膜联蛋白A11对人胃癌细胞株SGC7901 5-Fu耐药性影响及机制的研究

发布时间：2018-05-23 23:47

本文选题：膜联蛋白A11 + 胃癌　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:近年来,虽然全球范围内的胃癌发病率处于下降趋势,但我国的胃癌发病率却在逐年上升。胃癌的发病率与死亡率均仅次于肺癌,位于第二位。以手术为主的综合治疗是目前胃癌的主要治疗手段,但我国早期胃癌的诊断率不足10%,大多数患者就诊时往往失去了根治性手术的机会,这就使化疗在胃癌的综合治疗中占据重要的地位。自5-氟尿嘧啶(5-fluorouracil,5-Fu)问世以来,已过去50多年,但其仍是当前胃癌化疗方案的重要组成。由于肿瘤细胞对此类药物原发性和继发性耐药,导致其单药化疗有效率不到20%。因此,阐明5-Fu的耐药机制,缓解甚至逆转其耐药,成为当今亟待解决的问题。膜联蛋白家族(Annexins)是广泛表达于除酵母之外的真核细胞的细胞质及细胞核膜上的钙调节磷脂结合蛋白,其通过调节细胞膜表面蛋白与其他位于细胞内、细胞核膜、细胞外基质的蛋白之间相互作用而发挥功效。膜联蛋白家族共有A、B、C、D、E五个组,A组发现于哺乳动物,包括12个成员(A1-A11和A13)。目前研究表明膜联蛋白A11(Annexin A11,ANXA11)与肿瘤的发生及发展有着密切关系。既往在胃癌中研究发现膜联蛋白A11在胃癌组织中的阳性表达率和表达强度较正常胃组织中明显增高。ANXA11在转移的淋巴结中的阳性表达率和表达强度也较胃癌原发灶中明显增高。可见ANXA11与胃癌的发生、发展及转移密切相关。但目前关于ANXA11在不同胃癌细胞株中的表达,及其与胃癌细胞5-Fu耐药的关系尚未报道。本研究以人胃癌细胞株SGC7901为研究对象,应用RNA干扰技术抑制SGC7901细胞中ANXA11的表达,检测不同组对5-Fu敏感性,发现转染组较阴性对照组、空白对照组敏感性提高,并对其相关机制进行初步探讨。为阐明胃癌5-Fu耐药机制进一步提供理论基础,对于缓解乃至逆转胃癌的5-Fu耐药性及新治疗靶点的发掘具有重要意义。方法:培养人胃癌细胞株SGC7901、人胃癌细胞株BGC-823,应用实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction,q PCR)、蛋白免疫印迹(Western Blot)检测ANXA11 m RNA及蛋白在两种细胞株中的表达,并选取ANXA11高表达的人胃癌细胞株SGC7901。设计并合成针对ANXA11的特异性干扰序列si RNA及其阴性对照序列,应用Lipofectamine 2000转染试剂将ANXA11-si RNA及阴性对照序列瞬时转染入SGC7901细胞,转染后24h、48h、72h应用q PCR检测ANXA11 m RNA的表达,应用Western Blot检测蛋白的表达情况,检测干扰效果。应用CCK-8法检测转染后各组细胞对5-Fu的药物敏感性改变并计算出各组IC50(half maximal inhibitory concentration)与IC20值(考虑IC50浓度的5-Fu对细胞杀伤力较大,遂后续实验采用IC20浓度的5-Fu作用于ANXA11-si RNA-1转染组细胞)。实验分组为:ANXA11-si RNA-1转染组(以下简称为转染组)、ANXA11-si RNA-1转染组联合IC20浓度的5-Fu组(以下简称为联合组)、阴性对照组及空白对照组。应用流式细胞术检测各组凋亡率,应用q PCR、Western Blot检测中胸腺嘧啶脱氧核苷合成酶(Thymidine synthetase,TS)、Bax和Bcl-2 m RNA及蛋白水平表达的改变。结果:1 ANXA11 m RNA在人胃癌细胞株SGC7901、人胃癌细胞株BGC-823细胞中的相对表达量分别为:0.045±0.0012、0.022±0.004。ANXA11蛋白在人胃癌细胞株SGC7901、人胃癌细胞株BGC-823细胞中的相对表达量分别为:1.410±0.180、0.261±0.041。ANXA11 m RNA及蛋白表达量在人胃癌细胞株SGC7901中的表达显著高于人胃癌细胞株BGC823(P=0.033,P=0.000)。2 ANXA11-si RNA转染人胃癌细胞株SGC7901 24、48、72小时后,通过q PCR在m RNA水平检测ANXA11的抑制效果。结果发现,与阴性对照组及空白对照组相比,3条ANXA11-si RNA(ANXA11-si RNA-1、ANXA11-si RNA-2、ANXA11-si RNA-3)及3条ANXA11-si RNA的混合(ANXA11-si RNA123)对ANXA11的表达均有不同程度的抑制作用,其中转染后48小时ANXA11-si RNA-1的抑制效果最好(F=28.631,P=0.000)。采用不同浓度(20n M、40n M、60n M、80n M、100n M)的ANXA11-si RNA-1进行转染,24、48、72小时后,通过Western Blot在蛋白水平检测ANXA11的抑制效果。与阴性对照组及空白对照组相比,其对ANXA11的蛋白表达均有不同程度的抑制作用,其中转染后48小时40n M浓度的抑制效果最好(F=24.887,P=0.000)。3 CCK-8实验中,采用1μg/ml、10μg/ml、20μg/ml、40μg/ml、80μg/ml的5-Fu作用于转染后不同组人胃癌细胞株SGC7901 24、48、72、96h,其中48h时转染组与阴性、空白对照组OD值有明显差异(P0.05)。在48小时组,人胃癌细胞株SGC7901转染ANXA11-si RNA-1后对5-Fu的IC50为:6.827±0.770μg/ml,阴性对照组为:11.487±0.669μg/ml,空白对照组为12.053±0.720μg/ml。相应的IC20分别为0.086±0.011μg/ml、0.191±0.0188μg/ml、0.189±0.0153μg/ml。转染ANXA11-si RNA-1后人胃癌细胞株SGC7901对5-Fu的敏感性显著高于阴性对照组及空白对照组(F=47.500,P=0.000;F=45.523,P=0.000)。4人胃癌细胞株SGC7901转染ANXA11-si RNA-1后细胞凋亡率为(18.747±2.064)%,联合组为(30.153±2.344)%,阴性对照组为(6.657±0.976)%,空白对照组为(6.510±1.509)%。转染组、联合组凋亡率较阴性对照组及空白对照组显著升高(F=118.403,P=0.000),且联合组凋亡率较转染组凋亡率明显升高(P=0.000)。5人胃癌细胞株SGC7901转染ANXA11-si RNA-1后ANXA11蛋白的相对表达量为:0.523±0.091,联合组为0.167±0.060,阴性对照组为:0.887±0.075,空白对照组对照组为0.863±0.125。联合组及转染组蛋白表达显著降低(F=41.623,P=0.000)。联合组与转染组之间差异也具有统计学意义(P=0.001)。6应用q PCR检测转染ANXA11-si RNA-1及转染ANXA11-si RNA联合5-Fu对Bax、Bcl-2及TS m RNA表达的改变,发现与阴性对照组及空白对照组相比,转染组与联合组Bax的m RNA表达上调,而Bcl-2、TS的m RNA表达显著下调(F=35.608,P=0.000;F=30.048,P=0.000;F=22.874,P=0.000)。应用Western Blot检测上述基因蛋白表达的改变,发现与阴性对照组及空白对照组相比,转染组与联合组Bax的蛋白表达上调,而Bcl-2、TS的蛋白表达显著下调(F=24.396,P=0.000;F=33.890,P=0.000;F=29.630,P=0.000)。联合组与转染组之间差异也具有统计学意义(P0.05)。结论:1 ANXA11在人胃癌细胞株SGC7901中表达较高。抑制ANXA11表达可提高人胃癌细胞株SGC7901的凋亡率。2抑制ANXA11表达可提高5-Fu的杀伤力,继而增加人胃癌细胞株SGC7901对5-Fu敏感性,ANXA11可能是参与人胃癌细胞株SGC79015-Fu耐药的新的基因。3抑制ANXA11表达提高人胃癌细胞株SGC7901 5-Fu敏感性可能与抑制TS的表达有关,同时也与下调抑凋亡基因Bcl-2、上调促凋亡基因Bax的表达从而促使细胞凋亡有关。
[Abstract]:Objective: in recent years, although the incidence of gastric cancer in the world is declining, the incidence of gastric cancer in China is increasing year by year. The incidence and mortality of gastric cancer are second only to lung cancer, which is located in the second place. Comprehensive treatment based on surgery is the main treatment for gastric cancer at present, but the diagnosis rate of early gastric cancer in China is less than 10%. Most patients often lose the opportunity for radical surgery, which makes chemotherapy play an important role in the comprehensive treatment of gastric cancer. Since the advent of 5- fluorouracil (5-fluorouracil, 5-Fu), it has been over 50 years ago, but it is still an important component of the current chemotherapy regimen for gastric cancer. As a result of sexual resistance, the effective rate of single drug chemotherapy is less than 20%., so it is an urgent problem to clarify the mechanism of 5-Fu's resistance to drug resistance and even reverse its resistance. The membrane associated protein family (Annexins) is the cytoplasm and calcium regulating phospholipid binding protein of the eukaryotic cells, which are widely expressed in addition to the yeast, and it is regulated by the regulation of the protein. Cell membrane surface proteins interact with other proteins in cells, cell nuclear membranes, and extracellular matrix proteins. The annexin family consists of five groups of A, B, C, D, E. The A group is found in mammals, including 12 members (A1-A11 and A13). The present study shows the occurrence and development of the membrane associated protein A11 (Annexin A11, ANXA11) and tumor. The positive expression rate and expression intensity of annexin A11 in gastric cancer tissues were significantly higher than that in normal gastric tissue. The positive expression rate and expression intensity of.ANXA11 in the metastatic lymph nodes were also significantly higher than those in the primary gastric cancer. The occurrence, development and metastasis of ANXA11 and gastric cancer were also found. But at present, the expression of ANXA11 in different gastric cancer cell lines and the relationship with the drug resistance of gastric cancer cell 5-Fu have not been reported. In this study, the human gastric cancer cell line SGC7901 was used as the research object. The expression of ANXA11 in SGC7901 cells was suppressed by RNA interference technique, and the sensitivity of different groups to 5-Fu was detected, and the transfection group was found to be more than the negative control group. The sensitivity of the blank control group is improved and its related mechanism is preliminarily discussed. It provides a theoretical basis for clarifying the mechanism of 5-Fu resistance to gastric cancer, and is of great significance for alleviating and reversing the 5-Fu resistance of gastric cancer and the discovery of new therapeutic targets. Methods: the cultivation of human gastric cancer cell line SGC7901, human gastric cancer cell line BGC-823, is used in real time. Fluorescence quantitative polymerase chain reaction (quantitative real time polymerase chain reaction, Q PCR) and protein immunoblotting (Western Blot) were used to detect the expression of ANXA11 m and protein in two cell lines. The Lipofectamine 2000 transfection reagent was used to transfect ANXA11-si RNA and negative control sequence into SGC7901 cells instantaneously, and 24h, 48h and 72h were used to detect the expression of ANXA11 m RNA, and the expression of protein was detected by Q PCR, and the effect of interference was detected by using Q PCR. IC50 (half maximal inhibitory concentration) and IC20 values were calculated in each group. (5-Fu for IC50 concentration was more lethal than 5-Fu, then IC20 concentration 5-Fu was used in ANXA11-si RNA-1). Group 5-Fu group with IC20 concentration (hereinafter referred to as joint group), negative control group and blank control group. Flow cytometry was used to detect the apoptosis rate of each group. Q PCR, Western Blot detection of thymidine deoxynucleoside synthetase (Thymidine synthetase, TS), Bax and Bcl-2 m, and protein level expression changes. Results: 1 The relative expression of human gastric cancer cell line SGC7901 and human gastric cancer cell line BGC-823 cells are: 0.045 + 0.0012,0.022 + 0.004.ANXA11 protein in human gastric cancer cell line SGC7901, and the relative expression amount in human gastric cancer cell line BGC-823 cells is: 1.410 + 0.180,0.261 + 0.041.ANXA11 m RNA and protein expression in human gastric cancer cell strain SGC7 The expression in 901 was significantly higher than that of human gastric cancer cell line BGC823 (P=0.033, P=0.000).2 ANXA11-si RNA transfected to human gastric cancer cell line SGC7901 24,48,72 hours after SGC7901 24,48,72 hours, and the inhibitory effect was detected by Q PCR in M RNA. -si RNA-3) and the mixture of 3 ANXA11-si RNA (ANXA11-si RNA123) have different degrees of inhibition on the expression of ANXA11, and the inhibitory effect of ANXA11-si RNA-1 after 48 hours after transfection is the best (F=28.631, P=0.000). Blot was used to detect the inhibitory effect of ANXA11 at the protein level. Compared with the negative control group and the blank control group, the inhibition effect on the protein expression of ANXA11 was different. The inhibitory effect of 40n M at 48 hours after transfection was the best (F=24.887, P=0.000).3 CCK-8 experiment, using 1 Mu g/ml, 10 mu g/ml, 20 mu g/ml, 40 micron, 80 micron. SGC7901 24,48,72,96h in different groups of human gastric cancer cell line after transfection, in which 48h transfection group and negative control group were significantly different (P0.05). In the 48 hour group, the IC50 of human gastric cancer cell line SGC7901 transfected to 5-Fu was 6.827 + 0.770 mu g/ml, negative control group was 11.487 + 0.669 mu g/ml, and the blank control group was 1. The corresponding IC20 of 2.053 + 0.720 g/ml. was 0.086 + 0.011 mu g/ml, 0.191 + 0.0188 g/ml and 0.189 + 0.0153 mu g/ml. transfected to ANXA11-si RNA-1. The sensitivity of SGC7901 to 5-Fu was significantly higher than that of negative control group and blank control group (F=47.500, P=0.000; F=45.523, 0) The apoptosis rate was (18.747 + 2.064)%, the combined group was (30.153 + 2.344)%, the negative control group was (6.657 + 0.976)%, and the blank control group was (6.510 + 1.509)%. The apoptosis rate of the combined group was significantly higher than that of the negative control group and the blank control group (F=118.403, P=0.000), and the apoptosis rate of the combined group was significantly higher than that of the transfected group (P=0.000).5 human stomach. The relative expression of ANXA11 protein in the cancer cell line SGC7901 transfected with ANXA11-si RNA-1 was 0.523 + 0.091, the combined group was 0.167 + 0.060, and the negative control group was 0.887 + 0.075. The control group of the blank control group was 0.863 + 0.125. combined group and the transfection group was significantly reduced (F=41.623, P=0.000). The difference between the combined group and the transfected group was also statistically significant. P=0.001.6 applied Q PCR to detect the transfection of ANXA11-si RNA-1 and the transfection of ANXA11-si RNA with 5-Fu to Bax, Bcl-2 and TS. .874, P=0.000). Using Western Blot to detect the changes in the expression of the above gene protein, it was found that the protein expression of Bax in the transfected group and the combined group was up regulated compared to the negative control group and the blank control group, while the protein expression of the Bcl-2, TS was significantly down (F=24.396, P=0.000; F=33.890, P= 0, F=29.630,). Statistical significance (P0.05). Conclusion: 1 ANXA11 is highly expressed in human gastric cancer cell line SGC7901. Inhibition of ANXA11 expression can increase the apoptosis rate of SGC7901 in human gastric cancer cell line,.2 inhibition of ANXA11 expression can improve the viability of 5-Fu, and then increase the sensitivity of SGC7901 to 5-Fu in human gastric cancer cell line, ANXA11 may be involved in human gastric cancer cell strain SGC79015-Fu. The inhibition of ANXA11 expression by drug resistant new gene.3 enhances the sensitivity of SGC7901 5-Fu in human gastric cancer cell line, which may be related to inhibiting the expression of TS. It is also associated with down regulation of apoptosis gene Bcl-2, up regulation of the expression of apoptotic gene Bax and inducing apoptosis.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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