miR-421下调FOXO4促进鼻咽癌细胞增殖及凋亡抵抗
发布时间：2018-07-16 15:48
【摘要】：microRNA是最近研究发现的一类长度约为19-25个核苷酸的非编码单链小RNA分子,其通过与靶基因的3’端非翻译区互补配对结合的方法,从而调控内源基因的表达。miRNA作为生物体生长发育的重要调控基因,目前其主要研究方向是探明miRNAs与疾病的关系,尤其是miRNA与肿瘤的关系。鼻咽癌(Nasopharyngeal carcinoma, NPC)是广东地区常见的恶性肿瘤之一,在头颈部恶性肿瘤中占首位。鼻咽癌的发病人群以40-50岁的青壮年多见,一旦发病对社会、经济和家庭造成较大影响。鼻咽癌原发部位隐蔽,不易观察,且与鼻腔、鼻窦和颅内相毗邻,所以临床症状出现较晚而且各异；鼻咽癌也是头颈肿瘤中转移率最高的。这些都导致了鼻咽癌常常容易被误诊或漏诊,影响治疗效果。因此,鼻咽癌的早期诊断和治疗的研究一直是我国头颈外科学研究的重点之一。目前鼻咽癌的治疗还是以放射治疗为主。对放疗不敏感、放疗后复发以及中晚期的患者,可采取化学药物治疗,但这只是辅助性治疗或姑息性治疗。鼻咽癌的手术治疗也只在一些特殊情况下作为辅助方法采用。除了以上三种常规治疗,近年来也有作者尝试应用光动力疗法和微波热疗治疗鼻咽癌,但也只能作为辅助手段。重组DNA技术的发展使肿瘤的基因治疗成为可能。根据肿瘤发生机理制定的基因治疗方案已成为肿瘤治疗的热点。早期研究者主要运用反义核酸技术干预肿瘤相关基因的表达,取得了一定效果。近年来,RNA干扰技术在哺乳动物体内的应用,更为肿瘤的基因治疗开辟了一个新的视野。因此,我们意在探索性地研究miRNA在鼻咽癌发生发展中的调控作用。 我们通过Targetscan数据库,对差异表达miRNA的靶基因进行预测,并将预测结果输入GenMAPP软件进行生物学通路分析。在通路分析中,大部分靶基因富集度高的通路都涉及到信号转导及与癌症发生相关的通路,例如EGFR-1信号通路,MAPK信号通路等等。其中，，小GTP酶介导的信号转导通路最为显著。同时,Wnt信号通路的显著性,则与我们实验室过去在鼻咽癌cDNA表达谱的研究结果相吻合。继续深入研究这些差异]miRNA在生物学通路中的调控作用,以及寻找与鼻咽癌发生发展进程相关的临床诊断标记物,则是我们未来努力的方向。 microRNA (miRNA)即微小RNA,是一种存在于植物和动物基因组里的小分子RNA,约19～25nt(少数小于20nt),由一段具有发夹环结构、长度为70-80nt的单链RNA前体(Pre-miRNA)剪切后形成。它通过与其目标mRNA分子的3’端非编码区域(3'-untranslatedregion,3'UTR)完全或非完全互补匹配、参与基因转录后水平(蛋白表达)的调控,在生物体内各种生理和病理状态都发挥着十分重要的作用。 miRNA最早由Lee等在1993年对线虫(Caenothabditis elegans)胚胎发育研究过程中发现的一种具有调控功能的非编码RNA。首先,miRNA基因的初级转录产物(Pri-miRNA)在细胞核中被RNase (?) Drosha切割成为前体miRNA (pre-miRNA)。在最初的剪切后,Pre-miRNA在转运蛋白exPortin-5的作用下、由核内转到胞质中,然后由另一种RNase (?) Dicer进一步切割产生成熟的miRNA.这些成熟的miRNA与其他蛋白质一起组成RISC (RNA-induced sileneing complex)复合体,从而引起靶mRNA的降解或者翻译抑制。 microRNAs具有重要的组织特异功能,在基因表达中发挥着总体调控作用,其中miR-421在肿瘤进展调控,细胞增殖等方面起着重要的作用,目前对于鼻咽癌的调控作用尚缺少文献报道。本课题将探讨miR-421在鼻咽癌发生发展中的作用。本研究的目的是模拟天然miRNA构建表达miRNA前体的质粒,实现调控肿瘤相关基因miR-421的表达,开展对鼻咽癌的基因治疗研究。 首先是调控目标基因的miR-421表达质粒的构建和有效质粒筛选。然后进一步确定针对FOXO4基因的1niRNA的有效作用位点,观察构建的质粒对鼻咽癌中两个靶基因的调控效应,为后续的体外实验奠定基础。miRNA重组质粒调控FOX04表达能有效抑制肿瘤生长,为进一步的临床研究打下了基础。 第一部分 1、过表达miR-.421促进鼻咽癌细胞增殖和抗凋亡的作用 目的：检测过表达miR-421对于鼻咽癌细胞增殖和抗凋亡的能力。 方法：利用慢病毒感染技术,将miR-421转染到CNE2,观察体外miR-421对CNE2增殖作用,并利用Annexin V法及TUNEL检测细胞凋亡情况。 结果：软琼脂克隆形成实验表明,miR-421过表达细胞的增殖速度是对照组的3倍,表明miR-421上调可以显著增强鼻咽癌细胞的生长。此外,Annexin V法及TUNEL检测表明,miR-421的过表达可增强鼻咽癌细胞对于化疗药物顺铂的凋亡抵抗能力。这些结果表明,体外实验中,miR-421在鼻咽癌中扮演着调控细胞基因的作用。 结论：miR-421上调可促进入鼻咽癌细胞的增殖,提高细胞凋亡抵抗能力。 2、抑制miR-421减少鼻咽癌细胞增殖和诱导细胞凋亡的作用 目的：检测抑制miR-421对于鼻咽癌细胞增殖和凋亡的影响 方法：利用慢病毒感染技术,将抑制miR-421转染CNE2,观察体外miR-421对CNE2增殖作用,并利用Annexin V法及TUNEL检测细胞凋亡情况。 结果：软琼脂克隆形成实验表明,抑制miR-421后细胞的增殖速度降低,对照组是抑制组的3倍,表明抑制miR-421可以显著降低鼻咽癌细胞的生长。此外,Annexin V法及TUNEL检测表明,抑制miR-421可增强鼻咽癌细胞对于化疗药物顺铂的凋亡促进能力。这些结果表明,体外实验中,miR-421在鼻咽癌中扮演着调控细胞基因的作用。 结论：抑制miR-421可降低人鼻咽癌细胞的增殖,促进细胞凋亡。 第二部分 1、探讨miR-421对FOXO4信号途径的作用 目的：研究miR-421促进鼻咽癌细胞增殖及抗凋亡的内在分子机制是否通过FOXO4信号途径调节。 方法：构建针对耙基因FOXO4的荧光素酶报告基因载体,利用荧光照度计分别测定正常组、miR-421过表达组与miR-421抑制组的荧光值。并利用实时定量PCR及western blot检测FOXO4靶基因p21, p27, Bim和FasL的mRNA及蛋白表达情况。 结果：1.在荧光素酶活性检测中,FOXO4的荧光素酶活性在miR-421过表达的细胞中降低,而在miR-421抑制的细胞中表达增多。2.与此相一致的是,在正常组、miR-421过表达组与miR-421抑制组中我们发现,FOXO4四个经典靶点基因p21, p27, Bim和FasL的mRNA及蛋白表达在miR-421过表达组是下调,而在miR-421抑制的细胞中,上述基因及蛋白上调。 结论：通过构建荧光素酶以及过表达和抑制miR-421实验结果表明,miR-421可抑制FOXO4信号通路。 2、miR-421与FOXO4调控关系探讨 目的：探寻miR-421在基因调控中针对耙基因FOXO4调节作用。 方法：使用TargetScan软件工具mir-421目标预测分析表明,FOXO4是mir-421潜在目标,利用免疫印迹分析表明,在正常组、miR-421过表达组与miR-421抑制组中基因FOXO4的表达存在显著的差异性,过表达组蛋白表达低于正常组,而抑制组蛋白表达高于正常组。 为了进一步验证miR-421与FOXO4相互控制存在的关系,我们克隆了FOXO43'UTR片段,含有miR-421结合位点的psiCHECK-2荧光素酶载体,同时我们克隆了FOXO43'UTR突变载体,具体突变了二个碱基。 通过荧光素酶活性检测表明,miR-421过表达组低于正常组,而miR-421抑制组高于正常组。 另外,为了进一步分析miR-421与FOXO4在凋亡中的相互作用,在抑制miR-421的时,同时抑制FOXO4作对照,实验说明,抑制FOXO4基因后,细胞凋亡低于单纯抑制miR-421组。 结果：1、miR-421过表达与miR-421抑制组中,基因FOXO4表达存在差异性；2、荧光素酶检测表明,miR-421过表达与miR-421抑制组中荧光值存在差异性,而在突变体却无此差异性；3、同时抑制miR-421时,选择下调基因FOXO4,结果表明,凋亡细胞低于单纯抑制miR-421组。 结论：基因FOXO4是miR-421调控目的基因。 3. FOXO4是细胞生长和抗凋亡的关键基因 目的：探寻FOXO4在细胞生长和抗凋亡的调节作用。 方法：为了进一步分析miR-421与FOXO4在凋亡中的作用,我们选择将miR-421抑制时,然后再单纯抑制基因FOXO4来对照,检测数据显示,在同时抑制miR-421时,单纯抑制FOXO4后细胞凋亡数减少。 结果：同时抑制miR-421时,选择下调基因FOXO4,结果表明,凋亡细胞低于单纯抑制miR-421组。 结论：FOXO4是细胞生长和抗凋亡的关键基因。 4、在鼻咽癌组织中miR-421与FOXO4的临床相关性 目的：研究临床鼻咽癌患者的肿瘤标本中miR-421与FOXO4的关系。 方法：7例新鲜收集的人鼻咽癌样本组织,利用QPCR技术检测miR-421与FOXO4的表达量,同时采用免疫印迹法检测FOXO4表达水平,并研究两者之间的关系。 结果：在7例新鲜收集的鼻咽癌组织样本中miR-421表达量高者则其FOXO4表达量低,两者呈负相关(r=-0.798,P=0.032)。 结论：miR-421表达量在鼻咽癌组织样本中与FOXO4表达量呈负相关。 总之,miR-421上调可抑制FOXO4的表达,从而导致鼻咽癌细胞增殖和凋亡抵抗；反之,实验中阻遏miR-421的表达则可抑制鼻咽癌细胞增殖并促进其凋亡。
[Abstract]:MicroRNA is a non coding single strand RNA molecule of approximately 19-25 nucleotides, which is recently discovered, which is combined with the 3 'end non translation region of the target gene to regulate the expression of.MiRNA as an important regulatory gene for the growth and development of the organism. At present, the main research direction is to explore miRNAs The relationship with the disease, especially the relationship between miRNA and tumor. Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Guangdong area. It is the first in the head and neck malignant tumor. The incidence of nasopharyngeal carcinoma is more common in 40-50 years old, once the disease has a great influence on the society, economy and family. Nasopharyngeal carcinoma is the primary cancer. It is hidden, not easy to observe, and adjacent to the nasal cavity, sinus and intracranial phase, so the clinical symptoms are late and different, and nasopharyngeal carcinoma is also the highest metastatic rate in the head and neck tumor. All these cause nasopharyngeal carcinoma to be easily misdiagnosed or missed and affect the treatment effect. Therefore, the early diagnosis and treatment of nasopharyngeal carcinoma have been the research of our country. One of the key points in the scientific research of the head and neck. At present, the treatment of nasopharyngeal carcinoma is mainly radiation therapy. Chemotherapy is not sensitive to radiotherapy. Chemotherapy can be taken in patients with relapse and middle and late stage, but it is only adjuvant therapy or palliative treatment. The surgical treatment of nasopharyngeal carcinoma is also used only as an auxiliary method in some special cases. In addition to the three conventional treatments, the author has also tried to use photodynamic therapy and microwave thermotherapy in recent years to treat nasopharyngeal carcinoma, but it can only be used as an auxiliary means. The development of recombinant DNA technology makes it possible for tumor gene therapy. The gene therapy scheme based on the mechanism of tumor has become a hot spot in cancer treatment. The researchers mainly use antisense nucleic acid technology to interfere with the expression of tumor related genes, which has achieved some effect. In recent years, the application of RNA interference technology in mammals has opened up a new field of vision for the gene therapy of tumor. Therefore, we intend to explore the regulatory role of miRNA in the development of nasopharyngeal carcinoma.
We used the Targetscan database to predict the target gene for differentially expressed miRNA, and put the predicted results into the GenMAPP software for biological pathway analysis. In the pathway analysis, most of the high target gene enrichment pathways involved signal transduction and the pathway related to cancer, such as the EGFR-1 signaling pathway, the MAPK signaling pathway. And so on. Among them, small GTP enzyme mediated signal transduction pathway is most significant. At the same time, the significance of the Wnt signaling pathway is consistent with our laboratory's previous study of cDNA expression profiles in nasopharyngeal carcinoma. Further study of these differences in the regulatory role of these differences in the biological pathways and to search for the progression of nasopharyngeal carcinoma. Clinical diagnostic markers are the direction of our future efforts.
MicroRNA (miRNA), a small RNA, is a small molecule RNA in the genome of plants and animals, about 19 to 25nt (a few less than 20nt), formed by a single strand RNA precursor (Pre-miRNA) with a clip ring structure and a length of 70-80nt. It passes through the 3 'end non coding region of its target mRNA molecule (3'-untranslatedregion, 3'UTR). Complete or incomplete complementarity matches the regulation of the post transcriptional level (protein expression), which plays a very important role in various physiological and pathological conditions in the organism.
MiRNA first found by Lee and so on in the study of the Caenothabditis elegans embryo development in 1993, a non coded RNA., first of which the primary transcriptional product of the miRNA gene (Pri-miRNA) was cut into the precursor miRNA (pre-miRNA) by RNase (?) Drosha in the nucleus. After the initial shear, the Pre-miRNA was transported. Under the action of protein exPortin-5, it is transferred from the nucleus to the cytoplasm, and then another RNase (?) Dicer is further cut to produce mature miRNA., the mature miRNA, together with other proteins, to form a RISC (RNA-induced sileneing complex) complex, which causes the target mRNA degradation or translation inhibition.
MicroRNAs plays an important role in gene expression, which plays an important role in the regulation of gene expression, and miR-421 plays an important role in the regulation of tumor progression, cell proliferation, and so on. The role of miR-421 in the development of nasopharyngeal carcinoma is not reported. This study will discuss the role of this study in nasopharyngeal carcinoma. The aim is to simulate the construction of plasmid expressing miRNA precursor by natural miRNA, and to control the expression of tumor associated gene miR-421, and to carry out gene therapy for nasopharyngeal carcinoma.
The first is to regulate the construction of the miR-421 expression plasmid and the effective plasmid screening of the target gene, and then to further determine the effective action loci of the 1niRNA for the FOXO4 gene, and observe the regulatory effect of the constructed plasmid on the two target genes in nasopharyngeal carcinoma, which can effectively inhibit the expression of FOX04 expression by the recombinant plasmid based on the.MiRNA recombinant plasmid in the following in vitro experiment. Tumor growth has laid the foundation for further clinical research.
Part one
1, overexpression of miR-.421 promotes proliferation and anti apoptosis of nasopharyngeal carcinoma cells.
Objective: to detect the ability of over expression of miR-421 in nasopharyngeal carcinoma cell proliferation and apoptosis.
Methods: using the technique of lentivirus infection, miR-421 was transfected into CNE2, and the proliferation of CNE2 was observed by miR-421 in vitro, and the cell apoptosis was detected by Annexin V and TUNEL.
Results: the soft agar cloning experiments showed that the proliferation rate of miR-421 overexpressed cells was 3 times as high as that of the control group, indicating that up regulation of miR-421 could significantly enhance the growth of nasopharyngeal carcinoma cells. In addition, Annexin V and TUNEL tests showed that the overexpression of miR-421 could enhance the apoptosis resistance of nasopharyngeal carcinoma cells to cisplatin. The results showed that miR-421 played a role in regulating cell genes in nasopharyngeal carcinoma in vitro.
Conclusion: upregulation of miR-421 can promote the proliferation of nasopharyngeal carcinoma cells and enhance the ability of cell apoptosis.
2, inhibition of miR-421 reduces proliferation and induces apoptosis in nasopharyngeal carcinoma cells.
Objective: To investigate the effect of inhibiting miR-421 on proliferation and apoptosis of nasopharyngeal carcinoma cells.
Methods: using the technique of lentivirus infection, CNE2 was transfected by miR-421, and the proliferation of CNE2 was observed by miR-421 in vitro, and the cell apoptosis was detected by Annexin V and TUNEL.
Results: the soft agar clone formation experiment showed that the proliferation rate of miR-421 cells decreased after inhibition of miR-421, and the control group was 3 times as high as that of the inhibition group. It showed that inhibition of miR-421 could significantly reduce the growth of nasopharyngeal carcinoma cells. In addition, Annexin V and TUNEL detection showed that inhibition of miR-421 could enhance the apoptosis promoting energy of nasopharyngeal cancer cells to cisplatin. These results indicate that miR-421 plays a role in regulating cell genes in nasopharyngeal carcinoma in vitro.
Conclusion: inhibition of miR-421 can reduce the proliferation and promote apoptosis of NPC cells.
The second part
1, to explore the effect of miR-421 on the FOXO4 signal pathway
Objective: To investigate whether miR-421 promotes the proliferation and anti apoptosis of nasopharyngeal carcinoma cells through FOXO4 signaling pathway.
Methods: the luciferase reporter gene vector for the rake gene FOXO4 was constructed and the fluorescence values of the normal group, the miR-421 overexpression group and the miR-421 inhibition group were measured by the fluorescent illuminometer. The expression of the mRNA and protein of the FOXO4 target gene p21, p27, Bim and FasL was detected by real-time quantitative PCR and Western blot.
Results: 1. in the activity detection of luciferase activity, the luciferase activity of FOXO4 decreased in miR-421 overexpressed cells, and the expression of.2. in miR-421 inhibited cells was consistent with this phase. In the normal group, the miR-421 overexpression group and the miR-421 inhibition group, we found that the FOXO4 four classic target genes p21, p27, Bim and FasL mRNA. The protein expression was downregulated in the miR-421 overexpression group, while in the miR-421 inhibited cells, the above genes and proteins were upregulated.
Conclusion: by constructing luciferase, overexpression and inhibition of miR-421, the results show that miR-421 can inhibit FOXO4 signaling pathway.
2, study on the relationship between miR-421 and FOXO4
Objective: To explore the regulatory role of miR-421 in raking gene FOXO4 in gene regulation.
Methods: the TargetScan software tool mir-421 target prediction analysis showed that FOXO4 was a potential target of mir-421. The expression of FOXO4 in the miR-421 overexpressed group and the miR-421 inhibition group was significantly different in the normal group, and the expression of the overexpressed histone expression was lower than that in the normal group, and the expression of the inhibitory histone expression was higher than that in the normal group. Normal group.
In order to further verify the relationship between miR-421 and FOXO4, we cloned FOXO43'UTR fragment, psiCHECK-2 luciferase carrier containing miR-421 binding site, and we cloned the FOXO43'UTR mutant vector and mutated two bases.
The luciferase activity assay showed that the miR-421 overexpression group was lower than the normal group, while the miR-421 inhibition group was higher than the normal group.
In addition, in order to further analyze the interaction between miR-421 and FOXO4 in apoptosis and inhibition of miR-421 at the time of inhibiting the FOXO4 as control, the experiment showed that after the inhibition of the FOXO4 gene, the apoptosis was lower than that of the only inhibition of the miR-421 group.
Results: 1, the expression of gene FOXO4 in miR-421 overexpression and miR-421 inhibition group was different. 2, fluorescein detection showed that miR-421 overexpression was different from that in miR-421 inhibition group, but there was no difference in the mutant group; 3, at the same time inhibition of miR-421, the selective down gene FOXO4 was selected. The results showed that the apoptotic cells were lower than simple cells. Inhibition of group miR-421.
Conclusion: gene FOXO4 is the target gene for the regulation of miR-421.
3. FOXO4 is a key gene for cell growth and anti apoptosis
Objective: To explore the regulatory effect of FOXO4 on cell growth and anti apoptosis.
Methods: in order to further analyze the role of miR-421 and FOXO4 in apoptosis, we chose to inhibit miR-421 and then only inhibit gene FOXO4 to control, and the detection data showed that the number of apoptotic cells decreased after the inhibition of miR-421 at the same time.
Results: when miR-421 was inhibited, the FOXO4 gene was downregulated. The results showed that the apoptotic cells were lower than those of miR-421 alone.
Conclusion: FOXO4 is a key gene for cell growth and anti apoptosis.
4, the clinical correlation between miR-421 and FOXO4 in nasopharyngeal carcinoma tissues.
Objective: To study the relationship between miR-421 and FOXO4 in clinical specimens of nasopharyngeal carcinoma.
Methods: 7 samples of freshly collected human nasopharyngeal carcinoma samples were collected. The expression of miR-421 and FOXO4 was detected by QPCR technique. The expression of FOXO4 was detected by Western blot, and the relationship between them was studied.
Results: in 7 samples of nasopharyngeal carcinoma, the expression level of miR-421 was high, and the expression level of FOXO4 was low, and there was a negative correlation between them (r=-0.798, P=0.032).
Conclusion: the expression of miR-421 is negatively correlated with the expression of FOXO4 in nasopharyngeal carcinoma tissues.
In conclusion, up regulation of miR-421 can inhibit the expression of FOXO4, which leads to the proliferation and apoptosis resistance of nasopharyngeal carcinoma cells. Conversely, the inhibition of miR-421 expression in the experiment can inhibit the proliferation of nasopharyngeal carcinoma cells and promote its apoptosis.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63
本文编号：2126865
[Abstract]:MicroRNA is a non coding single strand RNA molecule of approximately 19-25 nucleotides, which is recently discovered, which is combined with the 3 'end non translation region of the target gene to regulate the expression of.MiRNA as an important regulatory gene for the growth and development of the organism. At present, the main research direction is to explore miRNAs The relationship with the disease, especially the relationship between miRNA and tumor. Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Guangdong area. It is the first in the head and neck malignant tumor. The incidence of nasopharyngeal carcinoma is more common in 40-50 years old, once the disease has a great influence on the society, economy and family. Nasopharyngeal carcinoma is the primary cancer. It is hidden, not easy to observe, and adjacent to the nasal cavity, sinus and intracranial phase, so the clinical symptoms are late and different, and nasopharyngeal carcinoma is also the highest metastatic rate in the head and neck tumor. All these cause nasopharyngeal carcinoma to be easily misdiagnosed or missed and affect the treatment effect. Therefore, the early diagnosis and treatment of nasopharyngeal carcinoma have been the research of our country. One of the key points in the scientific research of the head and neck. At present, the treatment of nasopharyngeal carcinoma is mainly radiation therapy. Chemotherapy is not sensitive to radiotherapy. Chemotherapy can be taken in patients with relapse and middle and late stage, but it is only adjuvant therapy or palliative treatment. The surgical treatment of nasopharyngeal carcinoma is also used only as an auxiliary method in some special cases. In addition to the three conventional treatments, the author has also tried to use photodynamic therapy and microwave thermotherapy in recent years to treat nasopharyngeal carcinoma, but it can only be used as an auxiliary means. The development of recombinant DNA technology makes it possible for tumor gene therapy. The gene therapy scheme based on the mechanism of tumor has become a hot spot in cancer treatment. The researchers mainly use antisense nucleic acid technology to interfere with the expression of tumor related genes, which has achieved some effect. In recent years, the application of RNA interference technology in mammals has opened up a new field of vision for the gene therapy of tumor. Therefore, we intend to explore the regulatory role of miRNA in the development of nasopharyngeal carcinoma.
We used the Targetscan database to predict the target gene for differentially expressed miRNA, and put the predicted results into the GenMAPP software for biological pathway analysis. In the pathway analysis, most of the high target gene enrichment pathways involved signal transduction and the pathway related to cancer, such as the EGFR-1 signaling pathway, the MAPK signaling pathway. And so on. Among them, small GTP enzyme mediated signal transduction pathway is most significant. At the same time, the significance of the Wnt signaling pathway is consistent with our laboratory's previous study of cDNA expression profiles in nasopharyngeal carcinoma. Further study of these differences in the regulatory role of these differences in the biological pathways and to search for the progression of nasopharyngeal carcinoma. Clinical diagnostic markers are the direction of our future efforts.
MicroRNA (miRNA), a small RNA, is a small molecule RNA in the genome of plants and animals, about 19 to 25nt (a few less than 20nt), formed by a single strand RNA precursor (Pre-miRNA) with a clip ring structure and a length of 70-80nt. It passes through the 3 'end non coding region of its target mRNA molecule (3'-untranslatedregion, 3'UTR). Complete or incomplete complementarity matches the regulation of the post transcriptional level (protein expression), which plays a very important role in various physiological and pathological conditions in the organism.
MiRNA first found by Lee and so on in the study of the Caenothabditis elegans embryo development in 1993, a non coded RNA., first of which the primary transcriptional product of the miRNA gene (Pri-miRNA) was cut into the precursor miRNA (pre-miRNA) by RNase (?) Drosha in the nucleus. After the initial shear, the Pre-miRNA was transported. Under the action of protein exPortin-5, it is transferred from the nucleus to the cytoplasm, and then another RNase (?) Dicer is further cut to produce mature miRNA., the mature miRNA, together with other proteins, to form a RISC (RNA-induced sileneing complex) complex, which causes the target mRNA degradation or translation inhibition.
MicroRNAs plays an important role in gene expression, which plays an important role in the regulation of gene expression, and miR-421 plays an important role in the regulation of tumor progression, cell proliferation, and so on. The role of miR-421 in the development of nasopharyngeal carcinoma is not reported. This study will discuss the role of this study in nasopharyngeal carcinoma. The aim is to simulate the construction of plasmid expressing miRNA precursor by natural miRNA, and to control the expression of tumor associated gene miR-421, and to carry out gene therapy for nasopharyngeal carcinoma.
The first is to regulate the construction of the miR-421 expression plasmid and the effective plasmid screening of the target gene, and then to further determine the effective action loci of the 1niRNA for the FOXO4 gene, and observe the regulatory effect of the constructed plasmid on the two target genes in nasopharyngeal carcinoma, which can effectively inhibit the expression of FOX04 expression by the recombinant plasmid based on the.MiRNA recombinant plasmid in the following in vitro experiment. Tumor growth has laid the foundation for further clinical research.
Part one
1, overexpression of miR-.421 promotes proliferation and anti apoptosis of nasopharyngeal carcinoma cells.
Objective: to detect the ability of over expression of miR-421 in nasopharyngeal carcinoma cell proliferation and apoptosis.
Methods: using the technique of lentivirus infection, miR-421 was transfected into CNE2, and the proliferation of CNE2 was observed by miR-421 in vitro, and the cell apoptosis was detected by Annexin V and TUNEL.
Results: the soft agar cloning experiments showed that the proliferation rate of miR-421 overexpressed cells was 3 times as high as that of the control group, indicating that up regulation of miR-421 could significantly enhance the growth of nasopharyngeal carcinoma cells. In addition, Annexin V and TUNEL tests showed that the overexpression of miR-421 could enhance the apoptosis resistance of nasopharyngeal carcinoma cells to cisplatin. The results showed that miR-421 played a role in regulating cell genes in nasopharyngeal carcinoma in vitro.
Conclusion: upregulation of miR-421 can promote the proliferation of nasopharyngeal carcinoma cells and enhance the ability of cell apoptosis.
2, inhibition of miR-421 reduces proliferation and induces apoptosis in nasopharyngeal carcinoma cells.
Objective: To investigate the effect of inhibiting miR-421 on proliferation and apoptosis of nasopharyngeal carcinoma cells.
Methods: using the technique of lentivirus infection, CNE2 was transfected by miR-421, and the proliferation of CNE2 was observed by miR-421 in vitro, and the cell apoptosis was detected by Annexin V and TUNEL.
Results: the soft agar clone formation experiment showed that the proliferation rate of miR-421 cells decreased after inhibition of miR-421, and the control group was 3 times as high as that of the inhibition group. It showed that inhibition of miR-421 could significantly reduce the growth of nasopharyngeal carcinoma cells. In addition, Annexin V and TUNEL detection showed that inhibition of miR-421 could enhance the apoptosis promoting energy of nasopharyngeal cancer cells to cisplatin. These results indicate that miR-421 plays a role in regulating cell genes in nasopharyngeal carcinoma in vitro.
Conclusion: inhibition of miR-421 can reduce the proliferation and promote apoptosis of NPC cells.
The second part
1, to explore the effect of miR-421 on the FOXO4 signal pathway
Objective: To investigate whether miR-421 promotes the proliferation and anti apoptosis of nasopharyngeal carcinoma cells through FOXO4 signaling pathway.
Methods: the luciferase reporter gene vector for the rake gene FOXO4 was constructed and the fluorescence values of the normal group, the miR-421 overexpression group and the miR-421 inhibition group were measured by the fluorescent illuminometer. The expression of the mRNA and protein of the FOXO4 target gene p21, p27, Bim and FasL was detected by real-time quantitative PCR and Western blot.
Results: 1. in the activity detection of luciferase activity, the luciferase activity of FOXO4 decreased in miR-421 overexpressed cells, and the expression of.2. in miR-421 inhibited cells was consistent with this phase. In the normal group, the miR-421 overexpression group and the miR-421 inhibition group, we found that the FOXO4 four classic target genes p21, p27, Bim and FasL mRNA. The protein expression was downregulated in the miR-421 overexpression group, while in the miR-421 inhibited cells, the above genes and proteins were upregulated.
Conclusion: by constructing luciferase, overexpression and inhibition of miR-421, the results show that miR-421 can inhibit FOXO4 signaling pathway.
2, study on the relationship between miR-421 and FOXO4
Objective: To explore the regulatory role of miR-421 in raking gene FOXO4 in gene regulation.
Methods: the TargetScan software tool mir-421 target prediction analysis showed that FOXO4 was a potential target of mir-421. The expression of FOXO4 in the miR-421 overexpressed group and the miR-421 inhibition group was significantly different in the normal group, and the expression of the overexpressed histone expression was lower than that in the normal group, and the expression of the inhibitory histone expression was higher than that in the normal group. Normal group.
In order to further verify the relationship between miR-421 and FOXO4, we cloned FOXO43'UTR fragment, psiCHECK-2 luciferase carrier containing miR-421 binding site, and we cloned the FOXO43'UTR mutant vector and mutated two bases.
The luciferase activity assay showed that the miR-421 overexpression group was lower than the normal group, while the miR-421 inhibition group was higher than the normal group.
In addition, in order to further analyze the interaction between miR-421 and FOXO4 in apoptosis and inhibition of miR-421 at the time of inhibiting the FOXO4 as control, the experiment showed that after the inhibition of the FOXO4 gene, the apoptosis was lower than that of the only inhibition of the miR-421 group.
Results: 1, the expression of gene FOXO4 in miR-421 overexpression and miR-421 inhibition group was different. 2, fluorescein detection showed that miR-421 overexpression was different from that in miR-421 inhibition group, but there was no difference in the mutant group; 3, at the same time inhibition of miR-421, the selective down gene FOXO4 was selected. The results showed that the apoptotic cells were lower than simple cells. Inhibition of group miR-421.
Conclusion: gene FOXO4 is the target gene for the regulation of miR-421.
3. FOXO4 is a key gene for cell growth and anti apoptosis
Objective: To explore the regulatory effect of FOXO4 on cell growth and anti apoptosis.
Methods: in order to further analyze the role of miR-421 and FOXO4 in apoptosis, we chose to inhibit miR-421 and then only inhibit gene FOXO4 to control, and the detection data showed that the number of apoptotic cells decreased after the inhibition of miR-421 at the same time.
Results: when miR-421 was inhibited, the FOXO4 gene was downregulated. The results showed that the apoptotic cells were lower than those of miR-421 alone.
Conclusion: FOXO4 is a key gene for cell growth and anti apoptosis.
4, the clinical correlation between miR-421 and FOXO4 in nasopharyngeal carcinoma tissues.
Objective: To study the relationship between miR-421 and FOXO4 in clinical specimens of nasopharyngeal carcinoma.
Methods: 7 samples of freshly collected human nasopharyngeal carcinoma samples were collected. The expression of miR-421 and FOXO4 was detected by QPCR technique. The expression of FOXO4 was detected by Western blot, and the relationship between them was studied.
Results: in 7 samples of nasopharyngeal carcinoma, the expression level of miR-421 was high, and the expression level of FOXO4 was low, and there was a negative correlation between them (r=-0.798, P=0.032).
Conclusion: the expression of miR-421 is negatively correlated with the expression of FOXO4 in nasopharyngeal carcinoma tissues.
In conclusion, up regulation of miR-421 can inhibit the expression of FOXO4, which leads to the proliferation and apoptosis resistance of nasopharyngeal carcinoma cells. Conversely, the inhibition of miR-421 expression in the experiment can inhibit the proliferation of nasopharyngeal carcinoma cells and promote its apoptosis.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63
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