Folate depletion causes chromosome instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5, 10 methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the parti-tioning of intracellular one carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to es-tablish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a Lentiviral vector; and (3) investigated the impacts of re-duced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein and enzy-matic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in in wildtype (Wt), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M; and cell population in the G2/M were inversely correlated with MTHFR gene level (r= -0.81, p<0.0001), MTHFR protein expression (r= -0.8; p=0.01), and MTHFR enzyme activity (r= -0.842; p=0.005). Folate depletion resulted in G2/M cell cycle arrest in Wt and Neg but not in shMTHFR cells. In addition, shMTHFR promoted the expression and transloca-tion of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate de-pendent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nu-clear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight on a significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies. (submitted 2021 Jul) 基因的遺傳多型性對於腫瘤的發生和預防扮演一個重要的角色。葉酸缺乏耗竭會增加 DNA 鏈斷裂、尿嘧啶錯誤摻入和修復缺陷導致染色體不穩定。葉酸介導的單碳代謝已被認為通過影響 DNA 完整性在肝細胞癌的癌變和進展中起關鍵作用。亞甲基四氫葉酸還原酶 (MTHFR) 是催化 5, 10 亞甲基四氫葉酸向 5-甲基四氫葉酸不可逆轉化的酶，可控制葉酸輔因子分佈並調節細胞內單碳部分的分配。過去研究對於 MTHFR基因多型行性肝癌 風險之間的關聯並不一致，在人群研究中仍然存在爭議。我們建立一個肝臟起源細胞的體外模型以闡明 MTHFR 功能、葉酸狀態和染色體穩定性之間的相互作用。 本研究探討肝癌患者中 MTHFR 的表達情況, 我們還 使用慢病毒載體遞送的髮夾 RNA 建立穩定抑制 MTHFR 的肝源細胞模型並進一步研究降低 MTHFR 和葉酸狀態對細胞週期、甲基基團穩態、核苷酸生物合成和 DNA 穩定性的影響，以上都是參與 DNA 完整性和修復的關鍵途徑，對人類腫瘤發生至關重要。通過次世代定序數據庫分析發現 MTHFR 較高的肝癌患者的生存率較差。 干擾 MTHFR 的 shRNA（shMTHFR）導致人肝癌細胞中MTHFR基因表達、MTHFR蛋白和酶活性均顯著降低。 shMTHFR 降低細胞內 S-腺苷甲硫氨酸 (SAM) 含量，但在缺乏葉酸情況下, 野生型 (Wt)、陰性對照細胞 (Neg) 和 shMTHFR 細胞中的 SAM 的減低 效果接近, 表明在肝臟來源的細胞中，shMTHFR 並不加劇葉酸枯竭造成的甲基供應不足現象。 shMTHFR 在 細胞週期 引起 G2/M 積累； G2/M 期和細胞群與 MTHFR 基因水平（r= -0.81，p<0.0001）、MTHFR 蛋白表達（r= -0.8；p=0.01）和 MTHFR 酶活性（r= -0.842； p=0.005）高度相關。葉酸耗竭導致 Wt 和 Neg 中的 G2/M 細胞週期停滯，但不會導致 shMTHFR 細胞 G2/M 細胞週期停滯。此外，shMTHFR 促進核胸苷合成酶複合物 SHMT1/DHFR/TYMS 的表達易位，並在葉酸限制下輔助葉酸依賴的從頭核苷酸生物合成。最後，shMTHFR 促進葉酸缺乏下的核 MLH1/p53 表達，並進一步減少葉酸缺乏下的微核形成和 DNA 尿嘧啶錯誤摻入。總之，在我們建立的細胞模式中, shMTHFR 誘導細胞週期停滯在 G2/M，這可能會促進核苷酸供應並幫助細胞防禦葉酸消耗誘導的染色體分離和 DNA 中的尿嘧啶錯誤嵌入。本研究為 MTHFR 功能對肝組織染色體穩定性的顯著影響提供了見解, 可能揭示 MTHFR 調節肝臟惡性腫瘤風險的潛在調節機制 (2020 Aug accepted).

Folate depletion causes chromosome instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5, 10 methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the parti-tioning of intracellular one carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to es-tablish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a Lentiviral vector; and (3) investigated the impacts of re-duced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein and enzy-matic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in in wildtype (Wt), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M; and cell population in the G2/M were inversely correlated with MTHFR gene level (r= -0.81, p<0.0001), MTHFR protein expression (r= -0.8; p=0.01), and MTHFR enzyme activity (r= -0.842; p=0.005). Folate depletion resulted in G2/M cell cycle arrest in Wt and Neg but not in shMTHFR cells. In addition, shMTHFR promoted the expression and transloca-tion of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate de-pendent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nu-clear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight on a significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies. (submitted 2021 Jul) 基因的多型性對於腫瘤的發生和預防扮演一個重要的角色。葉酸缺乏耗竭會增加 DNA 鏈斷裂、尿嘧啶錯誤摻入和修復缺陷導致染色體不穩定。葉酸介導的單碳代謝已被認為通過影響 DNA 完整性在肝細胞癌的癌變和進展中起關鍵作用。亞甲基四氫葉酸還原酶 (MTHFR) 是催化 5, 10 亞甲基四氫葉酸向 5-甲基四氫葉酸不可逆轉化的酶，可控制葉酸輔因子分佈並調節細胞內單碳部分的分配。過去研究對於 MTHFR基因多型行性肝癌 風險之間的關聯並不一致，在人群研究中仍然存在爭議。我們建立一個肝臟起源細胞的體外模型以闡明 MTHFR 功能、葉酸狀態和染色體穩定性之間的相互作用。 本研究探討肝癌患者中 MTHFR 的表達情況, 我們還 使用慢病毒載體遞送的髮夾 RNA 建立穩定抑制 MTHFR 的肝源細胞模型並進一步研究降低 MTHFR 和葉酸狀態對細胞週期、甲基基團穩態、核苷酸生物合成和 DNA 穩定性的影響，以上都是參與 DNA 完整性和修復的關鍵途徑，對人類腫瘤發生至關重要。通過次世代定序數據庫分析發現 MTHFR 較高的肝癌患者的生存率較差。 干擾 MTHFR 的 shRNA（shMTHFR）導致人肝癌細胞中MTHFR基因表達、MTHFR蛋白和酶活性均顯著降低。 shMTHFR 降低細胞內 S-腺苷甲硫氨酸 (SAM) 含量，但在缺乏葉酸情況下, 野生型 (Wt)、陰性對照細胞 (Neg) 和 shMTHFR 細胞中的 SAM 的減低 效果接近, 表明在肝臟來源的細胞中，shMTHFR 並不加劇葉酸枯竭造成的甲基供應不足現象。 shMTHFR 在 細胞週期 引起 G2/M 積累； G2/M 期和細胞群與 MTHFR 基因水平（r= -0.81，p<0.0001）、MTHFR 蛋白表達（r= -0.8；p=0.01）和 MTHFR 酶活性（r= -0.842； p=0.005）高度相關。葉酸耗竭導致 Wt 和 Neg 中的 G2/M 細胞週期停滯，但不會導致 shMTHFR 細胞 G2/M 細胞週期停滯。此外，shMTHFR 促進核胸苷合成酶複合物 SHMT1/DHFR/TYMS 的表達易位，並在葉酸限制下輔助葉酸依賴的從頭核苷酸生物合成。最後，shMTHFR 促進葉酸缺乏下的核 MLH1/p53 表達，並進一步減少葉酸缺乏下的微核形成和 DNA 尿嘧啶錯誤摻入。總之，在我們建立的細胞模式中, shMTHFR 誘導細胞週期停滯在 G2/M，這可能會促進核苷酸供應並幫助細胞防禦葉酸消耗誘導的染色體分離和 DNA 中的尿嘧啶錯誤嵌入。本研究為 MTHFR 功能對肝組織染色體穩定性的顯著影響提供了見解, 可能揭示 MTHFR 調節肝臟惡性腫瘤風險的潛在調節機制.

Genetic predisposition plays a significant role in tumorigenesis and cancer prevention. A common polymorphism 677C->T in methylenetetrahydrofolate reductase (MTHFR) is associated with altered risk of certain types of cancer, but the mechanism remains to be determined. MTHFR catalyzes the reduction of 5, 10-methylene- tetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the synthesis of methionine from homocysteine. The homozygous C677T mutation in the MTHFR gene results in reduction in enzyme activity and changes in cellular distribution of folates. In the present study we investigate if the folate form redistribution results in compromised homocysteine remethylation and elevated de novo purine synthesis, both of which may be potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were studied in Epstein-Barr virus transformed lymphoblasts expressing common polymorphic allele 677 C and 677T of the MTHFR gene. Using different stable isotopic tracers, specific enrichments in intracellular amino acid pool and nucleotides were measured by GCMS under various nutritional conditions. Our preliminary data indicated that when cellular folate was adequate, the MTHFR genotype did not affect the folate dependent homocysteine remethylation pathways. Only when cells became folate deficient, the folate dependent remethylation pathway in TT genotype was significantly reduced compared to the CC genotypes. Using 13C-serine as the tracer, de novo purine synthesis in TT genotype was found to be elevated when folate status is adequate, presumably due to increased availability of formylated folates. When folate was restricted, no difference in purine synthesis was found between the 2 genotypes, suggesting that the advantage in purine synthesis only exists when folate supply is adequate. Our data suggest that the impacts of MTHFR 677T polymorphism are closely related to nutritional conditions, and such alterations may modulate metabolic pathways involved in disease onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in certain type of cancers. These transformed cells are potential models for studying the consequences of human genetic variation in MTHFR and their relationship to pathways involved in cancer pathogenesis or progression. 基因的遺傳多型性對於腫瘤的發生和預防扮演一個重要的角色。亞甲基四氫葉酸還原酶的多型性和某些種類癌症發生的風險率有關，但是其機制仍然需要進一步的研究。已知亞甲基四氫葉酸還原酶多型性中TT同型合子的會導致酵素活性的下降並且改變細胞內葉酸形式的組成。在本研究我們研究此葉酸形式的重新分配是否會導致同半胱胺酸的再甲基化下降或/且提高嘌呤或胸腺嘧啶的生合成作用；而DNA甲基化及核酸生合成的調控和腫瘤的發生機制有密切關係。我們利用表現不同亞甲基四氫葉酸還原酶多型性的淋巴母細胞來研究同半胱胺酸的再甲基化和嘌呤及胸腺嘧啶的生合成作用及代謝改變情形。在不同的營養狀態下，以特定之穩定同位素分別追蹤代謝路徑，並利用氣相層析質譜儀來測定胞內胺基酸和核苷酸代謝路徑的變化。發現在葉酸狀態充足的情形下亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤的生合成作用其相對流量顯著較正常型為高；推測可能是因為可被利用的甲醯化型式的葉酸增加的緣故。 亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤生合成作用的優勢或許可以解釋何以在某些流行病學研究顯示此種基因型可降低某些癌症發生的風險，即此種基因型對於某些癌症發生具保護作用，但在其他的情形下如葉酸營養狀態不理想時，反而觀察到其可能有增加癌症發生風險。我們建立細胞模型研究人類亞甲基四氫葉酸還原酶基因變異和癌症發生及與營養狀況交互作用的相關代謝途徑。

Genetic predisposition plays a significant role in tumorigenesis and cancer prevention. A common polymorphism 677C->T in methylenetetrahydrofolate reductase (MTHFR) is associated with altered risk of certain types of cancer, but the mechanism remains to be determined. MTHFR catalyzes the reduction of 5, 10-methylene- tetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the synthesis of methionine from homocysteine. The homozygous C677T mutation in the MTHFR gene results in reduction in enzyme activity and changes in cellular distribution of folates. In the present study we investigate if the folate form redistribution results in compromised homocysteine remethylation and elevated de novo purine synthesis, both of which may be potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were studied in Epstein-Barr virus transformed lymphoblasts expressing common polymorphic allele 677 C and 677T of the MTHFR gene. Using different stable isotopic tracers, specific enrichments in intracellular amino acid pool and nucleotides were measured by GCMS under various nutritional conditions. Our preliminary data indicated that when cellular folate was adequate, the MTHFR genotype did not affect the folate dependent homocysteine remethylation pathways. Only when cells became folate deficient, the folate dependent remethylation pathway in TT genotype was significantly reduced compared to the CC genotypes. Using 13C-serine as the tracer, de novo purine synthesis in TT genotype was found to be elevated when folate status is adequate, presumably due to increased availability of formylated folates. When folate was restricted, no difference in purine synthesis was found between the 2 genotypes, suggesting that the advantage in purine synthesis only exists when folate supply is adequate. Our data suggest that the impacts of MTHFR 677T polymorphism are closely related to nutritional conditions, and such alterations may modulate metabolic pathways involved in disease onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in certain type of cancers. These transformed cells are potential models for studying the consequences of human genetic variation in MTHFR and their relationship to pathways involved in cancer pathogenesis or progression. 基因的遺傳多型性對於腫瘤的發生和預防扮演一個重要的角色。亞甲基四氫葉酸還原酶的多型性和某些種類癌症發生的風險率有關，但是其機制仍然需要進一步的研究。已知亞甲基四氫葉酸還原酶多型性中TT同型合子的會導致酵素活性的下降並且改變細胞內葉酸形式的組成。在本研究我們研究此葉酸形式的重新分配是否會導致同半胱胺酸的再甲基化下降或/且提高嘌呤或胸腺嘧啶的生合成作用；而DNA甲基化及核酸生合成的調控和腫瘤的發生機制有密切關係。我們利用表現不同亞甲基四氫葉酸還原酶多型性的淋巴母細胞來研究同半胱胺酸的再甲基化和嘌呤及胸腺嘧啶的生合成作用及代謝改變情形。在不同的營養狀態下，以特定之穩定同位素分別追蹤代謝路徑，並利用氣相層析質譜儀來測定胞內胺基酸和核苷酸代謝路徑的變化。發現在葉酸狀態充足的情形下亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤的生合成作用其相對流量顯著較正常型為高；推測可能是因為可被利用的甲醯化型式的葉酸增加的緣故。 亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤生合成作用的優勢或許可以解釋何以在某些流行病學研究顯示此種基因型可降低某些癌症發生的風險，即此種基因型對於某些癌症發生具保護作用，但在其他的情形下如葉酸營養狀態不理想時，反而觀察到其可能有增加癌症發生風險。我們建立細胞模型研究人類亞甲基四氫葉酸還原酶基因變異和癌症發生及與營養狀況交互作用的相關代謝途徑。

Aberrant metabolism and methylation are important risk factors for cancer occurrence and pro-gression. Transmethylation enzyme glycine N-methyltransferase (GNMT) and methionine ade-nosyltransferases (MATs), essential for homeostasis of the universal methyl donor S-adenosylmethionine (adoMet), are commonly diminished in human hepatocellular carcinoma (HCC). We performed comprehensive analyses using the Cancer Genome Atlas (TCGA) and discovered that HCC patients with defected MAT1A and GNMT expressions have poor survival. Using Gene Set Enrichment Analysis (GSEA), a novel pathway, REACTOME_TRANSLATION gene set, was identified in those with defected MAT1A and GNMT by the Venn Diagram. Cell models expressing various levels of MAT1A and GNMT demonstrated that expression of MAT1A and GNMT is associated with decreased cell proliferation and REAC-TOME_TRANSLATION genes, consistent with the better HCC prognosis. We demonstrated new findings that downregulation of MAT1A and GNMT enriches protein-associated translation process that may account for poor HCC prognosis.
異常代謝和甲基化是癌症發生和發展的重要危險因子。轉甲基酶甘氨酸 N-甲基轉移酶 (GNMT) 和甲硫氨酸腺苷轉移酶 (MAT) 對通用甲基供體 S-腺苷甲硫氨酸 (adoMet) 的穩態至關重要，此二個酵素在人類肝細胞癌 (HCC) 中通常會表現異常或減低。我們使用癌症基因組圖譜 進行綜合分析，發現具有 MAT1A 和 GNMT 表達缺陷的人類肝癌患者之存活率較差。利用基因集富集分析及維恩圖在 MAT1A 和 GNMT 缺陷者找出一新途徑: REACTOME_TRANSLATION 基因集。我們接著利用多株表達高低不同之 MAT1A 和 GNMT 細胞模型式發現, 正常 MAT1A 和 GNMT 表現與減少細胞增殖和 REACTOME_TRANSLATION 基因集連的降低有關，此與更好的肝癌預後一致。本研究展示新發現，即 MAT1A 和 GNMT 的下調會促進REACTOME_TRANSLATION 基因集，認為可能是MAT1A 和 GNMT 缺陷者 肝癌預後不良的原因之一。

(1) Background: Tumor hypoxia leads to metastasis and certain immune responses, and interferes with normal biological functions. It also affects glucose intake, down-regulates oxidative phosphorylation, and inhibits fatty-acid desaturation regulated by hypoxia-inducible factor 1α (HIF-1α). Although tumor hypoxia has been found to promote tumor metastasis, the roles of HIF-1α-regulated genes and their application are not completely integrated in clinical practice. (2) Methods: We examined the correlation between HIF-1α, metadherin (MTDH), and interleukin (IL)-10 mRNA expression, as well as their expression patterns in the prognosis of breast cancer using the Gene Expression Profiling Interactive Analysis (GEPIA) databases via a web interface; tissue microarrays (TMAs) were stained for MTDH and IL-10 protein expression using immunohistochemistry. (3) Results: HIF-1α, MTDH, and IL-10 mRNA expression are highly correlated and strongly associated with poor prognosis. MTDH and IL-10 protein expression of breast cancer patients usually harbored negative estrogen receptor (ER) or progesterone receptor (PR) status, and late-stage tumors have higher IL-10 expression. With regard to MTDH and IL-10 protein expression status for using univariate and multivariate analysis, the results showed that the protein expression of MTDH and IL-10 in ER-negative or PR-negative breast cancer patients have the worse prognosis. (4) Conclusions: we propose a new insight into hypoxia tumors in the metabolism and immune evidence for breast cancer therapy.
(1) 背景：腫瘤缺氧會導致轉移和某些免疫反應，並干擾正常的生物學功能。還會影響葡萄糖的攝入，下游調控氧化磷酸化，並抑制由缺氧誘導因子 1α (HIF-1α) 調節的脂肪酸去飽和。儘管已經發現腫瘤缺氧會促進腫瘤轉移，但是 HIF-1α 調控基因的作用及其應用尚未完全整合到臨床實踐中。(2) 方法：本研究使用基因表達譜交互式分析 (GEPIA) 藉由網絡界面的數據庫，確認 HIF-1α，間質素 (MTDH) 和白介素 (IL)-10 mRNA 表達之間的相關性，以及它們在乳腺癌預後中的表達模式；使用免疫組織化學對組織微陣列 (TMA) 的 MTDH 和 IL-10 蛋白表達進行染色。(3) 結果：HIF-1α、MTDH 及 IL-10 mRNA 表現高度相關性，並與不良預後密切相關。乳腺癌患者的 MTDH 和 IL-10 蛋白表達通常具有雌激素受體 (ER) 或孕激素受體 (PR) 陰性，晚期腫瘤的 IL-10 表達較高。對於單因素和多因素分析的 MTDH 和 IL-10 蛋白表達狀態，結果表明 ER 陰性或 PR 陰性乳腺癌患者 MTDH 和 IL-10 的蛋白表達預後較差。(4) 結論：本研究提出了對缺氧腫瘤的新見解，為乳腺癌治療提供了新陳代謝和免疫證據。

甲硫(1)Background: methionine cycle is not only essential for cancer cell proliferation but is also critical for metabolic reprogramming, a cancer hallmark. Hepatic and extrahepatic tissues methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A that catalyze the formation of S-adenosylmethionine (SAM), the principal biological methyl donor. Glycine N-methyltransferase (GNMT) further utilizes SAM for sarcosine formation thus it regulates the ratio of SAM:S-adenosylhomocysteine (SAH). (2) Methods: by analyzing the TCGA/GTEx da-tasets available within GEPIA2, we discovered that breast cancer patients with higher MAT2A had worse survival rate (P=0.0057). Protein expression pattern of MAT1AA, MAT2A and GNMT were investigated in the tissue microarray in our own cohort (n=252) by immunohistochemistry. MAT2A C/N expression ratio and cell invasion activity were further investigated in a panel of breast cancer cell-lines. (3) Results: GNMT and MAT1A were detected in the cytoplasm, whereas MAT2A showed both cytoplasmic and nuclear immunoreactivity. Neither GNMT nor MAT1A protein expression was associated with patient survival rate in our cohort. Kaplan-Meier survival curves showed that a higher cytoplasmic/nuclear (C/N) MAT2A protein expression ratio correlated with poor overall survival (5-year survival rate: 93.7% vs. 83.3%, C/N ratio ≥1.0 vs. C/N ratio <1.0, log-rank p=0.004). Accordingly, a MAT2A C/N expression ratio ≥1.0 was determined as an in-dependent risk factor by Cox regression analysis (hazard ratio = 2.771, P = 0.018, n=252). In vitro studies found that breast cancer cell-lines with a higher MAT2A C/N ratio were more invasive. (4) Conclusions: the subcellular localization of MAT2A may affect its functions, and elevated MAT2A C/N ratio in breast cancer cells is associated with in-creased invasiveness. MAT2A C/N expression ratio determined by IHC staining could serve as a novel independent prognostic marker for breast cancer. 甲硫氨酸循環不僅是細胞增殖所必需，且對代謝重編程亦至關重要。肝和肝外組織甲硫氨酸腺苷基轉移酶（MATs）是兩個基因MAT1A和MAT2A的產物，催化主要的生物甲基供體S-腺苷甲硫氨酸生成。甘氨酸N-甲基轉移酶（GNMT）進一步利用S-腺苷甲硫氨酸形成肌氨酸，因此可調節SAM：S-腺苷同型半胱氨酸（SAH）的比例。我們通過分析GEPIA2中可用的TCGA / GTEx數據集，發現MAT2A較高的乳腺癌患者的生存率較差（P = 0.0057)。通過免疫組織化學在隊列 (Cohort) 研究 (n = 252) 組織芯片中研究MAT1A，MAT2A和GNMT之蛋白表達情況。並探究多種乳腺癌細胞株中MAT2A細胞質與細胞核的分佈情況(C/N)和細胞侵襲性。在病患癌組織切片中檢測到GNMT和MAT1A主要位於細胞質，而抗體偵測到在細胞質和核均有表達MAT2A。GNMT和MAT1A蛋白的表達均與乳癌患者生存率無顯著相關。Kaplan-Meier生存曲線表明，病患有較高的胞質/核MAT2A蛋白表達比(C/N)其總體生存率較差（5年生存率：93.7％對83.3％, C/N比≥1.0對C / N比<1.0, p= 0.004)。通過 Cox 回歸分析確定MAT2A C/N表達比≥1.0為獨立危險因子(危險比= 2.771，P = 0.018，n = 252）。細胞研究發現，具較高MAT2A C/N比的乳腺癌細胞株具更高的侵襲性。本研究發現MAT2A的細胞定位分佈可能影響其功能，且乳腺癌細胞中MAT2A C / N比升高與侵襲力增加有關。通過病患缺片染色MAT2A C/N表達比率可作為乳腺癌的一種新的獨立預後標誌物。

RNA-Sequencing (RNA-Seq), the most commonly used sequencing application tool, is not only a method for measuring gene expression but also an excellent media to detect important structural variants such as single nucleotide variants (SNVs), insertion/deletion (Indels), or fusion transcripts. The Cancer Genome Atlas (TCGA) contains genomic data from a variety of cancer types and also provides the raw data generated by TCGA consortium. p53 is among the top 10 somatic mutations associated with hepatocellular carcinoma (HCC). The aim of the present study was to analyze concordant different gene profiles and the priori defined set of genes based on p53 mutation status in HCC using RNA-Seq data. In the study, expression profile of 11 799 genes on 42 paired tumor and adjacent normal tissues was collected, processed, and further stratified by the mutated versus normal p53 expression. Furthermore, we used a knowledge-based approach Gene Set Enrichment Analysis (GSEA) to compare between normal and p53 mutation gene expression profiles. The statistical significance (nominal P value) of the enrichment score (ES) genes was calculated. The ranked gene list that reflects differential expression between p53 wild-type and mutant genotypes was then mapped to metabolic process by KEGG, an encyclopedia of genes and genomes to assign functional meanings. These approaches enable us to identify pathways and potential target gene/pathways that are highly expressed in p53 mutated HCC. Our analysis revealed 2 genes, the hexokinase 2 (HK2) and Enolase 1 (ENO1), were conspicuous of red pixel in the heatmap. To further explore the role of these genes in HCC, the overall survival plots by Kaplan-Meier method were performed for HK2 and ENO1 that revealed high HK2 and ENO1 expression in patients with HCC have poor prognosis. These results suggested that these glycolysis genes are associated with mutated-p53 in HCC that may contribute to poor prognosis. In this proof-of-concept study, we proposed an approach for identifying novel potential therapeutic targets in human HCC with mutated p53. These approaches can take advantage of the massive next-generation sequencing (NGS) data generated worldwide and make more out of it by exploring new potential therapeutic targets.
癌症基因組圖譜（TCGA）包含來自多種癌症類型的基因組數據。 p53是與肝細胞癌(HCC) 相關的十大體細胞突變之一。本研究使用 RNA-Seq 數據分析肝細胞癌中 p53 突變狀態。這項研究收集42對配對的腫瘤和鄰近正常組織中的11799個基因的表達譜，將突變後的p53表達與正常p53表達進一步進行了分層。此外使用基因集富集分析（GSEA）來比較正常和 p53突變基因的表達譜。計算了富集得分（ES）基因的統計顯著性。然後通過 KEGG 將 p53 野生型和突變型基因型之間差異表達的排名基因列表與代謝路徑映射比對，賦予其功能含義。這些方法使我們能夠鑑定在 p53 突變的 HCC 中高度表達的途徑和潛在的靶基因/途徑。我們的分析顯示，己糖激酶 2（HK2）和烯醇酶1（ENO1）2個基因在熱圖中顯著紅色像素。接著進一步通過 Kaplan-Meier 對 HK2 和 ENO1 進行生存分析，顯示 HK2 和 ENO1 的高表達HCC 患者其預後較差。這些結果表明，這些糖酵解基因與 HCC 中的 p53突變有關，可能導致不良預後。在這項概念驗證研究中，我們提出了一種在突變的 p53人類 HCC中鑑定新的潛在治療靶標的方法。這些方法可以利用全球範圍內產生的大量下一代測序（NGS）數據，並通過探索新的潛在治療靶點來進一步利用在開發新的治療策略。