miR-29a通过靶向调节AKT3抑制甲状腺乳头状癌生长与转移的研究

发布时间：2018-05-12 00:08

本文选题：甲状腺乳头状癌 + miR-29a　；参考：《吉林大学》2016年博士论文

【摘要】：甲状腺癌是最常见的内分泌系统恶性肿瘤,占全部内分泌恶性肿瘤总数的94.5%,其发病率在头颈部恶性肿瘤中居首位。在过去的几十年间,其发病率一直呈稳步上升趋势。甲状腺乳头状癌(PTC)是一种公认的分化较好的恶性肿瘤,也是最常见的甲状腺恶性肿瘤,约占甲状腺癌总数的80%~90%。多数PTC患者预后良好,但总体复发率仍可高达35%,因为目前的手段还不能准确地预测其临床转归和生物学行为。因此,我们迫切的需要了解甲状腺乳头状癌发生发展的分子机制,并且找到一系列崭新的有助于PTC的及时诊断、判断预后和有效治疗的从宏观到微观的诊疗思路。微小RNA(mi RNAs)是一类非编码小RNA,长度约在19~25个核苷酸。可与信使RNA(m RNAs)的3’非编码区(3’-UTR)互补结合,在转录水平或转录后水平调节基因的表达。目前研究发现mi RNAs可能可以调节上千种人类基因的转录与表达,并且,越来越多的证据表明,mi RNAs还可以调节许多基础生理过程,如细胞的分化、增殖和存活,以及细胞凋亡、迁移和侵袭等。因为mi RNAs可以参与调节细胞增殖、分化与凋亡,所以认为它与癌症的发生与发展有着密切关系。进而,分析mi RNAs及其靶基因不但把我们对肿瘤发生发展的认识引入到了一个独特而崭新的视角,而且为PTC的诊断和治疗提供了新的切入点。mi R-29a是mi R-29s家族中的一员,这是一个保守的mi RNA家族,mi R-29s在一些癌症中表达减少,如胃癌、胰腺癌和前列腺癌等;恰恰相反,mi R-29s在另一些癌症中的表达是上升的,如乳腺癌、鼻咽癌、神经胶质瘤和急性髓性白血病等。这些研究表明,mi R-29a可能在不同的癌症中发挥着促进或抑制肿瘤发生发展的作用。然而,它在PTC中的临床意义、作用和潜在分子机制仍不十分清楚,在本研究中,我们分析了mi R-29a的表达与PTC患者临床病理特征之间的关系,并且通过一系列体外和体内实验来研究mi R-29a在PTC中发挥的作用和潜在的机制。目的:明确mi R-29a在甲状腺乳头状癌发生发展中的作用及机制方法:1.研究mi R-29a在PTC中的表达水平及其在增殖和迁移中的作用和机制(1)本实验通过q RT-PCR对30组PTC肿瘤患者组织标本及其邻近的正常组织中的mi R-29a的表达进行的分析,为了进一步研究mi R-29a表达水平在PTC患者中的临床病理学意义,我们选取中位数(0.59)为分界点,将30个PTC标本分为mi R-29a相对低水平组(0.59,14 cases)和mi R-29a相对高水平组(0.59,16 cases),然后将mi R-29a的表达与临床病理学参数使用卡方检验分析。(2)为了阐明mi R-29a在PTC中的生物学功能,我们将mi R-29a模拟物转染到K1细胞系中,并获得稳定表达mi R-29a的K1细胞系。经过q RT-PCR证实mi R-29a的表达水平在K1细胞系中确实增加,然后将mi R-29a模拟物和mi R-Ctrl分别转染到K1细胞系中用以检测细胞增殖、细胞周期和细胞凋亡。(3)通过伤口愈合实验和肿瘤细胞侵袭实验来研究mi R-29a调节PTC细胞侵袭和迁移的作用。2.mi R-29a通过调节AKT3而抑制PTC生长的机制研究(1)我们使用生物学信息数据库如Target Scan,mi Randa和Pic Tar对mi R-29a的潜在靶基因进行预测,AKT3被锁定为研究目标,因为它在PI3K/AKT信号通路中发挥重要作用并且涉及多种细胞功能。为了验证AKT3是否是mi R-29a在PTC中的直接目标,一个人类AKT3 3’UTR包含mi R-29a结合位点的片段或克隆了突变位点的PGL3载体,连同mi R-29a模拟物或mi R-Ctrl共转染进K1细胞,并孵育48h,然后检测荧光素酶的活性。通过q RT-PCR和western blotting实验分别从m RNA水平和蛋白质水平检测mi R-29a过表达对K1细胞中的AKT3表达水平的影响。(2)已经明确AKT3是mi R-29a的靶基因,我们继续使用q RT-PCR研究AKT3在PTC组织及其临近正常组织中的表达量。(3)为了进一步探究AKT3在PTC中的生物学作用,我们将si-AKT3和si-Ctrl分别转染进K1细胞,使用q RT-PCR和western blot验证AKT3的敲除效率。使用si-AKT3沉默AKT3基因后,继续使用MTT比色法检测细胞增殖、流式细胞术检测细胞凋亡、伤口愈合实验检测细胞迁移和肿瘤细胞侵袭实验检测细胞侵袭。(4)为了进一步研究AKT3针对于mi R-29a的相关功能,我们设计了一系列实验,对于当AKT3过表达时,是否会对mi R-29a在PTC细胞增殖、凋亡、迁移和侵袭等作用产生影响进行评估。为此,AKT3过表达质粒与mi R-29a模拟物或mi R-Ctrl一同转染到K1细胞中。接着使用MTT比色法检测细胞增殖、流式细胞术检测细胞凋亡、伤口愈合实验检测细胞迁移和肿瘤细胞侵袭实验检测细胞侵袭能力。3.mi R-29a在体内抑制PTC生长的实验研究体外实验显示miR-29a有抑制肿瘤生长的作用,我们进而对它的作用在体内进行了评估。为了这一目的,我们将稳定表达mi R-29a或mi R-Ctrl的K1细胞种植到裸鼠皮下,使其形成肿瘤。在注射后30天,将动物处死,剥取肿瘤组织。观察并测量两组肿瘤的大小和重量。此外,我们还通过q RT-PCR测定了mi R-29a和AKT3在肿瘤组织中的表达,接着通过q RT-PCR和western blotting实验分别从m RNA水平和蛋白质水平测定AKT3的表达水平。结果:1.mi R-29a在PTC中的表达水平及其在增殖和迁移中的作用及机制(1)mi R-29a在人PTC组织中表达下调PTC组织中的mi R-29a的相对表达量要明显低于其邻近正常组织(P0.01)。我们选取中位数(0.59)为分界点,将30个PTC标本分为mi R-29a相对低水平组(0.59,14 cases)和mi R-29a相对高水平组(0.59,16 cases),然后将mi R-29a的表达与临床病理学参数使用卡方检验分析。分析显示mi R-29a的表达与年龄和性别的相关性没有统计学意义,同时,mi R-29a的水平与TNM分期(P0.01)、肿瘤大小(P0.01)和淋巴结转移(P0.01)呈负相关趋势。(2)mi R-29a可抑制PTC细胞增殖并诱导凋亡经过q RT-PCR证实mi R-29a的表达水平在K1细胞系中确实增加(p0.01),MTT比色法显示mi R-29a可显著抑制K1细胞的增殖,与转染了mi R-Ctrl组对比,在48h时P0.05,而72h时P0.01,具有统计学意义。细胞周期分析显示,与转染了mi R-Ctrl组对比,恢复了mi R-29a的K1细胞中的S期的比例降低(P0.05),而G0/G1期比例显著升高(P0.01)。细胞凋亡实验显示在K1细胞系中转染了mi R-29a模拟物可明显促进细胞凋亡,与转染mi R-Ctrl组对比,P0.01。(3)miR-29a可抑制PTC细胞侵袭和迁移与mi R-Ctrl组比较,增加miR-29a的表达水平可显著抑制K1细胞的侵袭和转移的能力(p0.01)。2.mi R-29a通过调节AKT3而抑制PTC生长的机制研究(1)AKT3是mi R-29a的直接靶基因miR-29a过表达明显抑制了野生型AKT3位点的荧光素酶活性,但是突变型AKT3位点的荧光素酶活性并没有改变。这表明AKT3是mi R-29a的直接靶基因。然后通过q RT-PCR和western blotting实验证实mi R-29a过表达可彻底地抑制K1细胞中的AKT3表达水平,无论是从m RNA水平还是从蛋白质水平。另外,我们还发现,过表达的mi R-29a可抑制磷酸化AKT(p-AKT)的表达,这提示mi R-29a可以抑制PI3K/AKT信号转导通路的激活。以上结果表明,mi R-29a可直接与AKT3结合并抑制其表达。(2)在PTC组织中mi R-29a的表达与AKT3的表达是负性相关的与周围临近正常组织相比,AKT3的m RNA表达量在PTC组织中是升高的(P0.01)。并且,在PTC组织中,AKT3的表达量与mi R-29a的表达量是负性相关的(P0.01)。(3)下调AKT3的表达可模拟mi R-29a过表达的效果使用si-AKT3下调K1细胞系中的AKT3的表达可抑制细胞增殖并且诱导细胞凋亡,同时也可以抑制肿瘤细胞的迁徙和侵袭能力。换言之,减少AKT3的表达可模拟mi R-29a抑制肿瘤的效果。(4)AKT3过表达可逆转mi R-29a的作用AKT3过表达质粒可以在m RNA水平以及蛋白质水平上增加AKT3的表达。AKT3在K1细胞中过表达可以促进肿瘤细胞增殖、迁移和侵袭、抑制细胞凋亡,即AKT3过表达可逆转mi R-29a抑制肿瘤的作用。3.mi R-29a在体内抑制PTC生长与注射K1/miR-Ctrl组对比,注射K1/miR-29a组的肿瘤无论是在大小还是重量上都要明显减少(P0.01)。此外,肿瘤组织中的mi R-29a的表达水平要明显升高(P0.01),然而,AKT3的表达水平却显著地下降,无论是在m RNA水平(P0.01)还是蛋白质水平。结论:1.miR-29a的表达水平在PTC组织中是减少的,并且其表达水平与肿瘤的大小、TNM分期和是否有淋巴结转移有着密切的关系。2.mi R-29a过表达可显著抑制PTC细胞增殖、迁移和侵袭,并且促进PTC细胞凋亡,使细胞周期停滞在G0/G1期。体内实验证实,mi R-29a过表达可通过调节AKT3降低异种肿瘤移植裸鼠模型中的肿瘤生长。3.在PTC细胞中,miR-29a可与AKT3的3’非编码区直接结合,过表达的mi R-29a可显著抑制AKT3的表达,从而阻止(PI3K)/AKT信号转导途径的激活。4.在PTC组织中,AKT3的表达是增加的,并且其表达与miR-29a的表达是呈负相关的。使用si RNA降低AKT3的表达可模拟mi R-29a过表达的效果,反之,上调AKT3的表达可部分逆转mi R-29a抑制肿瘤生长的作用。
[Abstract]:Thyroid carcinoma is the most common malignant tumor of the endocrine system, accounting for 94.5% of the total number of endocrine malignant tumors. The incidence of thyroid cancer is the first in the head and neck malignant tumor. The incidence of thyroid cancer has been steadily increasing in the past several decades. Thyroid papillary carcinoma (PTC) is recognized as a well differentiated malignant tumor, and is the most frequent. Most of the 80%~90%. patients with thyroid malignant tumors, which account for the total number of thyroid cancer, have a good prognosis, but the overall recurrence rate can still be as high as 35%, because the current means can not accurately predict their clinical outcome and biological behavior. Therefore, we urgently need to understand the molecular mechanism of the development of thyroid papillary carcinoma and find out the molecular mechanism of the development of thyroid papillary carcinoma and find out the molecular mechanism of the development of thyroid papillary carcinoma. A new series of new diagnostics that contribute to the timely diagnosis of PTC to judge the prognosis and effective treatment from macro to microcosmic. The small RNA (MI RNAs) is a class of non coded small RNA with a length of about 19~25 nucleotides. The 3 'non coding region (3' -UTR) of the messenger RNA (m RNAs) can be combined with each other to regulate the gene table at the transcriptional level or post transcriptional level. Da. Current research has found that MI RNAs may regulate the transcription and expression of thousands of human genes, and more and more evidence suggests that MI RNAs can also regulate many basic physiological processes, such as cell differentiation, proliferation and survival, and cell apoptosis, migration and invasion, because mi RNAs can participate in regulating cell proliferation, differentiation and withering. It is believed that it is closely related to the occurrence and development of cancer. Then, the analysis of MI RNAs and its target genes not only introduce our understanding of tumor development to a unique and new perspective, but also provide a new entry point for the diagnosis and treatment of PTC,.Mi R-29a is a member of the Mi R-29s family, which is a conservative. The MI RNA family, MI R-29s, is expressed in some cancers, such as gastric cancer, pancreatic cancer, and prostate cancer. On the contrary, the expression of MI R-29s in other cancers is rising, such as breast cancer, nasopharyngeal carcinoma, glioma and acute myelogenous leukemia. These studies show that MI R-29a may play a role in the promotion or suppression of different cancers. However, its clinical significance, role and potential molecular mechanisms in PTC are still not very clear. In this study, we analyzed the relationship between the expression of MI R-29a and the clinicopathological features of PTC patients, and studied the role and potential of MI R-29a in PTC through a series of in vitro and in vivo tests. Objective: to clarify the role and mechanism of MI R-29a in the development of thyroid papillary carcinoma: 1. to study the expression level of MI R-29a in PTC and its role and mechanism in proliferation and migration (1) the expression of MI R-29a in 30 groups of PTC tumor tissue specimens and adjacent normal tissues by Q RT-PCR was carried out in this experiment. In order to further study the clinicopathological significance of MI R-29a expression level in PTC patients, we selected median (0.59) as the demarcation point and divided 30 PTC specimens into mi R-29a relative low level group (0.59,14 cases) and MI R-29a relative high level group (0.59,16 cases), and then used the card expression with the clinicopathological parameters. (2) in order to elucidate the biological function of MI R-29a in PTC, we transfected mi R-29a mimics into K1 cell lines and obtained a K1 cell line that stably expressed mi R-29a. To detect cell proliferation, cell cycle and apoptosis. (3) the effect of MI R-29a on the invasion and migration of PTC cells through wound healing experiments and tumor cell invasiveness experiments,.2.mi R-29a inhibits the growth of PTC by regulating AKT3 (1) we use biological information databases such as Target Scan, MI Randa, and Pic To predict the potential target gene of MI R-29a, AKT3 is locked as the research target because it plays an important role in the PI3K/AKT signaling pathway and involves a variety of cell functions. In order to verify whether AKT3 is the direct target of MI R-29a in PTC, a human AKT3 3 'UTR contains fragments of MI R-29a binding sites or cloned mutation sites. PGL3 vector, CO transfected with MI R-29a analogue or MI R-Ctrl into K1 cells, incubated 48h, and then detected the activity of luciferase. Through Q RT-PCR and Western blotting experiments, the effect of overexpression on the expression level in the cells was detected from the M proficiency level and protein level. (2) the target gene has been clearly defined. We continue to use Q RT-PCR to study the expression of AKT3 in PTC tissue and its adjacent normal tissues. (3) in order to further explore the biological effects of AKT3 in PTC, we transfect si-AKT3 and si-Ctrl into K1 cells respectively, and verify the knockout efficiency by Q RT-PCR and western. Cell proliferation was detected by colorimetric assay, cell apoptosis was detected by flow cytometry, cell migration and tumor cell invasiveness were detected by wound healing experiments. (4) in order to further study the function of AKT3 needle for MI R-29a, we designed a series of experiments on whether mi R-29a will increase in PTC cells when AKT3 is overexpressed. The effects of colonization, apoptosis, migration and invasion were evaluated. To this end, AKT3 overexpressed plasmids were transfected into K1 cells with MI R-29a analogue or MI R-Ctrl. Then MTT colorimetric assay was used to detect cell proliferation, flow cytometry was used to detect cell apoptosis, wound healing test was used to detect cell migration and tumor cell invasion test cells. The experimental study of the inhibitory effect of.3.mi R-29a on the growth of PTC in the body shows that miR-29a has the effect of inhibiting the growth of the tumor in vitro, and we then evaluate its role in the body. For this purpose, we will steadily express the K1 cells of MI R-29a or MI R-Ctrl into the subcutaneous of nude mice to form a tumor. 30 days after the injection. The animals were killed, and the tumor tissue was stripped. The size and weight of the two groups of tumors were observed and measured. In addition, we also measured the expression of MI R-29a and AKT3 in the tumor tissues by Q RT-PCR. Then, the expression level of AKT3 was measured by Q RT-PCR and Western blotting experiment. The expression level and its role and mechanism in proliferation and migration (1) the relative expression of MI R-29a in the expression of down regulated PTC in human PTC tissues by Mi R-29a is significantly lower than that of its adjacent normal tissue (P0.01). We select the median (0.59) as the demarcation point and divide the 30 PTC standard into the MI R-29a relative low level group (0.59,14 cases). 9A was compared with the high level group (0.59,16 cases), then the expression of MI R-29a and the clinicopathological parameters were analyzed with chi square test. The analysis showed that the expression of MI R-29a was not statistically significant to age and sex. At the same time, the level of MI R-29a was negatively correlated with TNM stages (P0.01), tumor size (P0.01) and lymph node metastasis. (2) mi R-29a inhibited PTC cell proliferation and induced apoptosis through Q RT-PCR to confirm that the expression level of MI R-29a increased in K1 cell lines (P0.01). MTT colorimetric assay showed that MI R-29a could significantly inhibit the proliferation of Q cells. Compared with the transfected mi R-Ctrl group, the proportion of S phase in the K1 cells of MI R-29a was reduced (P0.05), and the proportion of G0/G1 phase increased significantly (P0.01). Apoptosis experiments showed that the transfection of MI R-29a analog in K1 cell lines could significantly promote apoptosis. Compared with the MI R-Ctrl group, increasing the expression level of miR-29a significantly inhibited the ability of K1 cell invasion and metastasis (P0.01) the mechanism of.2.mi R-29a to inhibit PTC growth by regulating AKT3 (1) AKT3 is the direct target gene of MI R-29a, which obviously inhibited the luciferase activity of the wild type loci, but the mutant type The luciferase activity of the loci did not change. This indicates that AKT3 is a direct target gene for MI R-29a. Then, the Q RT-PCR and Western blotting experiments have demonstrated that MI R-29a overexpression can completely inhibit AKT3 expression in K1 cells, whether from m and protein levels. The expression of phosphorylated AKT (p-AKT) is inhibited, which suggests that MI R-29a can inhibit the activation of PI3K/AKT signal transduction pathway. The above results show that MI R-29a can be directly associated with AKT3 and inhibit its expression. (2) the expression of MI R-29a in PTC tissues is negatively related to the expression of AKT3. The expression of AKT3 was negatively correlated with the expression of MI R-29a in PTC tissue (P0.01). (3) down regulation of AKT3 expression can simulate the effect of MI R-29a overexpression using si-AKT3 to reduce the expression of AKT3 in K1 cell lines to inhibit cell proliferation and induce cell apoptosis, and also inhibit tumor finely. In other words, reducing the expression of AKT3 can simulate the effect of MI R-29a on the inhibition of tumor. (4) AKT3 overexpression can reverse the action of MI R-29a, AKT3 overexpression plasmid can increase the expression of AKT3 at m RNA level and protein level, and the expression of AKT3.AKT3 in K1 cells can promote tumor cell proliferation, migration and invasion, and inhibit the tumor cells. The effect of AKT3 overexpression reverses the inhibitory effect of MI R-29a on the tumor..3.mi R-29a inhibits the growth of PTC in the body and is compared with the injection K1/miR-Ctrl group. The tumor in the K1/miR-29a group is significantly reduced in both size and weight (P0.01). Furthermore, the expression level of MI R-29a in the tumor tissues should be significantly increased (P0.01), however. The expression level of AKT3 decreased significantly, whether at m RNA level (P0.01) or protein level. Conclusion: the expression level of 1.miR-29a is reduced in PTC tissue, and its expression level is closely related to the size of tumor, TNM staging and lymph node metastasis, and.2.mi R-29a overexpression can significantly inhibit the proliferation of PTC cells. Migration and invasion, and promoting apoptosis of PTC cells and stagnation of cell cycle in G0/G1 phase. In vivo experiments confirmed that MI R-29a overexpression can reduce the growth of tumor growth.3. in PTC cells by regulating AKT3 in xenotransplantation nude mice, miR-29a can be directly combined with the 3 'non coding region of AKT3, and the overexpressed Mi R-29a can inhibit the AKT3 table significantly. As a result, the expression of AKT3 was increased in PTC tissue, and the expression of AKT3 was negatively correlated with the expression of miR-29a in the PTC tissue, and the expression of.4. was negatively correlated with the expression of miR-29a. The expression of Si RNA to reduce the expression of AKT3 could simulate the effect of MI R-29a overexpression.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.1

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