【摘要】：葉酸(Folic acid,FA)是一個(gè)參與DNA合成和甲基化的核心微營養(yǎng)素,其代謝影響基因組穩(wěn)定性。當(dāng)胞內(nèi)葉酸代謝改變時(shí),MicroRNA可能產(chǎn)生響應(yīng)并影響下游靶基因的表達(dá),其失調(diào)還可作用于一碳代謝(OCM,or folate metabolism)可導(dǎo)致DNA合成、甲基化、氨基酸代謝核細(xì)胞增殖的失調(diào),這些事件往往與多種疾病或癌癥相關(guān)聯(lián)亞甲基四氫葉酸還原酶(Methylenetetrahydrofolate reductase,MTHFR)是作為一個(gè)關(guān)鍵的“開關(guān)”基因通過一碳代謝作用于DNA的甲基化和DNA合成,其異常與基因組不穩(wěn)定型和各種疾病或腫瘤的發(fā)生發(fā)展都有著密切的聯(lián)系。本研究旨在解析miR-22-3p/miR-149-5p是否響應(yīng)葉酸缺乏、隨后靶向進(jìn)而影響MTHFR的表達(dá),進(jìn)而影響其基因組穩(wěn)定性;并初步探究其可能存在的對癌細(xì)胞生長的抑制作用。本研究以葉酸缺乏的修飾RPMI 1640培養(yǎng)基干預(yù)人正常肝細(xì)胞(HL-7702)與人肝癌細(xì)胞(QGY-7703)21天;雙熒光素酶報(bào)告監(jiān)測系統(tǒng)分析受試miRNA與MTHFR之間的相互作用;Poly(A)加尾法RT-qPCR檢測miR-22-3p/miR-149-5p響應(yīng)葉酸缺乏時(shí)的表達(dá)改變;RT-qPCR和Western blotting分別在轉(zhuǎn)錄和翻譯水平檢測受試細(xì)胞MTHFR的表達(dá)量;CBMN-Cyt檢測基因組損傷情況;CCK-8法檢測肝癌細(xì)胞轉(zhuǎn)染miRNA后的增殖能力。結(jié)果顯示:miR-22-3p/miR-149-5p直接靶向MTHFR基因3’UTR;葉酸缺乏導(dǎo)致miR-22-3p/miR-149-5p在QGY-7703/HL-7702細(xì)胞中表達(dá)上調(diào),然而MTHFR的轉(zhuǎn)錄水平在QGY-7703細(xì)胞中下調(diào),在HL-7702細(xì)胞中上調(diào);Western blotting結(jié)果顯示葉酸缺乏導(dǎo)致QGY-7703細(xì)胞MTHFR蛋白水平下調(diào),但是在HL-7702細(xì)胞中保持不變;干預(yù)結(jié)果還顯示葉酸缺乏對肝正常細(xì)胞(抑癌基因mRNA水平下降)和肝癌細(xì)胞(抑癌基因mRNA水平上升)的產(chǎn)生不同的抑制增殖/抑癌效應(yīng);基因組損傷情況顯示HL-7702細(xì)胞對葉酸缺乏的抵抗能力強(qiáng)于QGY-7703細(xì)胞。miR-22-3p/miR-149-5p轉(zhuǎn)染QGY-7703細(xì)胞后,MTHFR mRNA水平下降,細(xì)胞增殖能力同時(shí)下降。我們的結(jié)果表明葉酸缺乏條件下miR-22-3p/miR-149-5p在人正常肝細(xì)胞和人肝癌細(xì)胞中對MTHFR的表達(dá)發(fā)揮不同的轉(zhuǎn)錄后調(diào)控作用。此結(jié)果暗示人肝癌細(xì)胞在葉酸缺乏條件下,miR-22-3p/miR-149-5p可能對肝癌細(xì)胞的生長起到抑制作用并促進(jìn)其基因組不穩(wěn)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.
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;R73-3

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