MicroRNA-21、PTEN及P-gp在大肠癌中的表达及三者关系的研究

发布时间：2018-03-06 19:36

本文选题：大肠癌　切入点：多药耐药　出处：《承德医学院》2017年硕士论文　论文类型：学位论文

【摘要】：大肠癌(colorectal cancer,CRC)是人类最常见的恶性肿瘤之一,严重危害着人类的健康。化疗是治疗中晚期大肠癌的一种重要策略,但多种原因导致的多药耐药(multidrug resistance,MDR)使得临床上化疗效果并不理想。MDR的机制非常复杂,但其中一个主要的原因是MDR基因MDR1过度表达P-糖蛋白(P-gp)。P-gp过表达可作为肿瘤化疗耐药的一个预测指标。任何能抑制或减少P-gp表达的因子均有助于提高化疗效果。张力蛋白同源的10号染色体缺失的磷酸酶(PTEN)基因是一个抑癌基因,促进野生型PTEN基因及蛋白的表达可以增加传统化疗药物的化疗效果,逆转肿瘤细胞多药耐药。检索Uni Hi数据库发现PTEN蛋白和P-gp之间存在相互关系。目前,已证实mi R-21是PTEN的调控基因,且多项研究报导mi R-21表达水平与细胞凋亡、增殖与迁移、预后、化疗耐药等密切相关,但mi R-21参与肿瘤耐药的机制目前尚不清楚。检索RNA 22发现,miR-21与MDR1基因有部分碱基序列互补配对,预测miR-21也可能是MDR1的调控基因,进而调控P-gp的表达。另外,福尔马林固定石蜡包埋是临床病理组织最常规的储存方式,石蜡标本包含有丰富的临床信息和病理资料,且石蜡组织中mi RNAs的分子结构和化学性质稳定,因此石蜡标本是进行分子生物学实验及人类疾病回顾性研究的现成资源。目的:本实验通过检测PTEN与P-gp在大肠癌组织及癌旁正常大肠黏膜组织中的表达,分析PTEN、P-gp与大肠癌临床病理学特征的关系及二者的相关性,通过石蜡组织提取mi R-21,分析mi R-21与大肠癌分化程度的关系及mi R-21与P-gp的相关性,探讨mi R-21是否与大肠癌多药耐药的形成有关,从而为评估大肠癌患者化疗耐药提供新的分子标志物和潜在的治疗靶点方法：收集2015年1月至2016年1月承德医学院附属医院术前均未行放化疗的大肠癌与癌旁正常黏膜石蜡标本各65例、新鲜标本各20例。采用免疫组化SP法检测65例石蜡组织和癌旁正常组织中PTEN蛋白、P-gp的表达,结合光学显微镜与MiVnt纤维图像分析系统对实验结果进行分析;采用Western Blot方法检测新鲜组织中PTEN蛋白、P-gp表达,运用Quantity One软件对条带灰度值进行量化分析;采用实时荧光定量PCR(q RT-PCR)检测并分析40例大肠癌石蜡组织与相应癌旁正常黏膜组织中mi R-21的表达。应用SPSS 19.0软件对实验数据进行统计分析。结果:1免疫组化实验结果1.1 PTEN蛋白与P-gp在大肠癌组织和癌旁正常组织中的表达PTEN蛋白在大肠癌组织中的阳性表达率(52.3%)显著低于癌旁正常组织(76.9%),差异具有统计学意义(χ2=8.613,P=0.003)。P-gp在大肠癌组织中阳性表达率(89.2%)显著高于癌旁正常组织(75.4%),差异具有统计学意义(χ2=4.279,P=0.039)1.2大肠癌组织中PTEN蛋白、P-gp表达与临床病理学特征的关系大肠癌组织中PTEN蛋白表达与分化程度、浸润深度、局部淋巴结转移有关,差异具有统计学意义(P0.05)。大肠癌组织中P-gp表达与分化程度和浸润深度有关,差异具有统计学意义(P0.05)。1.3大肠癌中PTEN蛋白表达与P-gp表达的相关性34例PTEN蛋白阳性表达的大肠癌组织中P-gp阳性表达占27例(79.4%),P-gp阴性表达占7例(20.6%);31例PTEN蛋白阴性表达的大肠癌组织中P-gp阳性表达占31例(100%)。Spearman相关性分析显示大肠癌组织中PTEN蛋白和P-gp的表达呈负相关(r=-0.332,P=0.007)。2 Western Blot实验结果大肠癌组织中PTEN蛋白的表达显著低于癌旁正常组织,差异具有统计学意义(t=-1.178,P=0.000);癌组织中P-gp表达显著高于癌旁正常组织,差异具有统计学意义(t=-3.275,P=0.004),且P-gp在中低分化组中的表达较高分化组中表达更高,差异具有统计学意义(t=7.905,P=0.000)。mi R-21在大肠癌组织中平均相对表达量较癌旁正常黏膜明显升高,差异具有统计学意义(P=0.000),且中、低分化组与各自相应的正常黏膜组织相比较,差异具有统计学意义(P中=0.030、P低=0.000)。在高、中、低分化组中,mi R-21的平均相对表达量分别是相应正常黏膜组织的1.25、1.69、2.04倍;组间比较:低分化组mi R-21表达较高分化组明显升高,差异有统计学意义(P0.05)。3.2大肠癌组织中mi R-21表达与P-gp表达的相关性Spearman相关分析显示miR-21表达与P-gp表达在大肠癌组织中呈正相关(r=0.444,P=0.004)。结论:1大肠癌组织中PTEN蛋白表达降低与分化程度、浸润深度、局部淋巴结转移密切相关。2大肠癌组织中P-gp表达升高与分化程度和浸润深度密切相关。3大肠癌组织中PTEN蛋白表达与P-gp表达呈负相关,PTEN与大肠癌多药耐药的关系可能与P-gp高表达有关。4大肠癌组织中miR-21表达与分化程度密切相关,与P-gp表达呈正相关,miR-21与大肠癌多药耐药的关系也可能与P-gp高表达有关。
[Abstract]:Colorectal cancer (colorectal cancer CRC) is one of the most common malignant tumor that seriously endangers human health. The chemotherapy is an important strategy in the treatment of advanced colorectal cancer, but multidrug resistance caused by various reasons (multidrug, resistance, MDR) the clinical treatment effect is not ideal.MDR mechanism is very complex however, one of the main reasons is the MDR gene overexpression of MDR1 glycoprotein P- (P-gp).P-gp expression as a predictor of tumor resistance to chemotherapy. Any can inhibit or reduce the expression of P-gp factor are helpful to enhance chemotherapy effect. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is a tumor suppressor gene, promote the expression of wild type PTEN gene and protein can increase the chemotherapeutic effect of traditional chemotherapy drugs, reversing the multidrug resistance of tumor cells. PTEN protein and P-gp Uni Hi database retrieval There is a correlation between. At present, it has been confirmed that MI R-21 is the PTEN gene, and the expression of a number of studies reported that MI R-21 level and cell apoptosis, proliferation and migration, prognosis, drug resistance is closely related, but the mechanism of MI R-21 in tumor resistance is unclear. The retrieval of RNA 22 found that miR-21 and MDR1 gene partial nucleotide sequence complementary, prediction of miR-21 may also be MDR1 gene, expression and regulation of P-gp. In addition, formalin fixed and paraffin embedded tissue is the most conventional clinical pathological storage, paraffin contains the clinical information and pathological data rich, molecular structure and chemical properties and stability of RNAs MI in paraffin embedded tissues therefore, is paraffin specimens of the molecular biology study and retrospective study of human diseases existing resources. Objective: through the detection of PTEN and P-gp in colorectal cancer tissue and normal The expression of intestinal mucosa in the analysis of PTEN, correlation between P-gp and clinicopathological features of colorectal cancer and two patients, through the paraffin tissue extraction of MI R-21 and MI R-21, the correlation analysis between P-gp MI and R-21 and differentiation degree of colorectal cancer, to investigate whether mi R-21 associated with the formation of multidrug resistance of colorectal cancer. In order to provide, and potential therapeutic targets for the assessment of new molecular markers in patients with colorectal cancer chemotherapy: from January 2015 to January 2016 in Affiliated Hospital of Chengde Medical College were treated without preoperative chemotherapy of colorectal cancer and adjacent normal mucosa paraffin specimens of 65 cases, 20 cases of fresh specimens. PTEN protein was detected by immunohistochemistry SP methods 65 cases of paraffin embedded tissues and adjacent normal tissues, the expression of P-gp and MiVnt fiber by optical microscope image analysis system was used to analyze the experimental results; using Western Blot method to detect the fresh group The fabric in the PTEN protein, the expression of P-gp, using the Quantity software of One band gray value quantitative analysis; by using real-time quantitative PCR (Q RT-PCR) to detect the expression and analysis of 40 cases of colorectal cancer tissue and adjacent normal mucosa tissues in MI R-21. SPSS 19 software was used for statistical analysis of experimental data. 1 immunohistochemistry results: the positive expression of PTEN protein in colorectal cancer tissues the expression rate of PTEN protein and P-gp in 1.1 colorectal cancer tissues and adjacent normal tissues (52.3%) was significantly lower than that in adjacent normal tissues (76.9%), the difference was statistically significant (2=8.613, P=0.003).P-gp in colorectal carcinoma the positive expression rate (89.2%) was significantly higher than that in adjacent normal tissues (75.4%), the difference was statistically significant (2=4.279, P=0.039) PTEN protein in 1.2 colorectal cancer tissues, P-gp expression of PTEN and clinicopathological features in colorectal cancer organization Protein expression and degree of differentiation, depth of invasion, lymph node metastasis, the difference was statistically significant (P0.05). The expression of P-gp and differentiation and depth of infiltration of colorectal carcinoma, the difference was statistically significant (P0.05) P-gp associated with the expression of P-gp in 34 cases of the positive expression of PTEN protein in colorectal carcinoma and the positive expression for in 27 cases the expression of PTEN protein in colorectal cancer.1.3 (79.4%), negative expression of P-gp accounted for 7 cases (20.6%); the positive expression of P-gp in colorectal carcinoma and 31 cases of negative expression of PTEN protein in 31 cases (100%) were negatively related to the expression of.Spearman protein and correlation analysis showed PTEN P-gp in colorectal cancer tissue (r=-0.332. The experimental results of.2 Western Blot P=0.007) expression of PTEN protein in colorectal carcinoma was significantly lower than that in normal tissues, the difference was statistically significant (t=-1.178, P=0.000); P-gp in cancer tissue was significantly higher than that in adjacent normal tissues, The difference was statistically significant (t=-3.275, P=0.004), and P-gp in low differentiation group the expression of high differentiation group was higher, the difference was statistically significant (t=7.905, P=0.000).Mi R-21 in colorectal cancer with average relative expression compared with adjacent normal mucosa was significantly increased, the difference was statistically significant (P=0.000). And in low differentiation group compared with their corresponding normal tissues, the difference was statistically significant (P =0.030, P low =0.000). In the high and low differentiation group, the average Mi of the relative expression of R-21 were 1.25,1.69,2.04 times that of normal mucosal tissues; comparison between groups: low differentiation group mi R-21 expression of high differentiation group was significantly increased, the difference was statistically significant (P0.05) between Spearman and P-gp expression correlation analysis showed that the expression of miR-21 in colorectal cancer tissues was positively correlated with the P-gp mi R-21 expression of.3.2 in colorectal carcinoma ( R=0.444, P=0.004). Conclusion: to reduce the depth of invasion and differentiation, expression of PTEN protein in 1 colorectal cancer tissues, the expression of P-gp was negatively correlated with P-gp protein expression of PTEN increased with the degree of differentiation and infiltration depth of.3 is closely related to colorectal carcinoma metastasis of.2 in colorectal carcinoma and regional lymph nodes, high expression of relationship drug resistant PTEN and colorectal cancer may be related to the expression of miR-21 and P-gp.4 in colorectal cancer tissue is closely related with the degree of differentiation was positively correlated with the expression of P-gp, the relationship of miR-21 with multidrug resistance of colorectal cancer may also be associated with high expression of P-gp.

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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