宫颈癌辐射抵抗相关miRNAs的筛选及初步功能研究

发布时间：2018-02-23 17:29

本文关键词： 辐射抵抗 miRNA miR-4778-3p 宫颈癌　出处：《第四军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景和研究目的宫颈癌(cervical carcinoma, CC)是发展中国家女性第一高发恶性肿瘤，，放射治疗是宫颈癌治疗的重要手段之一。然而，接受根治性放疗的局部晚期宫颈癌患者仍然有30%左右出现肿瘤复发和转移，导致治疗失败。其肿瘤细胞或肿瘤辐射抵抗特性可能是放疗后复发或转移的主要原因。研究表明引起肿瘤的辐射抵抗机制十分复杂，目前认为可能包括肿瘤细胞DNA损伤与修复异常、低氧环境和低氧诱导因子形成、肿瘤干细胞的存在、以及相关的microRNAs产生等多种因素。深入研究肿瘤辐射抵抗的形成机制或分子网络，对于有效改善肿瘤辐射治疗质量具有重要意义。 microRNAs（miRNAs）是近年来研究的热点，是真核生物体内一种内源性的非编码小RNA，在体内主要发挥基因沉默作用。大量研究显示，miRNA和多种恶性肿瘤的发生发展密切相关。已有报道显示异常表达的miRNA与白血病、肺腺癌、乳腺癌、胰腺癌等疾病的化疗抵抗、复发转移及预后密切相关。但是，miRNA异常表达与宫颈癌辐射敏感型或辐射抵抗的关系，尚有待研究阐明。本研究首先通过具有辐射敏感性差别的宫颈癌组织，筛选宫颈癌辐射抗性相关miRNAs，然后通过宫颈癌细胞培养和基因转染等技术，进行miRNA的功能验证和效应机制探讨，为临床预测宫颈癌放疗预后、逆转宫颈癌辐射抵抗、提高肿瘤治愈率提供新的思路和依据。材料和方法 1.选择我科八例宫颈癌患者组织标本，三例放疗后三年内无复发转移患者为对照组（辐射敏感组），三例局部复发、两例转移患者为实验组（辐射抵抗组）。采用美国Affymetrix公司microRNA芯片进行基因表达谱的差异性筛选，数据库版本和图像分析软件分别为miRBase Release20和GenePix pro V6.0。 2.采用实时荧光定量RT-PCR技术对基因芯片结果进行验证，并在宫颈癌辐射敏感及宫颈癌辐射抵抗的组织标本中，对所选差异表达miRNA表达水平进行验证。 3.采用基因转染方法，经特异miRNA前体和抑制物上调或下调HeLa及SiHa宫颈癌细胞miRNA表达水平，CCK-8、克隆形成、划痕实验、Transwell侵袭实验、流式细胞术等观察辐射后细胞增殖、侵袭能力、细胞凋亡及细胞周期变化。 4.采用多个生物信息网站对miR-4778-3p可能调控的下游靶基因进行初步预测，查阅相关文献，对miR-4778-3p调控辐射抵抗的可能机制进行初步探索。 5.研究结果采用SPSS17.0软件进行统计学处理，采用独立样本的t检验，P0.05差异有统计学意义。主要结果 1.通过miRNA芯片筛选，共获得32个与宫颈癌辐射抵抗相关的差异表达miRNA分子，差异性表达均在2倍以上(p＜0.05)；与辐射敏感组织相比，在复发患者中发生表达上调miRNA分子共10个，表达下调9个。在远转患者中表达上调9个，表达下调4个。其中，miR-4778-3p在复发及转移患者中的表达水平均明显下调。RT-PCR结果与芯片结果具有良好一致性，结果表明miR-4778-3p在复发转移患者肿瘤组织中表达降低（p＜0.01)。 2.使用特异性miR-4778-3p前体或抑制物转染宫颈癌细胞系HeLa及SiHa使其表达水平升高或降低后发现，与对照细胞相比，上调miR-4778-3p的表达显著抑制细胞辐射后的增殖活力、克隆形成和侵袭能力，而下调miR-4778-3p的表达水平后，宫颈癌细胞系的细胞活力显著增加。然而，与对照组相比，改变miR-4778-3p的表达水平，对辐照诱导的细胞凋亡和细胞周期无影响。 3.通过生物学靶基因预测分析，初步结果显示XRCC2、IRS2、IGF2BP2、CDH2、MEMO1、MYH7、ASH1及Slit1等靶基因可能受到miR-4778-3p的下游调控。但对于其确切调节作用和机制，需要进一步的实验验证。初步结论 1.本研究发现miR-4778-3p宫颈癌辐射敏感及辐射抵抗组织中表达水平有显著差异，细胞实验上调或下调miR-4778-3p能够降低或升高宫颈癌细胞辐照后细胞活力及增殖能力，提示miR-4778-3p可能是调节宫颈癌辐射抗性的重要因子。 2.初步表明miR-4778-3p可能通过靶向调控XRCC2、IRS2、IGF2BP2、CDH2、MEMO1、MYH7、ASH1及Slit1蛋白表达水平，影响宫颈癌细胞的辐射敏感性。但miR-4778-3p调控宫颈癌辐射抗性的确切下游机制问题，仍有待进一步深入研究。
[Abstract]:Background and research purposes
Cervical cancer (cervical carcinoma CC) is the most common malignant tumor of women in developing countries, radiotherapy is one of the important means of the treatment of cervical cancer. However, patients with locally advanced cervical cancer received radical radiotherapy still have tumor recurrence and metastasis 30%, leading to treatment failure. The tumor cells or tumor radiation resistance may be the main reason of recurrence or metastasis after radiotherapy. The research showed that the cancer causing mechanism of radiation resistance is considered to be very complex, including tumor cell DNA damage and repair of abnormal, hypoxia and hypoxia inducible factor formation, the existence of cancer stem cells, and the related factors. The formation mechanism of microRNAs or molecular network of tumor research radiation resistance, is of great significance to improve the quality of tumor radiation therapy.
MicroRNAs (miRNAs) is a research hotspot in recent years, is a kind of endogenous small non encoding RNA in eukaryotes. The in vivo plays an important role in gene silencing. Many studies show that closely related to the occurrence and development of malignant tumors and miRNA. Several reports have shown that the abnormal expression of miRNA and leukemia, lung cancer, breast cancer pancreatic cancer, disease resistance to chemotherapy, recurrence and prognosis. However, the relationship between the abnormal expression of miRNA in radiation sensitive or radiation resistance and cervical cancer remains to be elucidated. In this study, firstly by having different radiation sensitivity of cervical cancer, cervical cancer screening radiation resistance related miRNAs, followed by cervical cancer cell culture and gene transfection technique, to investigate the effect and mechanism of miRNA functional verification, for clinical radiotherapy of cervical cancer prognosis prediction of cervical cancer, reversal of radiation resistance, increase the cure rate of cancer Provide new ideas and basis.
Materials and methods
My 1. choice of eight cases of patients with cervical cancer tissue specimens, three cases after radiotherapy within three years without recurrence and metastasis in patients as control group (radiation sensitive group), three cases of local recurrence, two cases of metastatic patients into the experimental group (radiation resistant group). The American Affymetrix company microRNA chip gene expression difference spectrum screening the version and image analysis software were miRBase Release20 and GenePix database Pro V6.0.
2., real-time fluorescence quantitative RT-PCR technology was used to verify the results of gene chip, and the expression level of miRNA was detected in cervical cancer radiosensitivity and cervical cancer radiative resistance tissue samples.
3. by gene transfection method, the specific miRNA precursors and inhibitors or downregulation of HeLa and SiHa in cervical cancer cells the expression level of miRNA, CCK-8, clone formation, scratch test, Transwell invasion assay, cell proliferation, flow cytometry was observed after radiation invasion, apoptosis and cell cycle changes.
4., we used multiple bioinformatics websites to preliminarily predict the downstream target genes that might be regulated by miR-4778-3p. We consulted related literatures, and explored the possible mechanism of miR-4778-3p regulating radiation resistance.
5. the results of the study were statistically treated with SPSS17.0 software, and the independent sample t test was used, and the difference of P0.05 was statistically significant.
Main results
1. through miRNA chip screening, and obtained a total of 32 cervical cancer radiation resistance related differential expression of miRNA molecule expression were more than 2 times (P < 0.05); compared with the radiation sensitive tissue in patients with recurrent, upregulation of the expression of miRNA molecule is 10, down regulate the expression of 9. In the far patients in expression 9, expression of 4. Among them, the expression level of miR-4778-3p in patients with recurrence and metastasis were significantly reduced in good consistency with the results of.RT-PCR microarray results, the results show that the lower expression of miR-4778-3p in tumor tissue of patients with metastatic recurrence (P < 0.01).
2. the use of specific miR-4778-3p precursor or inhibitor transfected into cervical cancer cell lines HeLa and SiHa. The expression level increased or decreased after that, compared with the control cells, increase the expression of miR-4778-3p after irradiation significantly inhibited the cell proliferation activity, colony formation and invasion, and down regulate the expression level of miR-4778-3p, cervical carcinoma cell line the cell viability was significantly increased. However, compared with the control group, the level of the expression change of miR-4778-3p had no effect on cell apoptosis and cell cycle induced by irradiation.
3., through the prediction and analysis of biological target genes, preliminary results show that target genes such as XRCC2, IRS2, IGF2BP2, CDH2, MEMO1, MYH7, ASH1 and Slit1 may be regulated by miR-4778-3p downstream. However, the exact regulation mechanism and mechanism need further experimental validation.
Preliminary conclusion
1. this study found that miR-4778-3p radiation sensitive and radiation resistance of cervical cancer tissue expression levels had significant differences, or downregulation of miR-4778-3p cells to cell viability and proliferation of cervical cancer cells increased or decreased after irradiation, indicating that miR-4778-3p may be an important factor in the regulation of radiation resistance of cervical cancer.
2. preliminary showed that miR-4778-3p might be regulated by XRCC2, to target IRS2, IGF2BP2, CDH2, MEMO1, MYH7, ASH1 and Slit1 protein expression levels, effects of radiation sensitivity of cervical cancer cells. But the exact mechanism of miR-4778-3p in regulating downstream cervical cancer radiation resistance problem, still need further research.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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