本文關鍵詞： 輻射抵抗 miRNA miR-4778-3p 宮頸癌　出處：《第四軍醫(yī)大學》2014年碩士論文　論文類型：學位論文

【摘要】：背景和研究目的 宮頸癌(cervical carcinoma, CC)是發(fā)展中國家女性第一高發(fā)惡性腫瘤，，放射治療是宮頸癌治療的重要手段之一。然而，接受根治性放療的局部晚期宮頸癌患者仍然有30%左右出現腫瘤復發(fā)和轉移，導致治療失敗。其腫瘤細胞或腫瘤輻射抵抗特性可能是放療后復發(fā)或轉移的主要原因。研究表明引起腫瘤的輻射抵抗機制十分復雜，目前認為可能包括腫瘤細胞DNA損傷與修復異常、低氧環(huán)境和低氧誘導因子形成、腫瘤干細胞的存在、以及相關的microRNAs產生等多種因素。深入研究腫瘤輻射抵抗的形成機制或分子網絡，對于有效改善腫瘤輻射治療質量具有重要意義。 microRNAs（miRNAs）是近年來研究的熱點，是真核生物體內一種內源性的非編碼小RNA，在體內主要發(fā)揮基因沉默作用。大量研究顯示，miRNA和多種惡性腫瘤的發(fā)生發(fā)展密切相關。已有報道顯示異常表達的miRNA與白血病、肺腺癌、乳腺癌、胰腺癌等疾病的化療抵抗、復發(fā)轉移及預后密切相關。但是，miRNA異常表達與宮頸癌輻射敏感型或輻射抵抗的關系，尚有待研究闡明。本研究首先通過具有輻射敏感性差別的宮頸癌組織，篩選宮頸癌輻射抗性相關miRNAs，然后通過宮頸癌細胞培養(yǎng)和基因轉染等技術，進行miRNA的功能驗證和效應機制探討，為臨床預測宮頸癌放療預后、逆轉宮頸癌輻射抵抗、提高腫瘤治愈率提供新的思路和依據。 材料和方法 1.選擇我科八例宮頸癌患者組織標本，三例放療后三年內無復發(fā)轉移患者為對照組（輻射敏感組），三例局部復發(fā)、兩例轉移患者為實驗組（輻射抵抗組）。采用美國Affymetrix公司microRNA芯片進行基因表達譜的差異性篩選，數據庫版本和圖像分析軟件分別為miRBase Release20和GenePix pro V6.0。 2.采用實時熒光定量RT-PCR技術對基因芯片結果進行驗證，并在宮頸癌輻射敏感及宮頸癌輻射抵抗的組織標本中，對所選差異表達miRNA表達水平進行驗證。 3.采用基因轉染方法，經特異miRNA前體和抑制物上調或下調HeLa及SiHa宮頸癌細胞miRNA表達水平，CCK-8、克隆形成、劃痕實驗、Transwell侵襲實驗、流式細胞術等觀察輻射后細胞增殖、侵襲能力、細胞凋亡及細胞周期變化。 4.采用多個生物信息網站對miR-4778-3p可能調控的下游靶基因進行初步預測，查閱相關文獻，對miR-4778-3p調控輻射抵抗的可能機制進行初步探索。 5.研究結果采用SPSS17.0軟件進行統(tǒng)計學處理，采用獨立樣本的t檢驗，P0.05差異有統(tǒng)計學意義。 主要結果 1.通過miRNA芯片篩選，共獲得32個與宮頸癌輻射抵抗相關的差異表達miRNA分子，差異性表達均在2倍以上(p＜0.05)；與輻射敏感組織相比，在復發(fā)患者中發(fā)生表達上調miRNA分子共10個，表達下調9個。在遠轉患者中表達上調9個，表達下調4個。其中，miR-4778-3p在復發(fā)及轉移患者中的表達水平均明顯下調。RT-PCR結果與芯片結果具有良好一致性，結果表明miR-4778-3p在復發(fā)轉移患者腫瘤組織中表達降低（p＜0.01)。 2.使用特異性miR-4778-3p前體或抑制物轉染宮頸癌細胞系HeLa及SiHa使其表達水平升高或降低后發(fā)現，與對照細胞相比，上調miR-4778-3p的表達顯著抑制細胞輻射后的增殖活力、克隆形成和侵襲能力，而下調miR-4778-3p的表達水平后，宮頸癌細胞系的細胞活力顯著增加。然而，與對照組相比，改變miR-4778-3p的表達水平，對輻照誘導的細胞凋亡和細胞周期無影響。 3.通過生物學靶基因預測分析，初步結果顯示XRCC2、IRS2、IGF2BP2、CDH2、MEMO1、MYH7、ASH1及Slit1等靶基因可能受到miR-4778-3p的下游調控。但對于其確切調節(jié)作用和機制，需要進一步的實驗驗證。 初步結論 1.本研究發(fā)現miR-4778-3p宮頸癌輻射敏感及輻射抵抗組織中表達水平有顯著差異，細胞實驗上調或下調miR-4778-3p能夠降低或升高宮頸癌細胞輻照后細胞活力及增殖能力，提示miR-4778-3p可能是調節(jié)宮頸癌輻射抗性的重要因子。 2.初步表明miR-4778-3p可能通過靶向調控XRCC2、IRS2、IGF2BP2、CDH2、MEMO1、MYH7、ASH1及Slit1蛋白表達水平，影響宮頸癌細胞的輻射敏感性。但miR-4778-3p調控宮頸癌輻射抗性的確切下游機制問題，仍有待進一步深入研究。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

【參考文獻】

相關期刊論文 前1條

1 Andrii Vislovukh;Thaiz Rivera Vargas;Anna Polesskaya;Irina Groisman;;Role of 3'-untranslated region translational control in cancer development, diagnostics and treatment[J];World Journal of Biological Chemistry;2014年01期

本文編號：1526983