FAM172A在大肠癌转移的作用机制研究
发布时间：2019-04-04 08:25
【摘要】：大肠癌(Colorectal Cancer,CRC)作为常见的人类恶性肿瘤,全世界每年大约120万新发病例,同时至少60万患者死亡。转移是影响大肠癌患者预后的重要原因,大多数伴有转移的大肠癌患者不适合传统治疗方式。大肠癌转移主要与个体基因突变有关,大肠癌中基因突变不超过50%,故可认定大肠癌的转移尚有其它新的抑癌基因或癌基因在起作用。FAM172A是本课题组前期研究发现的一个新基因,我们最近的研究结果证实:1)FAM172A在CRC相关组织中表达下调,且显著抑制人大肠癌LoVo细胞增殖和促进凋亡;2)转录因子STAT1在转录水平调控FAM172A表达,增强后者功能。综上所述,我们推测:FAM172A基因是一个典型的抑癌基因,进一步研究其转录调控相关机制具有重要意义。基因表达调控除发生在转录水平,转录后调控也是关键环节,miRNAs是近年来发现参与转录后水平调控的重要因子。miRNAs是一种由内源基因编码的、长度大概为22个核苷酸的非编码单链小RNA分子。研究证实miRNA异常表达与大肠癌转移相关,如miR-21在大肠癌细胞中表达明显增高,并通过抑制PDCD4表达促进肿瘤细胞的侵袭。我们前期工作中利用生物信息学预测发现miR-27a可能靶向结合FAM172A,故我们推测FAM172A功能可能在转录后水平受到miR-27a的调控。本课题拟在前期工作基础上,在转录后水平进一步阐明FAM172A抑制大肠癌侵袭转移的调控机制,为大肠癌的基因治疗提供科学依据和实验基础。目的:1、明确FAM172A对大肠癌细胞迁移和侵袭能力的影响;2、生物信息学筛选和验证调控FAM172A的重要miRNA;3、明确大肠癌组织及细胞株中miR-27a表达水平及其相关临床意义;4、探讨miR-27a靶向FAM172A对大肠癌细胞迁移和侵袭的影响及其机制;方法:1、构建FAM172A重组质粒,通过细胞划痕及Transwell侵袭实验分别观察FAM172A对LoVo细胞迁移和侵袭的影响;2、生物信息学筛选靶向FAM172A的重要miRNA,Luciferase报告基因技术验证两者结合关系;3、RT-qPCR检测大肠癌细胞株及配对组织标本中miR-27a表达水平。4、构建稳定过表达miR-27a的LoVo细胞株;细胞划痕及Transwell明确miR-27a调控FAM172A对大肠细胞迁移和侵袭影响;western blot检测miR-27a调控FAM172A对侵袭转移相关重要信号通路分子的表达影响;5、数据统计采用SPSS 22.0软件分析,以均数±标准误表示。计量资料组间比较采用两独立样本t检验,多组间比较采用方差分析。P0.05为有统计学差异。所有实验均独立进行三次,取平均值进行统计分析。结果:1、过表达FAM172A减弱了LoVo细胞运动速度,引起LoVo细胞侵袭数量的减少,干扰FAM172A表达则加快了LoVo细胞运动速度,增加了其侵袭数量;2、FAM172A 3'UTR存在miR-27a结合位点且双荧光报告基因证实转染miR-27a能引起荧光素酶活性下降。3、在大肠癌相关组织中miR-27a含量明显高于正常癌旁组织,临床病例资料分析显示其表达水平与TNM分期、淋巴结转移和远处转移与否呈正相关;miR-27a在各人大肠癌细胞株的表达均高于正常人大肠上皮细胞。4、转染miR-27a导致LoVo细胞迁移加快和侵袭数量明显增加,干扰miR-27a表达则引起LoVo细胞迁移和侵袭数量减少;稳定过表达miR-27a的LoVo细胞在导入FAM172A过表达质粒后,LoVo细胞迁移减慢及侵袭细胞数量减少,且转移相关重要信号通路分子MMP-2、MMP-9和NF-κ B的蛋白表达降低。结论:1、FAM172A抑制大肠癌细胞的迁移和侵袭;2、miR-27a靶向结合FAM172A;3、miR-27a的表达变化对诊断和评估CRC患者病情具有一定的价值;4、FAM172A抑制大肠癌细胞株LoVo细胞迁移和侵袭活性的功能受到miR-27a负性调控,且可能通过下游NF-κ B信号通路而实现。
[Abstract]:Colorectal cancer (CRC), as a common human malignant tumor, is about 1.2 million new cases every year, and at least 600,000 patients die. Transfer is an important cause of the prognosis of patients with colorectal cancer. Most of the patients with large bowel cancer with metastasis are not suitable for the traditional treatment. The metastasis of colorectal cancer is mainly related to the individual gene mutation, and the gene mutation in the large intestine is not more than 50%. FAM172A is a new gene found in the previous study of the research group. Our recent results confirm that 1) the expression of FAM172A in CRC-related tissues is down-regulated, and the proliferation and apoptosis of human colorectal cancer LoVo cells are significantly inhibited; and 2) the transcription factor STAT1 regulates the expression of FAM172A at the transcription level, and enhances the latter function. In conclusion, we have speculated that the FAM172A gene is a typical tumor suppressor gene, and it is of great significance to further study the mechanism of transcription regulation. The regulation of gene expression is not only in the level of transcription, but also in the post-transcriptional regulation, and miRNAs are important factors that have been found to be involved in the post-transcriptional level regulation in recent years. MiRNAs is a non-coding single-stranded small-stranded RNA molecule encoded by an endogenous gene with a length of about 22 nucleotides. The study confirmed that the abnormal expression of miRNA was associated with the metastasis of colorectal cancer, such as the expression of miR-21 in colorectal cancer cells, and the inhibition of the invasion of tumor cells by inhibiting the expression of PDCD4. In our earlier work, using bioinformatics to predict that miR-27a may target the binding of FAAM172A, we assume that the FAM172A function may be regulated by miR-27a at the post-transcriptional level. In this paper, on the basis of the preliminary work, the regulation mechanism of FAM172A to inhibit the invasion and metastasis of large intestine cancer is further elucidated on the basis of the post-transcriptional level, and the scientific basis and the experimental basis for gene therapy of the large intestine cancer are provided. Objective:1. To clarify the effect of FAM172A on the migration and invasion of colorectal cancer cells;2. Bioinformatics to screen and verify the important miRNAs of FAM172A;3. To determine the expression level of miR-27a in colorectal cancer tissues and cell lines and its related clinical significance; The effects of miR-27a on the migration and invasion of colorectal cancer cells and its mechanism were discussed. Methods:1. The recombinant plasmid of FAM172A was constructed, and the effects of FAM172A on the migration and invasion of LoVo cells were observed by cell scratch and Transwell.2. The important miRNAs targeting the FAM172A were screened by bioinformatics. 3. The expression level of miR-27a in colorectal cancer cell lines and paired tissue samples was detected by RT-qPCR, and the LoVo cell strain with stable expression of miR-27a was constructed, and the cell scratch and Transwell's specific miR-27a control the migration and invasion of large intestine cells. Western blot was used to detect the effect of miR-27a on the expression of the important signaling pathway related to the invasion and metastasis. Two independent samples of t-test were used for the comparison between the data groups, and the variance of variance was used for many groups. There was a statistical difference between P0.05. All the experiments were carried out independently for three times, and the average value was taken for statistical analysis. Results:1. Overexpression of FAM172A decreased the movement speed of LoVo cells, resulting in a decrease in the invasion number of LoVo cells. in that presence of the miR-27a binding site in the FAM172A 3 'UTR and the double-fluorescent reporter gene confirmed that the transfection of the miR-27a can cause the luciferase activity to decrease.3, the content of the miR-27a in the colorectal cancer-related tissue is obviously higher than that of the normal adjacent tissue, and the clinical case data analysis shows the expression level and the TNM stage, There was a positive correlation between lymph node metastasis and distant metastasis. The expression of miR-27a in human colorectal cancer cell line was higher than that of normal human colorectal epithelial cell. LoVo cells stably overexpressing miR-27a, after the introduction of the FAM172A overexpression plasmid, decreased the migration of LoVo cells and decreased the number of invasive cells, and the protein expression of the related important signaling pathway molecules, MMP-2, MMP-9 and NF-EMAB, was reduced. Conclusion:1, FAM172A inhibits the migration and invasion of colorectal cancer cells;2, the expression of miR-27a in combination with FAM172A;3, the expression of miR-27a has a certain value in the diagnosis and evaluation of CRC patients; and 4, the function of the FAM172A to inhibit the migration and invasion activity of the LoVo cells of the colorectal cancer cell line is negatively regulated by the miR-27a, And may be implemented by a downstream NF-B signal path.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2453642
[Abstract]:Colorectal cancer (CRC), as a common human malignant tumor, is about 1.2 million new cases every year, and at least 600,000 patients die. Transfer is an important cause of the prognosis of patients with colorectal cancer. Most of the patients with large bowel cancer with metastasis are not suitable for the traditional treatment. The metastasis of colorectal cancer is mainly related to the individual gene mutation, and the gene mutation in the large intestine is not more than 50%. FAM172A is a new gene found in the previous study of the research group. Our recent results confirm that 1) the expression of FAM172A in CRC-related tissues is down-regulated, and the proliferation and apoptosis of human colorectal cancer LoVo cells are significantly inhibited; and 2) the transcription factor STAT1 regulates the expression of FAM172A at the transcription level, and enhances the latter function. In conclusion, we have speculated that the FAM172A gene is a typical tumor suppressor gene, and it is of great significance to further study the mechanism of transcription regulation. The regulation of gene expression is not only in the level of transcription, but also in the post-transcriptional regulation, and miRNAs are important factors that have been found to be involved in the post-transcriptional level regulation in recent years. MiRNAs is a non-coding single-stranded small-stranded RNA molecule encoded by an endogenous gene with a length of about 22 nucleotides. The study confirmed that the abnormal expression of miRNA was associated with the metastasis of colorectal cancer, such as the expression of miR-21 in colorectal cancer cells, and the inhibition of the invasion of tumor cells by inhibiting the expression of PDCD4. In our earlier work, using bioinformatics to predict that miR-27a may target the binding of FAAM172A, we assume that the FAM172A function may be regulated by miR-27a at the post-transcriptional level. In this paper, on the basis of the preliminary work, the regulation mechanism of FAM172A to inhibit the invasion and metastasis of large intestine cancer is further elucidated on the basis of the post-transcriptional level, and the scientific basis and the experimental basis for gene therapy of the large intestine cancer are provided. Objective:1. To clarify the effect of FAM172A on the migration and invasion of colorectal cancer cells;2. Bioinformatics to screen and verify the important miRNAs of FAM172A;3. To determine the expression level of miR-27a in colorectal cancer tissues and cell lines and its related clinical significance; The effects of miR-27a on the migration and invasion of colorectal cancer cells and its mechanism were discussed. Methods:1. The recombinant plasmid of FAM172A was constructed, and the effects of FAM172A on the migration and invasion of LoVo cells were observed by cell scratch and Transwell.2. The important miRNAs targeting the FAM172A were screened by bioinformatics. 3. The expression level of miR-27a in colorectal cancer cell lines and paired tissue samples was detected by RT-qPCR, and the LoVo cell strain with stable expression of miR-27a was constructed, and the cell scratch and Transwell's specific miR-27a control the migration and invasion of large intestine cells. Western blot was used to detect the effect of miR-27a on the expression of the important signaling pathway related to the invasion and metastasis. Two independent samples of t-test were used for the comparison between the data groups, and the variance of variance was used for many groups. There was a statistical difference between P0.05. All the experiments were carried out independently for three times, and the average value was taken for statistical analysis. Results:1. Overexpression of FAM172A decreased the movement speed of LoVo cells, resulting in a decrease in the invasion number of LoVo cells. in that presence of the miR-27a binding site in the FAM172A 3 'UTR and the double-fluorescent reporter gene confirmed that the transfection of the miR-27a can cause the luciferase activity to decrease.3, the content of the miR-27a in the colorectal cancer-related tissue is obviously higher than that of the normal adjacent tissue, and the clinical case data analysis shows the expression level and the TNM stage, There was a positive correlation between lymph node metastasis and distant metastasis. The expression of miR-27a in human colorectal cancer cell line was higher than that of normal human colorectal epithelial cell. LoVo cells stably overexpressing miR-27a, after the introduction of the FAM172A overexpression plasmid, decreased the migration of LoVo cells and decreased the number of invasive cells, and the protein expression of the related important signaling pathway molecules, MMP-2, MMP-9 and NF-EMAB, was reduced. Conclusion:1, FAM172A inhibits the migration and invasion of colorectal cancer cells;2, the expression of miR-27a in combination with FAM172A;3, the expression of miR-27a has a certain value in the diagnosis and evaluation of CRC patients; and 4, the function of the FAM172A to inhibit the migration and invasion activity of the LoVo cells of the colorectal cancer cell line is negatively regulated by the miR-27a, And may be implemented by a downstream NF-B signal path.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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