote the proliferation of glioma cells in vitro and in vivo, which is related to the inhibition of MEK/ ERK and AKT signals. However, Rothschild et al. found that the expression of mi R-381 in lung adenocarcinoma was significantly reduced, and the low mi R-381 expression level was associated with poor prognosis. In addition, mi R-381 enhances the chemotherapy sensitivity of 5-FU in renal cancer cells by up-regulating the activity of Cdc2. The expression of mi R-381 was also negatively correlated with the expression of MDR1 gene and the development of multiple drug resistance. The results show that the role of mi R-381 in cancer cells is different from that of the tumor. Therefore, it is necessary to study the biological function of mi R-381 in the colon cancer and to open up a new research path for the in-depth understanding of the occurrence and development of colon cancer. Objective: To study the effect of mi R-381 on the growth, proliferation and invasion of colon cancer cells, and then to predict and verify the target gene of mi R-381. The role of mi R-381 in the development of colon cancer is described, and the experimental basis for the further understanding of the potential molecular mechanism of colon cancer and the development of new effective molecular therapy target is provided. Methods: The expression of mi R-381 and the expression of mi R-381 in different clinical stages and their relationship with the clinicopathological features were observed by RT-PCR. The effects of mi R-381 on the growth, proliferation and invasion of colon cancer cell lines were observed by using the RNA interference technique to silence the expression of mi R-381. The potential target gene of mi R-381 was analyzed by using the mi-RNA target gene prediction software, Target Scan, mi rana and mi RWalk, and the mi R-381 was found to be able to target the ACAUAUA sequence of LRH-1 3 'UTR, and verified whether the mi R-381 was specifically bound to it by the double-luciferase reporter gene analysis. The effects of mi R-381 and mi R-381 on the expression of LRH-1 in human colon cancer cell line SW480 and HCT116 were studied. The effect of mi R-381 on the expression of human colon cancer cell line LRH-1 was observed, and the effect of mi R-381 on the expression of mi R-381 was observed by using the RNA interference technique to silence the expression of LRH-1. Results: The specific results of the three parts of the subject are as follows: The first part mi R-381 is the expression of the first part mi R-381 in the colon cancer tissue and the adjacent tissue, and the expression of the mi R-381 in the clinical resection of the colon cancer specimen and the adjacent tissue is observed by the fluorescence quantitative RT-PCR method. The results are as follows:1 mi R-381 expression in colon cancer tissue RT-PCR results show that the relative expression level of mi R-381 in the colon cancer tissue (0.50-0.08) is significantly reduced, and is 0.5 times that of the adjacent tissue (1.00-0.13). The relative expression of mi R-381 in 20 patients with stage III and 5 patients with stage IV colon cancer (0.28-0.06) was significantly lower than that of 3 patients with stage I and 7 cases of colon cancer (1.00-0.12), P 0.001. The expression of mi R-381 was related to the depth of tumor invasion, the degree of histological differentiation, distant metastasis and other factors (P0.05), but not related to the patient's sex and age, regardless of the number of lymph node metastasis. The effects of the second part of mi R-381 on the growth, proliferation and invasion of colon cancer cells were mainly used to detect the growth, proliferation and invasion of the colon cancer cells after the silent mi R-381 was detected by the methods of gene transfection, MTT colorimetric method, Brd U method, Transwell chamber invasion experiment and in vivo tumorigenesis. The results are as follows:1. The effect of the inhibition of mi R-381 on the growth, proliferation and invasion of the cells in vitro is the effect of down-regulation of mi R-381 in the colon cancer, and the antisense nucleus of the cells is transfected into the SW480 and HCT116 cells, and then the growth, proliferation and invasion ability of the cells are analyzed. MTT and BrdU incorporation experiments showed that the inhibition of mi R-381 significantly promoted the growth and proliferation of cells. In addition, the infiltration capacity of both cells was also enhanced. The results showed that the down-regulation of mi R-381 could promote the growth, proliferation and invasion of colon cancer cells cultured in vitro. As a result, the inhibition of mi R-381 significantly promoted the growth of cancer cells in the mice, and the size and weight of the tumor significantly increased. The third part mi R-381, by targeting the growth, proliferation and invasion of colon cancer cells by targeting LRH-1, studies the potential target genes of mi R-381 by using the mi-RNA target gene prediction software, Target Scan, mi rana and mi RWk, and found that mi R-381 is capable of targeting the ACAUAUA sequence in combination with LRH-13 'UTR, The expression of mi R-381 and mi R-381 in human colon cancer cell line SW480 and HCT116 and Western blot were used to detect the regulatory effects of mi R-381 on the target gene LRH-1. And the growth, proliferation and invasion ability of colon cancer cells after the expression of the silent LRH-1. The results are as follows:1 mi R-381 targeting regulatory gene in colon cancer cells has passed 3 bioinformatics software, and we predict and identify the regulatory target of mi R-381 and reveal its potential molecular mechanism. A potential mi R-381 binding site is present in the 3 'UTR region of the gene encoding the hepatic receptor analog-1 (LRH-1). Western blot showed that the inhibition of mi R-381 resulted in an increase in the protein level of LRH-1 in SW480 and HCT116 cells. In addition, the overexpression of mi R-381 mimics the expression of LRH-1 in both cells. Therefore, the non-coding sequence of the 3 '-UTR of the LRH-1 gene is successfully cloned, and the mutant type 3'-UTR non-coding sequence is further constructed to be inserted into the luciferase reporter gene vector for studying whether the mi R-381 can be specifically combined with the 3 '-UTR non-coding sequence of the LRH-1 gene. The results showed that the expression of mi R-381 in the colon cancer cell line enhanced the luciferase activity carrying the wild type 3 '-UTR, but could not enhance the luciferase activity carrying the mutant 3'-UTR. The effect of the silence LRH-1 on the anti-cancer of the mi R-381 was after the LRH-1si RNA was transfected into the SW480 cell, The endogenous LRH-1 gene expression was silent. The endogenous deletion of LRH-1 inhibits the growth, proliferation and invasion of mi R-381 antisense, and suggests that LRH-1 plays a very important role in cell growth, proliferation and invasion. Conclusion: The conclusion is as follows:1 mi R-381 is significantly down-regulated in human colon cancer tissue, and the expression of mi R-381 is associated with the clinical stage of colon cancer. In vitro and in vivo studies have further shown that the inhibition of mi R-381 promotes the growth, proliferation and invasion of colon cancer cells, 3LRH-1 is the target gene of mi R-381, and the down-regulation of mi R-381 plays an important role in the development of colon cancer.4 mi R-381 is an important biomarker in the development of colon cancer, and has the potential to target LRH-1 for cancer.
【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】：R692
本文编号：2484499
[Abstract]:Colon cancer is a common malignant tumor, which is an individual disease of multiple gene expression disorders that occur under the effects of internal and external factors, which is a serious threat to the health of human life. Although a large number of studies have confirmed that diet and genetic factors play an important role in the occurrence and development of colon cancer, the key factors for malignant transformation of colon cancer and the proliferation and invasion of tumor cells are still to be further studied and determined. MicroRNAs (micro RNAs, mi RNA) are a class of RNA with a length of about 19-22 nucleotides and regulate the post-transcriptional expression by complementary matching with the 3 '-UTR non-coding region of the target gene m RNA. This interaction results in translation repression, in most cases the level of expression of m-RNA. The study shows that the expression of multiple mi-RNA in human tumors is down-regulated or up-regulated, including colon cancer, which provides a new molecular basis for tumorigenesis. Tang et al. first reported the role of mi R-381 in glioma cells. The results show that mi R-381 can promote the proliferation of glioma cells in vitro and in vivo, which is related to the inhibition of MEK/ ERK and AKT signals. However, Rothschild et al. found that the expression of mi R-381 in lung adenocarcinoma was significantly reduced, and the low mi R-381 expression level was associated with poor prognosis. In addition, mi R-381 enhances the chemotherapy sensitivity of 5-FU in renal cancer cells by up-regulating the activity of Cdc2. The expression of mi R-381 was also negatively correlated with the expression of MDR1 gene and the development of multiple drug resistance. The results show that the role of mi R-381 in cancer cells is different from that of the tumor. Therefore, it is necessary to study the biological function of mi R-381 in the colon cancer and to open up a new research path for the in-depth understanding of the occurrence and development of colon cancer. Objective: To study the effect of mi R-381 on the growth, proliferation and invasion of colon cancer cells, and then to predict and verify the target gene of mi R-381. The role of mi R-381 in the development of colon cancer is described, and the experimental basis for the further understanding of the potential molecular mechanism of colon cancer and the development of new effective molecular therapy target is provided. Methods: The expression of mi R-381 and the expression of mi R-381 in different clinical stages and their relationship with the clinicopathological features were observed by RT-PCR. The effects of mi R-381 on the growth, proliferation and invasion of colon cancer cell lines were observed by using the RNA interference technique to silence the expression of mi R-381. The potential target gene of mi R-381 was analyzed by using the mi-RNA target gene prediction software, Target Scan, mi rana and mi RWalk, and the mi R-381 was found to be able to target the ACAUAUA sequence of LRH-1 3 'UTR, and verified whether the mi R-381 was specifically bound to it by the double-luciferase reporter gene analysis. The effects of mi R-381 and mi R-381 on the expression of LRH-1 in human colon cancer cell line SW480 and HCT116 were studied. The effect of mi R-381 on the expression of human colon cancer cell line LRH-1 was observed, and the effect of mi R-381 on the expression of mi R-381 was observed by using the RNA interference technique to silence the expression of LRH-1. Results: The specific results of the three parts of the subject are as follows: The first part mi R-381 is the expression of the first part mi R-381 in the colon cancer tissue and the adjacent tissue, and the expression of the mi R-381 in the clinical resection of the colon cancer specimen and the adjacent tissue is observed by the fluorescence quantitative RT-PCR method. The results are as follows:1 mi R-381 expression in colon cancer tissue RT-PCR results show that the relative expression level of mi R-381 in the colon cancer tissue (0.50-0.08) is significantly reduced, and is 0.5 times that of the adjacent tissue (1.00-0.13). The relative expression of mi R-381 in 20 patients with stage III and 5 patients with stage IV colon cancer (0.28-0.06) was significantly lower than that of 3 patients with stage I and 7 cases of colon cancer (1.00-0.12), P 0.001. The expression of mi R-381 was related to the depth of tumor invasion, the degree of histological differentiation, distant metastasis and other factors (P0.05), but not related to the patient's sex and age, regardless of the number of lymph node metastasis. The effects of the second part of mi R-381 on the growth, proliferation and invasion of colon cancer cells were mainly used to detect the growth, proliferation and invasion of the colon cancer cells after the silent mi R-381 was detected by the methods of gene transfection, MTT colorimetric method, Brd U method, Transwell chamber invasion experiment and in vivo tumorigenesis. The results are as follows:1. The effect of the inhibition of mi R-381 on the growth, proliferation and invasion of the cells in vitro is the effect of down-regulation of mi R-381 in the colon cancer, and the antisense nucleus of the cells is transfected into the SW480 and HCT116 cells, and then the growth, proliferation and invasion ability of the cells are analyzed. MTT and BrdU incorporation experiments showed that the inhibition of mi R-381 significantly promoted the growth and proliferation of cells. In addition, the infiltration capacity of both cells was also enhanced. The results showed that the down-regulation of mi R-381 could promote the growth, proliferation and invasion of colon cancer cells cultured in vitro. As a result, the inhibition of mi R-381 significantly promoted the growth of cancer cells in the mice, and the size and weight of the tumor significantly increased. The third part mi R-381, by targeting the growth, proliferation and invasion of colon cancer cells by targeting LRH-1, studies the potential target genes of mi R-381 by using the mi-RNA target gene prediction software, Target Scan, mi rana and mi RWk, and found that mi R-381 is capable of targeting the ACAUAUA sequence in combination with LRH-13 'UTR, The expression of mi R-381 and mi R-381 in human colon cancer cell line SW480 and HCT116 and Western blot were used to detect the regulatory effects of mi R-381 on the target gene LRH-1. And the growth, proliferation and invasion ability of colon cancer cells after the expression of the silent LRH-1. The results are as follows:1 mi R-381 targeting regulatory gene in colon cancer cells has passed 3 bioinformatics software, and we predict and identify the regulatory target of mi R-381 and reveal its potential molecular mechanism. A potential mi R-381 binding site is present in the 3 'UTR region of the gene encoding the hepatic receptor analog-1 (LRH-1). Western blot showed that the inhibition of mi R-381 resulted in an increase in the protein level of LRH-1 in SW480 and HCT116 cells. In addition, the overexpression of mi R-381 mimics the expression of LRH-1 in both cells. Therefore, the non-coding sequence of the 3 '-UTR of the LRH-1 gene is successfully cloned, and the mutant type 3'-UTR non-coding sequence is further constructed to be inserted into the luciferase reporter gene vector for studying whether the mi R-381 can be specifically combined with the 3 '-UTR non-coding sequence of the LRH-1 gene. The results showed that the expression of mi R-381 in the colon cancer cell line enhanced the luciferase activity carrying the wild type 3 '-UTR, but could not enhance the luciferase activity carrying the mutant 3'-UTR. The effect of the silence LRH-1 on the anti-cancer of the mi R-381 was after the LRH-1si RNA was transfected into the SW480 cell, The endogenous LRH-1 gene expression was silent. The endogenous deletion of LRH-1 inhibits the growth, proliferation and invasion of mi R-381 antisense, and suggests that LRH-1 plays a very important role in cell growth, proliferation and invasion. Conclusion: The conclusion is as follows:1 mi R-381 is significantly down-regulated in human colon cancer tissue, and the expression of mi R-381 is associated with the clinical stage of colon cancer. In vitro and in vivo studies have further shown that the inhibition of mi R-381 promotes the growth, proliferation and invasion of colon cancer cells, 3LRH-1 is the target gene of mi R-381, and the down-regulation of mi R-381 plays an important role in the development of colon cancer.4 mi R-381 is an important biomarker in the development of colon cancer, and has the potential to target LRH-1 for cancer.
【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】：R692
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