miR-22-3p和miR-149-5p响应叶酸缺乏并调控人肝细胞亚甲基四氢叶酸还原酶的表达及其对细胞增殖抑制的机制
发布时间：2019-06-19 13:14
【摘要】：叶酸(Folic acid,FA)是一个参与DNA合成和甲基化的核心微营养素,其代谢影响基因组稳定性。当胞内叶酸代谢改变时,MicroRNA可能产生响应并影响下游靶基因的表达,其失调还可作用于一碳代谢(OCM,or folate metabolism)可导致DNA合成、甲基化、氨基酸代谢核细胞增殖的失调,这些事件往往与多种疾病或癌症相关联亚甲基四氢叶酸还原酶(Methylenetetrahydrofolate reductase,MTHFR)是作为一个关键的“开关”基因通过一碳代谢作用于DNA的甲基化和DNA合成,其异常与基因组不稳定型和各种疾病或肿瘤的发生发展都有着密切的联系。本研究旨在解析miR-22-3p/miR-149-5p是否响应叶酸缺乏、随后靶向进而影响MTHFR的表达,进而影响其基因组稳定性;并初步探究其可能存在的对癌细胞生长的抑制作用。本研究以叶酸缺乏的修饰RPMI 1640培养基干预人正常肝细胞(HL-7702)与人肝癌细胞(QGY-7703)21天;双荧光素酶报告监测系统分析受试miRNA与MTHFR之间的相互作用;Poly(A)加尾法RT-qPCR检测miR-22-3p/miR-149-5p响应叶酸缺乏时的表达改变;RT-qPCR和Western blotting分别在转录和翻译水平检测受试细胞MTHFR的表达量;CBMN-Cyt检测基因组损伤情况;CCK-8法检测肝癌细胞转染miRNA后的增殖能力。结果显示:miR-22-3p/miR-149-5p直接靶向MTHFR基因3’UTR;叶酸缺乏导致miR-22-3p/miR-149-5p在QGY-7703/HL-7702细胞中表达上调,然而MTHFR的转录水平在QGY-7703细胞中下调,在HL-7702细胞中上调;Western blotting结果显示叶酸缺乏导致QGY-7703细胞MTHFR蛋白水平下调,但是在HL-7702细胞中保持不变;干预结果还显示叶酸缺乏对肝正常细胞(抑癌基因mRNA水平下降)和肝癌细胞(抑癌基因mRNA水平上升)的产生不同的抑制增殖/抑癌效应;基因组损伤情况显示HL-7702细胞对叶酸缺乏的抵抗能力强于QGY-7703细胞。miR-22-3p/miR-149-5p转染QGY-7703细胞后,MTHFR mRNA水平下降,细胞增殖能力同时下降。我们的结果表明叶酸缺乏条件下miR-22-3p/miR-149-5p在人正常肝细胞和人肝癌细胞中对MTHFR的表达发挥不同的转录后调控作用。此结果暗示人肝癌细胞在叶酸缺乏条件下,miR-22-3p/miR-149-5p可能对肝癌细胞的生长起到抑制作用并促进其基因组不稳定性。
[Abstract]:Folic acid (Folic acid,FA) is a core micronutrient involved in DNA synthesis and methylation, and its metabolism affects genome stability. When intracellular folic acid metabolism changes, MicroRNA may respond and affect the expression of downstream target genes, and its imbalance can also act on one carbon metabolism (OCM,or folate metabolism) can lead to DNA synthesis, methylated, amino acid metabolic cell proliferation disorders, these events are often associated with a variety of diseases or cancer associated with methylenetetrahydrofolate reductase (Methylenetetrahydrofolate reductase,. MTHFR), as a key "switch" gene, acts on the methylation and DNA synthesis of DNA through a carbon metabolism. Its abnormality is closely related to genomic instability and the occurrence and development of various diseases or tumors. The purpose of this study was to analyze whether miR-22-3p/miR-149-5p responds to folic acid deficiency, and then targets to affect the expression of MTHFR and then affect its genomic stability, and to explore its possible inhibitory effect on the growth of cancer cells. In this study, human normal hepatocytes (HL-7702) and human hepatocellular carcinoma cells (QGY-7703) were pretreated with folic acid deficient modified RPMI 1640 medium for 21 days, and the interaction between miRNA and MTHFR was analyzed by double luciferase report monitoring system. The expression of miR-22-3p/miR-149-5p in response to folic acid deficiency was detected by; Poly (A) plus tail RT-qPCR. The expression of MTHFR in the tested cells was detected by RT-qPCR and Western blotting at transcriptional and translation levels, the genomic damage was detected by CBMN-Cyt, and the proliferation of HCC cells after miRNA was detected by CCK-8 assay. The results showed that miR-22-3p/miR-149-5p directly targeted MTHFR gene 3 / UTR. folic acid deficiency led to the up-regulation of miR-22-3p/miR-149-5p expression in QGY-7703/HL-7702 cells. However, the transcription level of MTHFR was down-regulated in QGY-7703 cells. The up-regulation of; Western blotting in HL-7702 cells showed that folic acid deficiency led to the down-regulation of MTHFR protein level in QGY-7703 cells, but remained unchanged in HL-7702 cells. The results also showed that folic acid deficiency had different inhibitory / antitumor effects on normal liver cells (the decrease of tumor inhibitor mRNA level) and liver cancer cells (the increase of tumor inhibitor mRNA level). Genomic damage showed that the resistance of HL-7702 cells to folic acid deficiency was stronger than that of QGY-7703 cells. The, MTHFR mRNA level of QGY-7703 cells was decreased and the proliferation ability of QGY-7703 cells decreased at the same time. Our results suggest that miR-22-3p/miR-149-5p plays a different post-transcriptional role in the expression of MTHFR in human normal hepatocytes and human hepatocellular carcinoma cells under folic acid deficiency. These results suggest that miR-22-3p/miR-149-5p may inhibit the growth of HCC cells and promote their genomic instability under folic acid deficiency.
【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】：Q78;R73-3
[Abstract]:Folic acid (Folic acid,FA) is a core micronutrient involved in DNA synthesis and methylation, and its metabolism affects genome stability. When intracellular folic acid metabolism changes, MicroRNA may respond and affect the expression of downstream target genes, and its imbalance can also act on one carbon metabolism (OCM,or folate metabolism) can lead to DNA synthesis, methylated, amino acid metabolic cell proliferation disorders, these events are often associated with a variety of diseases or cancer associated with methylenetetrahydrofolate reductase (Methylenetetrahydrofolate reductase,. MTHFR), as a key "switch" gene, acts on the methylation and DNA synthesis of DNA through a carbon metabolism. Its abnormality is closely related to genomic instability and the occurrence and development of various diseases or tumors. The purpose of this study was to analyze whether miR-22-3p/miR-149-5p responds to folic acid deficiency, and then targets to affect the expression of MTHFR and then affect its genomic stability, and to explore its possible inhibitory effect on the growth of cancer cells. In this study, human normal hepatocytes (HL-7702) and human hepatocellular carcinoma cells (QGY-7703) were pretreated with folic acid deficient modified RPMI 1640 medium for 21 days, and the interaction between miRNA and MTHFR was analyzed by double luciferase report monitoring system. The expression of miR-22-3p/miR-149-5p in response to folic acid deficiency was detected by; Poly (A) plus tail RT-qPCR. The expression of MTHFR in the tested cells was detected by RT-qPCR and Western blotting at transcriptional and translation levels, the genomic damage was detected by CBMN-Cyt, and the proliferation of HCC cells after miRNA was detected by CCK-8 assay. The results showed that miR-22-3p/miR-149-5p directly targeted MTHFR gene 3 / UTR. folic acid deficiency led to the up-regulation of miR-22-3p/miR-149-5p expression in QGY-7703/HL-7702 cells. However, the transcription level of MTHFR was down-regulated in QGY-7703 cells. The up-regulation of; Western blotting in HL-7702 cells showed that folic acid deficiency led to the down-regulation of MTHFR protein level in QGY-7703 cells, but remained unchanged in HL-7702 cells. The results also showed that folic acid deficiency had different inhibitory / antitumor effects on normal liver cells (the decrease of tumor inhibitor mRNA level) and liver cancer cells (the increase of tumor inhibitor mRNA level). Genomic damage showed that the resistance of HL-7702 cells to folic acid deficiency was stronger than that of QGY-7703 cells. The, MTHFR mRNA level of QGY-7703 cells was decreased and the proliferation ability of QGY-7703 cells decreased at the same time. Our results suggest that miR-22-3p/miR-149-5p plays a different post-transcriptional role in the expression of MTHFR in human normal hepatocytes and human hepatocellular carcinoma cells under folic acid deficiency. These results suggest that miR-22-3p/miR-149-5p may inhibit the growth of HCC cells and promote their genomic instability under folic acid deficiency.
【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】：Q78;R73-3
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