SLC5A8基因转染对大肠癌细胞株SW480增殖和侵袭力影响的实验研究

发布时间：2018-04-23 09:25

本文选题：SLC5A8 + 大肠癌　；参考：《河北大学》2017年硕士论文

【摘要】：背景大肠癌(colorectal cancer,CRC)包括结肠癌和直肠癌,是最常见的消化道恶性肿瘤之一,全世界每年约有120万新发病例,约60万人死于该疾病。中国医学科学院肿瘤医院/国家癌症中心在CA Cancer J Clin(临床肿瘤)杂志发表的2015年中国癌症统计数据显示我国大肠癌发病率、死亡率在全部恶性肿瘤中均位居前5位,其中新发病例37.6万,死亡病例19.1万,已成为严重危害中国人民健康的疾病。随着我国经济的发展,人民生活水平的不断提高及饮食结构的改变,进食过多脂肪、肉类,而少膳食纤维,体育锻炼较少,精神压力过大,导致大肠癌的发病率、病死率逐年上升,同时基因的改变也导致了个体对大肠肿瘤的易感性。肿瘤的发生发展是一个复杂的病理、生理过程,涉及到多种基因改变的复杂的异质性疾病。通常,基因组在复制、转录、翻译等各个水平的改变均会影响其生物学行为的改变。因此对大肠癌相关分子机制的深入研究,将对大肠癌的预防和治疗具有十分重要的意义。我们前期的研究发现,SLC5A8基因在大肠癌组织中表达下降或缺失,提示该基因的沉默与大肠癌的发生存在一定关系。但是对SLC5A8基因在大肠癌中沉默的分子机制、作用底物、信号通路、与其他蛋白的相互作用等还不是很了解,仍需我们进一步的深入研究。溶质载体家族第5家族第8员(solute carrier family 5 member 8,SLC5A8)是2002年Rodriguez等人发现的新的候选抑癌基因,位于人类染色体12q13,属于Na+/葡萄糖共转运蛋白家族成员,是短链脂肪酸(SCFA)如,醋酸、丙酸、丁酸、丙酮酸和乳酸的Na+-偶联转运蛋白。国内外学者发现SLC5A8在人类的食管癌、贲门癌、乳腺癌、甲状腺癌、肺癌、胰腺癌、脑胶质瘤、前列腺癌、急性髓系白血病等多种肿瘤组织中表达降低或缺失,说明其可能参与了肿瘤的发生、转移、侵袭过程。研究发现,SLC5A8基因在肿瘤组织中表达降低或缺失的原因是其GPG岛的甲基化及组蛋白的去乙酰化。丁酸作为SLC5A8基因的转运底物之一,是由细菌发酵膳食纤维产生的。丁酸除了作为β-氧化和柠檬酸循环产生代谢能量的底物,同样也是组蛋白去乙酰化酶抑制剂(HDAC),尤其是HDAC1和HDAC3亚型。这些内源性HDAC抑制剂存在于正常结肠腔中,SLC5A8介导它们进入结肠上皮细胞,并抑制HDACs。由于SLC5A8基因的表达减少或缺失,使丁酸不能进入结肠腔中,这可能成为大肠癌发生的原因之一。目的探讨SLC5A8基因转染对大肠癌细胞株SW480增殖和侵袭力的影响。方法实验分三组,实验组:将含SLC5A8基因的p EX-4质粒转染到大肠癌细胞株SW480;空载对照组:将空载质粒p EX-4转染到大肠癌细胞株SW480;空白对照组:正常大肠癌细胞株SW480。(1)运用q RT-PCR法对细胞中SLC5A8 m RNA表达水平进行分析;(2)采用MTS法检测细胞增值抑制率;(3)Transwell细胞侵袭实验检测SLC5A8基因转染SW480细胞后侵袭力的变化;(4)通过细胞划痕实验观察转染SLC5A8基因对SW480细胞迁移能力的影响。统计方法:所有数据应用SPSS19.0统计软件分析;计量数据采用均数±标准差(sx±)表示;组间比较采用t检验和ANOVA方差分析,检验水准P0.05为差异有统计学意义。结果(1)荧光显微镜显示:转染p EX-4-SLC5A8、p EX-4空载质粒的SW480细胞均显示高强度的绿色荧光。SLC5A8扩增产物溶解曲线呈现单峰,引物特异性良好,扩增产物及扩增循环数(CT值)均可用。q RT-PCR结果显示,转染后细胞中SLC5A8 m RNA表达水平显著增高,表明转染效率较高,实验方法及条件可靠。(2)SLC5A8基因转染后,通过MTS法检测发现,当培养基中加入丁酸时,实验组细胞SW480的增殖抑制率高于对照组,差异具有统计学意义(P=0.013)。当培养基中没有丁酸,实验组细胞SW480的增殖抑制率与对照组之间差异无统计学意义(P=0.957)。实验组在培养基中有丁酸时对细胞SW480的增殖抑制率高于培养基中无丁酸,差异具有统计学意义(P=0.017)。无论培养基中有、无丁酸,对照组对细胞SW480的增值抑制率差异不具有统计学意义(P=0.930)。MTS实验结果说明实验组SW480细胞在有丁酸时增殖明显受到抑制。(3)Transwell体外侵袭实验结果显示,实验组的透膜细胞数低于空载对照组,差异有统计学意义(t=14.072,P=0.000)。实验组的透膜细胞数低于空白对照组,差异有统计学意义(t=17.710,P=0.000)。空载对照组透膜细胞数与空白对照组之间的差异无统计学意义(t=1.540,P=0.162)。Transwell体外侵袭实验以透膜细胞数作为侵袭力的评价指标。说明SLC5A8基因在丁酸的作用下可以抑制大肠癌细胞株SW480的侵袭能力。(4)应用细胞划痕实验观察SLC5A8基因对细胞迁移的影响,划痕后24小时,实验组细胞迁移率低于空载对照组,差异有统计学意义(t=13.617,P=0.000)。实验组细胞迁移率低于空白对照组,差异有统计学意义(t=15.997,P=0.000)。空载对照组细胞迁移率与空白对照组之间的差异无统计学意义(t=1.421,P=0.193)。同样,划痕后48小时,实验组细胞迁移率低于空载对照组,差异有统计学意义(t=29.526,P=0.000)。实验组细胞迁移率低于空白对照组,差异有统计学意义(t=48.936,P=0.000)。空载对照组细胞迁移率与空白对照组之间的差异无统计学意义(t=1.988,P=0.082)。说明SLC5A8基因在丁酸的作用下可以抑制大肠癌细胞株SW480的迁徙能力。结论1.SLC5A8基因在大肠癌细胞株SW480中表达下降或缺失。2.本实验所采用基因转染技术成功将SLC5A8基因转染到大肠癌细胞株SW480中。3.SLC5A8基因抑制大肠癌细胞株SW480的增殖、侵袭及迁徙是通过丁酸介导的,丁酸是其转运底物之一。4.本实验为大肠癌的基因治疗提供新的方法。
[Abstract]:Background colorectal cancer (CRC), including colon and rectal cancer, is one of the most common digestive malignant tumors. There are about 120 million new cases in the world each year. About 600 thousand people die of the disease. The cancer hospital / National Cancer Center of the Chinese Academy of Medical Sciences in the 2015 magazine of the CA Cancer J Clin (clinical cancer), China Cancer system The data show the incidence of colorectal cancer in China, and the mortality rate is the top 5 in all malignant tumors, of which 376 thousand of the new cases and 191 thousand of the deaths have become a serious disease which seriously endangering the health of the Chinese people. With the development of China's economy, the people's living standards are constantly raised and the diet structure changes, eating too much fat and meat, With less dietary fiber, less physical exercise and excessive mental stress, the incidence of large intestine cancer and the mortality rate are increasing year by year. At the same time, the change of genes also leads to the susceptibility to colorectal cancer. The development of the tumor is a complex pathological process, involving complex heterogeneous diseases of various gene changes. It is of great significance for the prevention and treatment of colorectal cancer. Our previous studies have found that the expression of SLC5A8 gene in colorectal cancer is reduced or missing, which suggests that the gene in colorectal cancer can be reduced or missing. There is a certain relationship between the gene silencing and the occurrence of colorectal cancer. However, the molecular mechanism of the silence of the SLC5A8 gene in colorectal cancer, the role of the substrate, the signaling pathway, and the interaction with other proteins are not well understood. We still need further in-depth study. The eighth members of the fifth family of the solute carrier family (solute carrier family 5 member 8, SL C5A8) is a new candidate tumor suppressor gene found by Rodriguez et al. In 2002, located in the human chromosome 12q13, belonging to the Na+/ glucose co transporter family. It is the Na+- coupling transporter of short chain fatty acid (SCFA), acetic acid, propionic acid, butyric acid, pyruvic acid and lactic acid. Domestic and foreign scholars found SLC5A8 in human esophageal, cardiac and breast cancer. The reduction or deletion of the expression of the SLC5A8 gene in the tumor, the reduction or deletion of the gene in the tumor tissue is the methylation of the GPG island and the histone removal. Acetylation. As one of the transport substrates of SLC5A8 gene, butyric acid is produced by bacterial fermentation of dietary fiber. Butyric acid is also the substrate for producing metabolic energy as a beta oxidation and citric acid cycle. It is also a histone deacetylase inhibitor (HDAC), especially the HDAC1 and HDAC3 subtypes. These endogenous HDAC inhibitors exist in the normal colon. In the cavity, SLC5A8 mediates their entry into the colonic epithelial cells and inhibits the decrease or loss of the expression of HDACs. because of the SLC5A8 gene expression, which may not enter the colon cavity. This may be one of the reasons for the occurrence of colorectal cancer. Objective to investigate the effect of SLC5A8 gene transfection on the proliferation and invasiveness of colorectal cancer cell line SW480. Method experiments were divided into three groups. Test group: transfection of P EX-4 plasmid containing SLC5A8 gene into colorectal cancer cell line SW480; empty load control group: transfection of empty plasmid P EX-4 into colorectal cancer cell line SW480; blank control group: normal colorectal cancer cell strain SW480. (1) used Q RT-PCR method to analyze the expression level of SLC5A8 m in cells; (2) the inhibition of cell increment inhibition was detected by the method. Rate; (3) Transwell cell invasion test was used to detect the invasiveness of SLC5A8 gene transfected to SW480 cells; (4) the effect of transfection on the migration ability of SW480 cells by transfection of cells through cell scratch test. Statistical methods: all data were analyzed by SPSS19.0 statistical software; the count data were expressed by mean number + standard deviation (SX +); comparison was taken between groups. T test and ANOVA analysis of variance showed that the level of P0.05 was statistically significant. Results (1) the fluorescence microscope showed that the SW480 cells transfected with P EX-4-SLC5A8 and P EX-4 empty plasmid showed a single peak of high intensity green fluorescence.SLC5A8 product dissolution curve, and the primer specificity was good, the amplification products and the number of amplification cycles (CT values) were all The results of.Q RT-PCR showed that the expression level of SLC5A8 m RNA in the transfected cells was significantly higher, indicating that the transfection efficiency was higher and the experimental methods and conditions were reliable. (2) after the transfection of SLC5A8 gene, the MTS method was used to detect the proliferation and the inhibitory rate of SW480 in the experimental group was higher than that of the control group, and the difference was statistically significant. P=0.013). When there was no butyric acid in the medium, there was no significant difference between the proliferation inhibition rate of SW480 in the experimental group and the control group (P=0.957). The inhibition rate of the proliferation of SW480 in the experimental group was higher than that in the medium (P=0.017). The difference was statistically significant (P=0.017). The difference of the inhibitory rate of SW480 in the group was not statistically significant (P=0.930).MTS experimental results showed that the proliferation of SW480 cells in the experimental group was obviously inhibited when there was butyric acid. (3) the experimental results of Transwell invasion in vitro showed that the number of membrane cells in the experimental group was lower than that of the empty control group, the difference was statistically significant (t=14.072, P=0.000). The number of membrane cells in the group was lower than that in the blank control group. The difference was statistically significant (t=17.710, P=0.000). There was no significant difference between the number of membrane cells in the empty control group and the blank control group (t=1.540, P=0.162).Transwell in the invasion experiment in vitro using the number of transmembrane cells as the evaluation index of the emplacement force. The effect of SLC5A8 gene in butyric acid was explained. The invasion ability of SW480 was inhibited. (4) the effect of SLC5A8 gene on cell migration was observed with the cell scratch test. The cell migration rate of the experimental group was lower than that of the control group at 24 hours after scratch (t=13.617, P=0.000). The cell migration rate in the experimental group was lower than that in the blank control group. The difference was statistically significant T=15.997 (P=0.000). There was no significant difference between the cell migration rate and the blank control group in the control group (t=1.421, P=0.193). Also, the cell migration rate of the experimental group was lower than that of the control group at 48 hours after the scratch (t=29.526, P=0.000). The cell migration rate of the experimental group was lower than that of the blank control group, and the difference was statistically significant Significance (t=48.936, P=0.000). There was no significant difference in the cell migration rate between the blank control group and the blank control group (t=1.988, P=0.082). It indicated that the SLC5A8 gene could inhibit the migration of SW480 in the colorectal cancer cell line under the action of butyric acid. Conclusion the 1.SLC5A8 gene was expressed in the colon cancer cell line SW480 to decrease or lose the.2. based experiment. The gene transfection technique successfully transfected the SLC5A8 gene into the colorectal cancer cell line SW480.3.SLC5A8 gene to inhibit the proliferation of colorectal cancer cell line SW480. Invasion and migration are mediated by butyric acid, and butyric acid is one of its transport substrates. This experiment provides a new method for the gene therapy of colorectal cancer.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

【参考文献】