【摘要】：目的：腫瘤細(xì)胞代謝發(fā)生根本性改變,與正常細(xì)胞相比,腫瘤細(xì)胞攝取糖類急劇增加,并且利用糖類方式也發(fā)生改變。經(jīng)大量研究及前期實(shí)驗(yàn)發(fā)現(xiàn)肝癌(HCC,hepatocellular carcinoma)中糖分解代謝主要依賴糖酵解及磷酸戊糖途徑為主,而氧化磷酸化處于“失用”狀態(tài)。線粒體是執(zhí)行氧化磷酸化的主要細(xì)胞器,為細(xì)胞供能,線粒體基因編碼合成氧化磷酸化組成蛋白,線粒體功能障礙或基因改變是否導(dǎo)致氧化磷酸化的“失用”目前尚不清楚。細(xì)胞中miRNA (microRNA,微小RNA)大量存在并且廣泛調(diào)控基因轉(zhuǎn)錄,抑制靶基因表達(dá)水平,那么miRNA是否參與調(diào)控磷酸戊糖途徑基因,從而影響肝癌磷酸戊糖途徑的代謝?snoRNA (small nucleolar RNA,小核仁RNA)作為蛋白合成器核糖體中rRNA的重要調(diào)節(jié)因素,是否在肝癌中異常表達(dá),從而影響蛋白合成?因此本研究目的在于：1.從基因水平,探索肝癌線粒體基因組突變情況及對(duì)氧化磷酸化的影響；2.從轉(zhuǎn)錄后水平,探索miR-1 (microRNA-1)對(duì)肝癌磷酸戊糖途徑通路關(guān)鍵酶TKT(transketolase,轉(zhuǎn)酮醇酶)、G6PD (glucose-6-phosphate dehydrogenase,6-磷酸葡萄糖脫氫酶)基因的調(diào)控作用及其對(duì)磷酸戊糖途徑的影響；3.從翻譯水平,探索肝癌SNORD78(small nucleolar RNA78,小核仁RNA78)表達(dá)水平及對(duì)肝癌細(xì)胞生物學(xué)行為的影響,并初步建立莖環(huán)法逆轉(zhuǎn)錄SNORD78的方法。方法；1.采用線粒體全基因組擴(kuò)增、Sanger直接測(cè)序篩查及克隆測(cè)序驗(yàn)證的方法,篩查肝癌中線粒體基因體細(xì)胞突變；采用大片段基因合成法合成野生型及突變型COX3 (cytochrome c oxidase 3,細(xì)胞色素c氧化酶3)基因,并亞克隆至pcDNA3.1-Mito載體中,轉(zhuǎn)染至肝癌細(xì)胞HepG2中,檢測(cè)細(xì)胞產(chǎn)生ATP、乳酸水平,流式細(xì)胞術(shù)分析細(xì)胞凋亡及ROS水平。2.生物信息學(xué)軟件預(yù)測(cè)miR-1調(diào)控磷酸戊糖途徑關(guān)鍵酶TKT及G6PD,實(shí)時(shí)熒光定量(real-time PCR, RT-PCR)分析肝癌中miR-1表達(dá)水平。在肝癌細(xì)胞HepG2中共轉(zhuǎn)染miR-1-3p及靶基因后,雙熒光素酶報(bào)告實(shí)驗(yàn)檢測(cè)熒光強(qiáng)度,Western blot檢測(cè)靶基因蛋白水平以驗(yàn)證miR-1-3p調(diào)控靶基因TKT及G6PD。肝癌細(xì)胞HepG2過表達(dá)miR-1后,流式細(xì)胞術(shù)分析細(xì)胞凋亡及ROS水平；CCK-8實(shí)驗(yàn)檢測(cè)細(xì)胞活力；顯色法檢測(cè)細(xì)胞產(chǎn)生乳酸、NADPH水平。3. RT-PCR分析肝癌組織及肝癌血漿中SNORD78表達(dá)水平。siRNA下調(diào)肝癌細(xì)胞SK-Hep-1中SNORD78表達(dá),采用RT-PCR驗(yàn)證SNORD78及其宿主基因GAS5(growth arrest-specific transcript 5,生長抑制特異性轉(zhuǎn)錄物5)表達(dá)水平,流式細(xì)胞術(shù)分析細(xì)胞凋亡及細(xì)胞周期,CCK-8法檢測(cè)細(xì)胞活力,Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移與侵襲。采用莖環(huán)法逆轉(zhuǎn)錄SNORD78, Taqman探針法檢測(cè)及digitalPCR驗(yàn)證肝癌血漿中SNORD78表達(dá)水平。結(jié)果：1.肝癌線粒體基因體細(xì)胞突變(1)檢測(cè)56對(duì)肝癌組織及對(duì)應(yīng)白細(xì)胞線粒體全基因序列,對(duì)比癌組織與白細(xì)胞序列,共篩選出51種肝癌線粒體基因體細(xì)胞突變,在肝癌中發(fā)生率約為(27/56)48.2%；其中非同義突變共有11種,在肝癌中發(fā)生率約為(6/56)10.7%,非同義突變?cè)诰�粒體編碼基因均勻分布且突變率較低,未發(fā)現(xiàn)突變熱點(diǎn)；異質(zhì)性突變?cè)诟伟┙M織及白細(xì)胞中均可見,發(fā)生率分別為21.4%(12/56)、12.5%(7/56)。(2)COX3是人類線粒體基因組高度保守基因,因此挑選該基因G9267A突變作為探索肝癌線粒體基因突變功能的模型。(3)G9267A突變型較野生型合成ATP水平減少、ROS水平增加,且隨突變率升高,細(xì)胞合成ATP減少、ROS增加(P for trend=0.0152,0.001); G9267A突變型較野生型產(chǎn)生乳酸水平增多,隨突變率升高而增多(P for trend=0.0461).另外,G9267A突變型較野生型發(fā)生細(xì)胞凋亡現(xiàn)象增多,凋亡率隨著突變率升高而降低(P for trend=0.0005)。實(shí)驗(yàn)顯示G9267A突變損傷肝癌細(xì)胞線粒體氧化磷酸化能力、增強(qiáng)糖酵解活性,并且隨G9267A突變率升高,氧化磷酸化能力降低、糖酵解能力增強(qiáng)。2.肝癌miR-1表達(dá)水平及其對(duì)磷酸戊糖途徑的影響(1)肝癌組織miR-1-3p水平較癌旁組織降低(P=0.001),且與TNM分期呈負(fù)相關(guān)(P=0.043)。(2)轉(zhuǎn)染miR-1-3p較陰性對(duì)照顯著抑制熒光素酶基因的活性(P0.001),并且顯著下調(diào)TKT與G6PD的mRNA水平及蛋白水平(P0.001,0.001),明確miR-1-3p對(duì)G6PD及TKT基因具有靶向調(diào)控作用。(3)肝癌細(xì)胞HepG2過表達(dá)miR-1-3p可促進(jìn)細(xì)胞周期停滯在S期,阻止細(xì)胞有絲分裂；抑制HepG2細(xì)胞增殖；促進(jìn)細(xì)胞產(chǎn)生ROS及細(xì)胞凋亡；抑制細(xì)胞合成NAPDH,促進(jìn)乳酸合成(P均0.05)。表明miR-1不僅影響肝癌細(xì)胞的生長、增殖與凋亡,而且負(fù)性調(diào)控肝癌細(xì)胞磷酸戊糖途徑的關(guān)鍵酶TKT、G6PD基因,致磷酸戊糖途徑活性增強(qiáng)。3.肝癌SNORD78表達(dá)水平及其對(duì)肝癌細(xì)胞生物學(xué)行為的影響(1)肝癌組織中SNORD78表達(dá)水平較癌旁組織高(P=0.004),且與肝癌腫瘤個(gè)數(shù)、分期及遠(yuǎn)處轉(zhuǎn)移成正比(P=0.02,0.014,0.01)；Kaplan-Meier生存曲線分析顯示,高表達(dá)SNORD78肝癌患者總體生存期和無瘤生存期顯著低于低表達(dá)患者(P=0.023,0.014)。暗示SNORD78表達(dá)可作為評(píng)估肝癌預(yù)后的潛在生物學(xué)指標(biāo)。(2)下調(diào)肝癌細(xì)胞SK-Hep-1中的SNORD78后,兩干擾組均與陰性對(duì)照組的SNORD78表達(dá)顯著降低(P均0.05),GAS5表達(dá)無顯著差異。下調(diào)SNORD78水平顯著促進(jìn)SK-Hep-1細(xì)胞凋亡(P均0.05),使細(xì)胞停滯在G0/G1期(P均0.05),阻滯細(xì)胞進(jìn)入S期(P均0.05),同時(shí)抑制SK-Hep-1細(xì)胞增殖(P均0.05),并抑制細(xì)胞遷移與侵襲能力(P均0.05)。(3)肝癌患者血漿中SNORD78水平較正常對(duì)照組降低(P=0.0138),與肝硬化患者無差異(P=0.1281)；且與肝癌腫瘤個(gè)數(shù)、分期成反比(P=0.016,0.039)。(4)莖環(huán)法逆轉(zhuǎn)錄與加“A”法、特異法逆轉(zhuǎn)錄檢測(cè)SNORD78的相關(guān)性分別是(R2=0.7626,P=0.0015；R2=0.8198,P=0.00103)。采用莖環(huán)法逆轉(zhuǎn)錄并Taqman探針法檢測(cè)肝癌血漿中SNORD78表達(dá)較正常人降低(P0.05),digital PCR驗(yàn)證肝癌血漿中SNORD78表達(dá),與Taqman探針法相符。因此,本文改良的莖環(huán)法逆轉(zhuǎn)錄SNORD78是一種穩(wěn)定可靠的方法。結(jié)論：1.肝癌線粒體基因體細(xì)胞COX3-G9267A突變損傷肝癌細(xì)胞氧化磷酸化能力,并增強(qiáng)糖酵解能力。2.肝癌中低表達(dá)的miR-1通過靶向調(diào)控磷酸戊糖途徑關(guān)鍵酶G6PD及TKT基因,抑制靶基因轉(zhuǎn)錄,導(dǎo)致肝癌中磷酸戊糖途徑活性增強(qiáng)。3. SNORD78高表達(dá)與肝癌細(xì)胞增殖、遷移和侵襲能力相關(guān),可作為評(píng)估肝癌預(yù)后的潛在生物學(xué)指標(biāo)。并初步建立改良莖環(huán)法逆轉(zhuǎn)錄SNORD78的方法。
[Abstract]:AIM: Metabolism of tumor cells has undergone fundamental changes. Compared with normal cells, the uptake of carbohydrates by tumor cells has increased dramatically, and the use of carbohydrates has also changed. Mitochondria are the main organelles that perform oxidative phosphorylation and provide energy for cells. Mitochondrial genes encode and synthesize oxidative phosphorylated constituent proteins. Whether mitochondrial dysfunction or genetic alterations lead to oxidative phosphorylation is unclear. And regulate gene transcription extensively, inhibit the expression of target genes, so does microRNA participate in the regulation of pentose phosphate pathway genes, thereby affecting the metabolism of pentose phosphate pathway in hepatocellular carcinoma? Therefore, the purpose of this study is: 1. to explore the mitochondrial genomic mutations and their effects on oxidative phosphorylation in hepatocellular carcinoma at the gene level; 2. to explore the effects of microRNA-1 on the key enzymes TKT (transketolase), G6PD (glucose-6-phosphate dehydrogenase, 6) in the pentose phosphate pathway of hepatocellular carcinoma at the post-transcriptional level. 3. To explore the expression level of small nucleolar RNA 78 (SNORD 78) and its effect on the biological behavior of hepatocellular carcinoma cells from the translation level, and to establish a stem-loop method for the reverse transcription of SNORD 78. Mitochondrial somatic mutations in hepatocellular carcinoma were screened by genomic amplification, Sanger direct sequencing and cloning and sequencing. Wild and mutant COX3 (cytochrome c oxidase 3, cytochrome c oxidase 3) genes were synthesized by large fragment gene synthesis and subcloned into pcDNA3.1-Mito vector and transfected into hepatocellular carcinoma HepG2 cells. Bioinformatics software predicted the key enzymes of the pentose phosphate pathway, TKT and G6PD. Real-time PCR (RT-PCR) was used to analyze the expression of microRNA-1 in hepatocellular carcinoma. After co-transfection of microRNA-1-3p and target gene in HepG2 cells, double fluorescence was detected. The fluorescence intensity was detected by the photoenzyme report assay, and the target gene protein level was detected by Western blot to verify that the target genes TKT and G6PD were regulated by microRNAs-1-3p. After overexpression of microRNAs-1 in HepG2 cells, the apoptosis and ROS levels were analyzed by flow cytometry; the cell viability was detected by CCK-8 assay; the lactic acid production and NADPH levels were detected by color rendering assay.3. RT-PCR analysis. The expression of SNORD78 was down-regulated by siRNA. The expression of SNORD78 and its host gene GAS5 were detected by RT-PCR. Cell apoptosis and cell cycle were analyzed by flow cytometry. Cell viability was detected by CCK-8 assay. Transwell assay was used to detect the migration and invasion of hepatocellular carcinoma cells. The expression of SNORD78 in the plasma of hepatocellular carcinoma was detected by stem-ring reverse transcription SNORD78, Taqman probe assay and digital PCR. Results: 1. Mitochondrial somatic mutation of hepatocellular carcinoma (1) The whole mitochondrial gene sequences of 56 pairs of hepatocellular carcinoma tissues and corresponding leukocytes were detected, and the cancer tissues and leukocytes were compared. Fifty-one kinds of mitochondrial gene somatic mutations were screened out, the incidence of which was about (27/56) 48.2%. Among them, 11 kinds of non-synonymous mutations were found, and the incidence of non-synonymous mutations was about (6/56) 10.7%. The non-synonymous mutations were uniformly distributed in mitochondrial coding genes with low mutation rate and no mutation hotspot was found. The incidence of COX3 was 21.4% (12/56) and 12.5% (7/56), respectively. (2) COX3 was a highly conserved gene in human mitochondrial genome, so G9267A mutation was selected as a model to explore the function of mitochondrial gene mutation in hepatocellular carcinoma. (3) The level of ATP synthesis in G9267A mutant was lower than that in wild type, and the level of ROS increased with the increase of mutation rate. Cell ATP synthesis decreased and ROS increased (P for trend = 0.0152, 0.001); G9267A mutant produced more lactic acid than wild type, and increased with the increase of mutation rate (P for trend = 0.0461). In addition, G9267A mutant had more apoptosis than wild type, and the apoptosis rate decreased with the increase of mutation rate (P for trend = 0.0005). 67A mutation impairs mitochondrial oxidative phosphorylation and enhances glycolytic activity in hepatocellular carcinoma cells. With the increase of G9267A mutation rate, oxidative phosphorylation ability decreases and glycolytic ability increases. There was a negative correlation (P = 0.043). (2) Mi-1-3p transfection significantly inhibited the activity of luciferase gene (P 0.001), and significantly decreased the mRNA and protein levels of TKT and G6PD (P 0.001, 0.001). It was clear that Mi-1-3p had a targeted regulatory effect on G6PD and TKT genes. (3) Overexpression of Mi-1-3p by HepG2 could promote cell cycle arrest in S cells. Phase I, inhibiting cell mitosis, inhibiting HepG2 cell proliferation, promoting ROS production and apoptosis, inhibiting cell synthesis of NAPDH and promoting Lactate Synthesis (P The expression level of SNORD78 in hepatocellular carcinoma and its effect on the biological behavior of hepatocellular carcinoma cells (1) The expression level of SNORD78 in hepatocellular carcinoma tissues was higher than that in adjacent tissues (P = 0.004), and was proportional to the number, stage and distant metastasis of hepatocellular carcinoma (P = 0.02, 0.014, 0.01); Kaplan-Meier survival curve analysis showed that the expression of SNORD78 in hepatocellular carcinoma tissues was higher than that in adjacent tissues (P = 0.004). The overall survival and tumor-free survival were significantly lower than those in the low-expression group (P = 0.023, 0.014). It suggested that SNORD78 expression could be used as a potential biological marker for evaluating the prognosis of HCC. (2) After down-regulating SNORD78 in SK-Hep-1 cells, the expression of SNORD78 in both interference groups was significantly lower than that in the negative control group (P = 0.05), and the expression of GAS5 was not significantly different. Adjusting the level of SNORD78 significantly promoted the apoptosis of SK-Hep-1 cells (all P 0.05), arrested the cells in G0/G1 phase (all P 0.05), blocked the cells entering S phase (all P 0.05), inhibited the proliferation of SK-Hep-1 cells (all P 0.05), and inhibited the migration and invasion of SK-Hep-1 cells (all P 0.05). (3) The level of SNORD78 in plasma of patients with hepatocellular carcinoma was lower than that of normal control group (P = 0.0138), and inhibited the proliferation of SK-Hep-1 cells (all P 0.05). There was no significant difference between cirrhosis patients (P = 0.1281), and it was inversely proportional to the number of hepatocellular carcinoma (P = 0.016, 0.039). (4) The expression of SNORD78 in hepatocellular carcinoma plasma was detected by stem-loop reverse transcription and Taqman probe, respectively (R2 = 0.7626, P = 0.0015; R2 = 0.8198, P = 0.00103). The expression of SNORD78 in hepatocellular carcinoma plasma was confirmed by digital PCR, which was consistent with Taqman probe method. Therefore, the improved stem-ring method was a stable and reliable method for the reverse transcription of SNORD78. Mi-1, a key enzyme in the pentose-phosphate pathway, inhibits the transcription of target genes by targeting the G6PD and TKT genes. 3. The high expression of SNORD78 is associated with the proliferation, migration and invasion of hepatocellular carcinoma cells, which may serve as a potential biological marker for evaluating the prognosis of hepatocellular carcinoma. Stem loop method for reverse transcription of SNORD78.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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