线粒体基因突变及miR-1与肝癌相关性研究
发布时间：2018-08-27 16:07
【摘要】：目的：肿瘤细胞代谢发生根本性改变,与正常细胞相比,肿瘤细胞摄取糖类急剧增加,并且利用糖类方式也发生改变。经大量研究及前期实验发现肝癌(HCC,hepatocellular carcinoma)中糖分解代谢主要依赖糖酵解及磷酸戊糖途径为主,而氧化磷酸化处于“失用”状态。线粒体是执行氧化磷酸化的主要细胞器,为细胞供能,线粒体基因编码合成氧化磷酸化组成蛋白,线粒体功能障碍或基因改变是否导致氧化磷酸化的“失用”目前尚不清楚。细胞中miRNA (microRNA,微小RNA)大量存在并且广泛调控基因转录,抑制靶基因表达水平,那么miRNA是否参与调控磷酸戊糖途径基因,从而影响肝癌磷酸戊糖途径的代谢?snoRNA (small nucleolar RNA,小核仁RNA)作为蛋白合成器核糖体中rRNA的重要调节因素,是否在肝癌中异常表达,从而影响蛋白合成?因此本研究目的在于：1.从基因水平,探索肝癌线粒体基因组突变情况及对氧化磷酸化的影响；2.从转录后水平,探索miR-1 (microRNA-1)对肝癌磷酸戊糖途径通路关键酶TKT(transketolase,转酮醇酶)、G6PD (glucose-6-phosphate dehydrogenase,6-磷酸葡萄糖脱氢酶)基因的调控作用及其对磷酸戊糖途径的影响；3.从翻译水平,探索肝癌SNORD78(small nucleolar RNA78,小核仁RNA78)表达水平及对肝癌细胞生物学行为的影响,并初步建立茎环法逆转录SNORD78的方法。方法；1.采用线粒体全基因组扩增、Sanger直接测序筛查及克隆测序验证的方法,筛查肝癌中线粒体基因体细胞突变；采用大片段基因合成法合成野生型及突变型COX3 (cytochrome c oxidase 3,细胞色素c氧化酶3)基因,并亚克隆至pcDNA3.1-Mito载体中,转染至肝癌细胞HepG2中,检测细胞产生ATP、乳酸水平,流式细胞术分析细胞凋亡及ROS水平。2.生物信息学软件预测miR-1调控磷酸戊糖途径关键酶TKT及G6PD,实时荧光定量(real-time PCR, RT-PCR)分析肝癌中miR-1表达水平。在肝癌细胞HepG2中共转染miR-1-3p及靶基因后,双荧光素酶报告实验检测荧光强度,Western blot检测靶基因蛋白水平以验证miR-1-3p调控靶基因TKT及G6PD。肝癌细胞HepG2过表达miR-1后,流式细胞术分析细胞凋亡及ROS水平；CCK-8实验检测细胞活力；显色法检测细胞产生乳酸、NADPH水平。3. RT-PCR分析肝癌组织及肝癌血浆中SNORD78表达水平。siRNA下调肝癌细胞SK-Hep-1中SNORD78表达,采用RT-PCR验证SNORD78及其宿主基因GAS5(growth arrest-specific transcript 5,生长抑制特异性转录物5)表达水平,流式细胞术分析细胞凋亡及细胞周期,CCK-8法检测细胞活力,Transwell实验检测细胞迁移与侵袭。采用茎环法逆转录SNORD78, Taqman探针法检测及digitalPCR验证肝癌血浆中SNORD78表达水平。结果：1.肝癌线粒体基因体细胞突变(1)检测56对肝癌组织及对应白细胞线粒体全基因序列,对比癌组织与白细胞序列,共筛选出51种肝癌线粒体基因体细胞突变,在肝癌中发生率约为(27/56)48.2%；其中非同义突变共有11种,在肝癌中发生率约为(6/56)10.7%,非同义突变在线粒体编码基因均匀分布且突变率较低,未发现突变热点；异质性突变在肝癌组织及白细胞中均可见,发生率分别为21.4%(12/56)、12.5%(7/56)。(2)COX3是人类线粒体基因组高度保守基因,因此挑选该基因G9267A突变作为探索肝癌线粒体基因突变功能的模型。(3)G9267A突变型较野生型合成ATP水平减少、ROS水平增加,且随突变率升高,细胞合成ATP减少、ROS增加(P for trend=0.0152,0.001); G9267A突变型较野生型产生乳酸水平增多,随突变率升高而增多(P for trend=0.0461).另外,G9267A突变型较野生型发生细胞凋亡现象增多,凋亡率随着突变率升高而降低(P for trend=0.0005)。实验显示G9267A突变损伤肝癌细胞线粒体氧化磷酸化能力、增强糖酵解活性,并且随G9267A突变率升高,氧化磷酸化能力降低、糖酵解能力增强。2.肝癌miR-1表达水平及其对磷酸戊糖途径的影响(1)肝癌组织miR-1-3p水平较癌旁组织降低(P=0.001),且与TNM分期呈负相关(P=0.043)。(2)转染miR-1-3p较阴性对照显著抑制荧光素酶基因的活性(P0.001),并且显著下调TKT与G6PD的mRNA水平及蛋白水平(P0.001,0.001),明确miR-1-3p对G6PD及TKT基因具有靶向调控作用。(3)肝癌细胞HepG2过表达miR-1-3p可促进细胞周期停滞在S期,阻止细胞有丝分裂；抑制HepG2细胞增殖；促进细胞产生ROS及细胞凋亡；抑制细胞合成NAPDH,促进乳酸合成(P均0.05)。表明miR-1不仅影响肝癌细胞的生长、增殖与凋亡,而且负性调控肝癌细胞磷酸戊糖途径的关键酶TKT、G6PD基因,致磷酸戊糖途径活性增强。3.肝癌SNORD78表达水平及其对肝癌细胞生物学行为的影响(1)肝癌组织中SNORD78表达水平较癌旁组织高(P=0.004),且与肝癌肿瘤个数、分期及远处转移成正比(P=0.02,0.014,0.01)；Kaplan-Meier生存曲线分析显示,高表达SNORD78肝癌患者总体生存期和无瘤生存期显著低于低表达患者(P=0.023,0.014)。暗示SNORD78表达可作为评估肝癌预后的潜在生物学指标。(2)下调肝癌细胞SK-Hep-1中的SNORD78后,两干扰组均与阴性对照组的SNORD78表达显著降低(P均0.05),GAS5表达无显著差异。下调SNORD78水平显著促进SK-Hep-1细胞凋亡(P均0.05),使细胞停滞在G0/G1期(P均0.05),阻滞细胞进入S期(P均0.05),同时抑制SK-Hep-1细胞增殖(P均0.05),并抑制细胞迁移与侵袭能力(P均0.05)。(3)肝癌患者血浆中SNORD78水平较正常对照组降低(P=0.0138),与肝硬化患者无差异(P=0.1281)；且与肝癌肿瘤个数、分期成反比(P=0.016,0.039)。(4)茎环法逆转录与加“A”法、特异法逆转录检测SNORD78的相关性分别是(R2=0.7626,P=0.0015；R2=0.8198,P=0.00103)。采用茎环法逆转录并Taqman探针法检测肝癌血浆中SNORD78表达较正常人降低(P0.05),digital PCR验证肝癌血浆中SNORD78表达,与Taqman探针法相符。因此,本文改良的茎环法逆转录SNORD78是一种稳定可靠的方法。结论：1.肝癌线粒体基因体细胞COX3-G9267A突变损伤肝癌细胞氧化磷酸化能力,并增强糖酵解能力。2.肝癌中低表达的miR-1通过靶向调控磷酸戊糖途径关键酶G6PD及TKT基因,抑制靶基因转录,导致肝癌中磷酸戊糖途径活性增强。3. SNORD78高表达与肝癌细胞增殖、迁移和侵袭能力相关,可作为评估肝癌预后的潜在生物学指标。并初步建立改良茎环法逆转录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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
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