叶酸缺乏对不同状态结肠细胞FOLR1表达和染色体不稳定性的影响
发布时间：2019-04-02 20:36
【摘要】：随着人们生活习性和膳食结构的改变,结直肠癌发生率已在全球范围位居第三。叶酸对结直肠癌在内的多种肿瘤有一定防范作用,且能抑制结直肠癌个体术后黏膜细胞增生。FOLR1基因编码叶酸受体蛋白α(Folate Receptorα,FRα),该受体蛋白与叶酸的亲和力及其自身的表达量对叶酸摄取起决定性作用。FOLR1蛋白能与叶酸特异性地结合并将其转运至细胞内,进入细胞的叶酸通过一碳单位代谢参与DNA的合成、损伤修复和甲基化。当叶酸缺乏和代谢异常时,通常引起DNA结构和基因表达异常等基因组稳定性下降事件,从而提高神经系统发育异常、多种退行性疾病及肿瘤发生风险。为探讨不同氧化态叶酸缺乏对不同状态结肠细胞FOLR1表达和细胞基因组稳定性的影响与差异,本研究从叶酸对人正常/结肠癌细胞FOLR1转录与翻译的影响及差异、叶酸对二受试细胞染色体不稳定性(Chromosome Instability,CIN)的影响二个层面进行分析,以RPMI-1640培养基为对照(叶酸2266nmol/L),选取还原态的5-methyltetrahydrofolate(5-MeTHF)和氧化态的Folic Acid(FA)缺乏浓度(22.66 nmol/L)、维持基因组结构稳定的充足浓度(226.6 nmol/L)干预培养人结肠腺癌细胞COLO205、人正常结肠上皮细胞CCD-841-CoN(CCD)28天,取培养第14天和第28天细胞提取总RNA和总蛋白,用RT-qPCR及2-ΔΔCT法分析受试细胞二个时段FOLR1基因转录表达情况;用Western Blotting检测二个时段干预后FOLR1蛋白表达情况;在干预培养的第9、18、27天,用胞质分裂阻断微核试验(Cytokinesis-Block Micronucleus assay,CBMN)检测二受试细胞株的CIN水平。研究结果显示:(1)在自然状态下,CCD细胞的FOLR1蛋白相对表达显著低于COLO205细胞(P0.001)。(2)二种状态的叶酸干预14和28天,COLO205细胞FOLR1转录和蛋白表达变化趋势一致;叶酸浓度降低,FOLR1转录(P0.001~0.01)和蛋白(P0.01~0.05)表达显著升高;且在叶酸缺乏(22.66 nmol/L)时,CIN率显著高于对照(2266nmol/L,P0.01)。在CCD细胞中,叶酸缺乏(22.66 nmol/L)时,14天干预时FOLR1转录(P0.001)和蛋白(P0.01~0.05)较对照组显著升高;而干预28天时,叶酸缺乏引起CCD细胞的FOLR1转录(P0.001)和蛋白(P0.01~0.05)表达显著低于对照,同时,叶酸缺乏引起CCD细胞的CIN率显著高于对照组(P0.01~0.05)。(3)不同氧化态叶酸干预对COLO205和CCD细胞FOLR1转录影响程度存在差异,在叶酸缺乏浓度(22.66 nmol/L)下,5-MeTHF对二种受试细胞FOLR1转录表达上调作用显著强于FA组(P0.01)。结果提示,叶酸缺乏在正常细胞和结肠腺癌细胞中均引起FOLR1 mRNA及蛋白表达上调,CIN升高;但在正常细胞中,叶酸持续缺乏导致后期出现FOLR1mRNA和蛋白表达下降,推测叶酸缺乏诱导染色体损伤并促使细胞生长停滞或凋亡,基因表达下滑;而肿瘤细胞自身具有永生化及FOLR1高表达的特性,其响应叶酸缺乏及其细胞生长阻滞效应的能力低于正常细胞。研究还提示,叶酸缺乏导致FOLR1表达上升,5-MeTHF对基因转录上调作用强于FA。
[Abstract]:With the change of people's living habits and dietary patterns, the incidence of colorectal cancer has become the third in the world. Folic acid plays an important role in the prevention of colorectal cancer and inhibits the proliferation of mucosal cells in colorectal cancer patients after operation. The folate receptor protein 伪 (Folate Receptor 伪 (FR 伪) is encoded by the FOLR1 gene, which encodes folate receptor protein 伪-folate receptor 伪 (FR 伪). The affinity of the receptor protein with folic acid and its own expression level play a decisive role in folic acid uptake. FOLR1 protein can specifically bind to folic acid and transport it to cells. Folic acid entering the cells is involved in the synthesis of DNA through the metabolism of one carbon unit. Damage repair and methylation. When folic acid is deficient and abnormal metabolism, it usually leads to genomic stability decline events such as abnormal structure and gene expression of DNA, so as to improve the risk of neurodevelopmental abnormalities, various degenerative diseases and tumorigenesis. In order to investigate the effects and differences of different oxidized folic acid deficiency on the expression of FOLR1 and the stability of cell genome in human normal / colon cancer cells, the effects of folic acid on the transcription and translation of FOLR1 in human normal / colon cancer cells were studied in this study. The effects of folic acid on chromosome instability (Chromosome Instability,CIN) of two tested cells were analyzed on two levels. The RPMI-1640 medium was used as control (folic acid 2266nmol/L). The reduced 5-methyltetrahydrofolate (5-MeTHF) and oxidized Folic Acid (FA) deficiency concentration (22.66 nmol/L) were selected to maintain the stability of genomic structure (226.6 nmol/L) in human colon adenocarcinoma cell line COLO205,. Human normal colon epithelial cells were cultured for 28 days. Total RNA and total protein were extracted from human normal colon epithelial cells on day 14 and day 28. The expression of FOLR1 gene was analyzed by RT-qPCR and 2-螖 CT methods. The expression of FOLR1 protein was detected by Western Blotting, and the CIN level of the two tested cell lines was detected by cytoplasmic mitosis blocking micronucleus test (Cytokinesis-Block Micronucleus assay,CBMN) on the 9th, 18th and 27th day after intervention. The results showed that: (1) under natural condition, the relative expression of FOLR1 protein in CCD cells was significantly lower than that in COLO205 cells (P0.001). (2), and the changes of FOLR1 transcription and protein expression in COLO205 cells were consistent at 14 and 28 days after treatment with folic acid. When folic acid concentration decreased, the expression of FOLR1 transcription (P0.001) and protein (P0.01) increased significantly, and the rate of CIN in folic acid deficiency (22.66 nmol/L) was significantly higher than that in control (2266 nmol / L, P0.01). In CCD cells, FOLR1 transcription (P0.001) and protein (P0.01) at 14 days after folic acid deficiency (22.66 nmol/L) were significantly higher than those of the control group. At 28 days after intervention, folic acid deficiency induced FOLR1 transcription (P0.001) and protein (P0.01) expression in CCD cells significantly lower than that in the control group. The CIN rate of CCD cells induced by folic acid deficiency was significantly higher than that of control group (P0.01 / 0.05). (3). There was a significant difference in the effect of different oxidized folic acid intervention on FOLR1 transcription in COLO205 and CCD cells. At folic acid deficiency concentration (22.66 nmol/L), The upregulated effect of 5-MeTHF on the expression of FOLR1 in the two tested cells was significantly stronger than that in the FA group (P0.01). The results suggested that folic acid deficiency induced the up-regulation of FOLR1 mRNA and protein expression and the increase of CIN in both normal cells and colon adenocarcinoma cells. However, in normal cells, continuous deficiency of folic acid resulted in decreased expression of FOLR1mRNA and protein at the later stage, suggesting that folic acid deficiency induced chromosome damage and cell growth arrest or apoptosis, and decreased gene expression. Tumor cells had immortalization and high expression of FOLR1, and their ability to respond to folic acid deficiency and cell growth arrest was lower than that of normal cells. It is also suggested that folic acid deficiency leads to the increase of FOLR1 expression, and the up-regulation of gene transcription by 5-MeTHF is stronger than that of FA..
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
本文编号：2452901
[Abstract]:With the change of people's living habits and dietary patterns, the incidence of colorectal cancer has become the third in the world. Folic acid plays an important role in the prevention of colorectal cancer and inhibits the proliferation of mucosal cells in colorectal cancer patients after operation. The folate receptor protein 伪 (Folate Receptor 伪 (FR 伪) is encoded by the FOLR1 gene, which encodes folate receptor protein 伪-folate receptor 伪 (FR 伪). The affinity of the receptor protein with folic acid and its own expression level play a decisive role in folic acid uptake. FOLR1 protein can specifically bind to folic acid and transport it to cells. Folic acid entering the cells is involved in the synthesis of DNA through the metabolism of one carbon unit. Damage repair and methylation. When folic acid is deficient and abnormal metabolism, it usually leads to genomic stability decline events such as abnormal structure and gene expression of DNA, so as to improve the risk of neurodevelopmental abnormalities, various degenerative diseases and tumorigenesis. In order to investigate the effects and differences of different oxidized folic acid deficiency on the expression of FOLR1 and the stability of cell genome in human normal / colon cancer cells, the effects of folic acid on the transcription and translation of FOLR1 in human normal / colon cancer cells were studied in this study. The effects of folic acid on chromosome instability (Chromosome Instability,CIN) of two tested cells were analyzed on two levels. The RPMI-1640 medium was used as control (folic acid 2266nmol/L). The reduced 5-methyltetrahydrofolate (5-MeTHF) and oxidized Folic Acid (FA) deficiency concentration (22.66 nmol/L) were selected to maintain the stability of genomic structure (226.6 nmol/L) in human colon adenocarcinoma cell line COLO205,. Human normal colon epithelial cells were cultured for 28 days. Total RNA and total protein were extracted from human normal colon epithelial cells on day 14 and day 28. The expression of FOLR1 gene was analyzed by RT-qPCR and 2-螖 CT methods. The expression of FOLR1 protein was detected by Western Blotting, and the CIN level of the two tested cell lines was detected by cytoplasmic mitosis blocking micronucleus test (Cytokinesis-Block Micronucleus assay,CBMN) on the 9th, 18th and 27th day after intervention. The results showed that: (1) under natural condition, the relative expression of FOLR1 protein in CCD cells was significantly lower than that in COLO205 cells (P0.001). (2), and the changes of FOLR1 transcription and protein expression in COLO205 cells were consistent at 14 and 28 days after treatment with folic acid. When folic acid concentration decreased, the expression of FOLR1 transcription (P0.001) and protein (P0.01) increased significantly, and the rate of CIN in folic acid deficiency (22.66 nmol/L) was significantly higher than that in control (2266 nmol / L, P0.01). In CCD cells, FOLR1 transcription (P0.001) and protein (P0.01) at 14 days after folic acid deficiency (22.66 nmol/L) were significantly higher than those of the control group. At 28 days after intervention, folic acid deficiency induced FOLR1 transcription (P0.001) and protein (P0.01) expression in CCD cells significantly lower than that in the control group. The CIN rate of CCD cells induced by folic acid deficiency was significantly higher than that of control group (P0.01 / 0.05). (3). There was a significant difference in the effect of different oxidized folic acid intervention on FOLR1 transcription in COLO205 and CCD cells. At folic acid deficiency concentration (22.66 nmol/L), The upregulated effect of 5-MeTHF on the expression of FOLR1 in the two tested cells was significantly stronger than that in the FA group (P0.01). The results suggested that folic acid deficiency induced the up-regulation of FOLR1 mRNA and protein expression and the increase of CIN in both normal cells and colon adenocarcinoma cells. However, in normal cells, continuous deficiency of folic acid resulted in decreased expression of FOLR1mRNA and protein at the later stage, suggesting that folic acid deficiency induced chromosome damage and cell growth arrest or apoptosis, and decreased gene expression. Tumor cells had immortalization and high expression of FOLR1, and their ability to respond to folic acid deficiency and cell growth arrest was lower than that of normal cells. It is also suggested that folic acid deficiency leads to the increase of FOLR1 expression, and the up-regulation of gene transcription by 5-MeTHF is stronger than that of FA..
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
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