非小細胞肺癌中EGFR表達及其啟動子甲基化的臨床意義研究
發(fā)布時間：2019-06-21 05:30
【摘要】：EGFR基因位于第7號染色體的p1 1.2,編碼橫跨細胞膜表面的糖蛋白受體。EGFR為酪氨酸激酶受體,是蛋白激酶家族重要一員。EGFR與其配體結(jié)合成二聚體,隨后酪氨酸自身磷酸化啟動下游各通路,從而調(diào)節(jié)細胞的增殖,分化,凋亡等。在多種癌癥中EGFR信號通路的改變導致了癌細胞更難以被清除。大量文獻報道EGFR蛋白的高表達與多種腫瘤的分期和預(yù)后相關(guān)。近年來表觀遺傳學的調(diào)控機制已經(jīng)成為研究的熱點,表觀遺傳學信息是指基因的DNA序列未發(fā)生變化而表型發(fā)生了改變,并且這種改變在發(fā)育和細胞增殖過程中能穩(wěn)定遺傳。DNA甲基化是表觀遺傳學調(diào)控機制之一。本研究旨在研究非小細胞肺癌中EGFR表達與臨床各指標間的聯(lián)系,并探討EGFR啟動子甲基化調(diào)控的改變與EGFR表達間的相關(guān)性。[目的]1.應(yīng)用免疫印跡法檢測肺癌組織和正常組織樣本中EGFR蛋白表達,并研究樣本中EGFR表達水平與組織分類,臨床分期,樣本病理等臨床資料的相關(guān)性。2.應(yīng)用甲基化特異性PCR檢測肺癌組織和正常組織樣本中EGFR基因啟動子區(qū)CpG島甲基化情況,并研究樣品中EGFR啟動子甲基化水平與EGFR的表達差異間的相關(guān)性。[方法]1.收集2015年12月至2016年5月間云南省腫瘤醫(yī)院收治的手術(shù)切除的50例非小細胞肺癌患者的癌組織與正常組織樣本。入選標準為:①手術(shù)切除標本經(jīng)病理診斷為非小細胞肺癌②術(shù)前未經(jīng)過任何抗腫瘤治療③腫瘤經(jīng)手術(shù)切除,切緣為陰性。④年齡≥30歲,≤75歲。2.將病人標本收回后,用液氮冰凍后至-80℃冰箱中保存。組織研磨后用試劑盒提取DNA,用Ripa提取蛋白,并測定其吸光度和濃度。提取后放回-80℃冰箱中保存待后續(xù)處理。3.BCA測定蛋白濃度,并使用Western-blot測定EGFR的蛋白表達。4.提取的DNA進行急速重亞硫酸鹽處理,并用甲基化引物進行擴增,運用瓊脂糖凝膠電泳,觀測其表達差異。5.Western-blot 結(jié)果采用圖像分析處理軟件(ImageJ,National Institutes of Health)進行半定量分析。6.應(yīng)用SPSS20.0軟件包的分析和統(tǒng)計,各組間實驗數(shù)據(jù)進行正態(tài)分布,方差齊性檢驗,非正態(tài)分布數(shù)據(jù)之間的差異用卡方(chi-square test)和秩和(Wilcoxon signed ranks test)檢驗法,應(yīng)用非線性相關(guān)分析進行相關(guān)性分析。[結(jié)果]1.在非小細胞肺癌中癌和正常組織中EGFR的表達有統(tǒng)計學差異(p=0.009),EGFR在癌中高表達。2.EGFR的表達與淋巴結(jié)轉(zhuǎn)移有統(tǒng)計學意義(p=0.008) , EGFR表達越高,淋巴結(jié)轉(zhuǎn)移的可能性增加,兩者呈正相關(guān)。3.EGFR的表達與EGFR啟動子區(qū)甲基化具有統(tǒng)計學意義(p=0.025),甲基化程度降低時EGFR的表達上調(diào),兩者呈負相關(guān)。[結(jié)論]1.EGFR在肺癌組織中相對高表達,且EGFR在肺癌的癌組織中相對表達與淋巴結(jié)轉(zhuǎn)移相關(guān),說明EGFR的表達可能成為臨床上判斷疾病進展的指標和治療的靶點。2.EGFR的表達與甲基化的改變具有相關(guān)性,肺癌的癌組織中EGFR啟動子相對低甲基化時EGFR表達有增高趨勢,EGFR啟動子相對高甲基化時EGFR表達有降低趨勢,所以EGFR的表達可能與啟動子甲基化狀態(tài)相關(guān),為以EGFR為靶點的腫瘤靶向治療提供了新的思路。
[Abstract]:The EGFR gene is located at p1 1.2 of chromosome 7 and encodes a glycoprotein receptor across the surface of the cell membrane. EGFR is a tyrosine kinase receptor and is an important member of the protein kinase family. EGFR binds to its ligand to form a dimer, followed by tyrosine autophosphorylation to initiate downstream pathways, thereby modulating cell proliferation, differentiation, apoptosis, and the like. The change in the EGFR signaling pathway in a variety of cancers leads to a more difficult removal of cancer cells. A large number of literature reports that the high expression of the EGFR protein is associated with the staging and prognosis of a variety of tumors. In recent years, the control mechanism of epigenetics has become the hot spot of the research, and the epigenetic information is that the DNA sequence of the gene has not changed and the phenotype has changed, and the change can be stably inherited in the course of development and cell proliferation. DNA methylation is one of the mechanisms of epigenetic control. The purpose of this study was to study the relationship between EGFR expression in non-small cell lung cancer and the clinical indicators, and to explore the relationship between the change of EGFR promoter methylation and the expression of EGFR. [Objective] 1. The expression of EGFR in lung cancer tissues and normal tissue samples was detected by immunoblotting method, and the correlation of the expression level of EGFR in the samples with the clinical data such as tissue classification, clinical stage and sample pathology was studied. Methylation-specific PCR was used to detect the methylation of the CpG island in the promoter region of the EGFR gene in the lung cancer tissue and the normal tissue sample, and the relationship between the methylation level of the EGFR promoter and the expression of EGFR in the sample was studied. [Method] 1. To collect 50 non-small cell lung cancer patients with non-small cell lung cancer and normal tissue samples from December 2015 to May 2016. The inclusion criteria were as follows: the surgical resection of the specimens was performed by the pathologic diagnosis of non-small cell lung cancer without any anti-tumor treatment, and the tumor was removed by operation and the cut edge was negative. She is 30 years old and 75 years of age. After the patient's specimen was recovered, the patient was frozen with liquid nitrogen and stored in a-80.degree. C. refrigerator. After the tissue was ground, the DNA was extracted with the kit, and the protein was extracted with the Ripa and its absorbance and concentration were determined. And the protein concentration of the EGFR was measured by Western-blot, and the protein expression of the EGFR was determined by Western-blot. The extracted DNA was subjected to rapid bisulfite treatment and amplified by a methylation primer, and the expression difference was observed by agarose gel electrophoresis. Based on the analysis and statistics of the SPSS10.0 software package, the normal distribution, the homogeneity test and the non-normal distribution data between the groups were analyzed by the chi-square test and the Wilcoxon signed test, and the correlation analysis was carried out with the non-linear correlation analysis. [Results] 1. The expression of EGFR in non-small cell lung cancer and normal tissue was statistically different (p = 0.009), and EGFR was highly expressed in cancer.2. The expression of EGFR and lymph node metastasis were of statistical significance (p = 0.008), and the higher the expression of EGFR, the possibility of lymph node metastasis was increased. There was a positive correlation between the expression of EGFR and the methylation of EGFR promoter region (p = 0.025). [Conclusion] 1. EGFR is relatively highly expressed in lung cancer tissues, and the relative expression of EGFR in the cancer tissues of lung cancer is related to lymph node metastasis, and it is indicated that the expression of EGFR may be a target for clinical judgment of disease progression and the target of treatment.2. The expression of EGFR is related to the change of methylation. In the case of lung cancer, the expression of EGFR is increased in the case of relatively low methylation of the EGFR promoter, and the expression of EGFR in the case of relatively high methylation of the EGFR promoter tends to decrease, so the expression of EGFR may be related to the methylation state of the promoter, and a new idea is provided for the target therapy of the tumor targeting EGFR.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
本文編號：2503826
[Abstract]:The EGFR gene is located at p1 1.2 of chromosome 7 and encodes a glycoprotein receptor across the surface of the cell membrane. EGFR is a tyrosine kinase receptor and is an important member of the protein kinase family. EGFR binds to its ligand to form a dimer, followed by tyrosine autophosphorylation to initiate downstream pathways, thereby modulating cell proliferation, differentiation, apoptosis, and the like. The change in the EGFR signaling pathway in a variety of cancers leads to a more difficult removal of cancer cells. A large number of literature reports that the high expression of the EGFR protein is associated with the staging and prognosis of a variety of tumors. In recent years, the control mechanism of epigenetics has become the hot spot of the research, and the epigenetic information is that the DNA sequence of the gene has not changed and the phenotype has changed, and the change can be stably inherited in the course of development and cell proliferation. DNA methylation is one of the mechanisms of epigenetic control. The purpose of this study was to study the relationship between EGFR expression in non-small cell lung cancer and the clinical indicators, and to explore the relationship between the change of EGFR promoter methylation and the expression of EGFR. [Objective] 1. The expression of EGFR in lung cancer tissues and normal tissue samples was detected by immunoblotting method, and the correlation of the expression level of EGFR in the samples with the clinical data such as tissue classification, clinical stage and sample pathology was studied. Methylation-specific PCR was used to detect the methylation of the CpG island in the promoter region of the EGFR gene in the lung cancer tissue and the normal tissue sample, and the relationship between the methylation level of the EGFR promoter and the expression of EGFR in the sample was studied. [Method] 1. To collect 50 non-small cell lung cancer patients with non-small cell lung cancer and normal tissue samples from December 2015 to May 2016. The inclusion criteria were as follows: the surgical resection of the specimens was performed by the pathologic diagnosis of non-small cell lung cancer without any anti-tumor treatment, and the tumor was removed by operation and the cut edge was negative. She is 30 years old and 75 years of age. After the patient's specimen was recovered, the patient was frozen with liquid nitrogen and stored in a-80.degree. C. refrigerator. After the tissue was ground, the DNA was extracted with the kit, and the protein was extracted with the Ripa and its absorbance and concentration were determined. And the protein concentration of the EGFR was measured by Western-blot, and the protein expression of the EGFR was determined by Western-blot. The extracted DNA was subjected to rapid bisulfite treatment and amplified by a methylation primer, and the expression difference was observed by agarose gel electrophoresis. Based on the analysis and statistics of the SPSS10.0 software package, the normal distribution, the homogeneity test and the non-normal distribution data between the groups were analyzed by the chi-square test and the Wilcoxon signed test, and the correlation analysis was carried out with the non-linear correlation analysis. [Results] 1. The expression of EGFR in non-small cell lung cancer and normal tissue was statistically different (p = 0.009), and EGFR was highly expressed in cancer.2. The expression of EGFR and lymph node metastasis were of statistical significance (p = 0.008), and the higher the expression of EGFR, the possibility of lymph node metastasis was increased. There was a positive correlation between the expression of EGFR and the methylation of EGFR promoter region (p = 0.025). [Conclusion] 1. EGFR is relatively highly expressed in lung cancer tissues, and the relative expression of EGFR in the cancer tissues of lung cancer is related to lymph node metastasis, and it is indicated that the expression of EGFR may be a target for clinical judgment of disease progression and the target of treatment.2. The expression of EGFR is related to the change of methylation. In the case of lung cancer, the expression of EGFR is increased in the case of relatively low methylation of the EGFR promoter, and the expression of EGFR in the case of relatively high methylation of the EGFR promoter tends to decrease, so the expression of EGFR may be related to the methylation state of the promoter, and a new idea is provided for the target therapy of the tumor targeting EGFR.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
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