International Journal of Molecular Sciences Article MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA Ming-Tsung Wu 1,2, Wei-Ting Ye 1, Yi-Cheng Wang 1, Po-Ming Chen 1, Jun-You Liu 1, Chien-Kuo Tai 3 , Feng-Yao Tang 4, Jian-Rong Li 5, Chun-Chi Liu 5 and En-Pei Isabel Chiang 1,6,* 1 Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; [email protected] (M.-T.W.); [email protected] (W.-T.Y.); [email protected] (Y.-C.W.); [email protected] (P.-M.C.); [email protected] (J.-Y.L.) 2 Department of Civil and Environmental Engineering, South Kensington Campus, Imperial College London, London SW7 2AZ, UK 3 Department of Biomedical Sciences, National Chung Cheng University, Chia-Yi 62102, Taiwan; [email protected] 4 Department of Nutrition, China Medical University, Taichung 40402, Taiwan; [email protected] 5 Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 40227, Taiwan; [email protected] (J.-R.L.); [email protected] (C.-C.L.) 6 Innovation and Development Center of Sustainable Agriculture (IDCSA), National Chung Hsing University, Taichung 40227, Taiwan * Correspondence: [email protected] Citation: Wu, M.-T.; Ye, W.-T.; Wang, Y.-C.; Chen, P.-M.; Liu, J.-Y.; Tai, C.-K.; Abstract: Folate depletion causes chromosomal instability by increasing DNA strand breakage, Tang, F.-Y.; Li, J.-R.; Liu, C.-C.; Chiang, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been E.-P.I. MTHFR Knockdown Assists suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) Cell Defense against Folate Depletion through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme Induced Chromosome Segregation catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate and Uracil Misincorporation in DNA. that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon Int. J. Mol. Sci. 2021, 22, 9392. moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains https://doi.org/10.3390/ijms22179392 controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the Academic Editors: Jihane Basbous, present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of Cyril Ribeyre and Antoine Aze liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl Received: 2 July 2021 Accepted: 19 August 2021 group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved Published: 30 August 2021 in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR Publisher’s Note: MDPI stays neutral had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene with regard to jurisdictional claims in expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR published maps and institutional affil- tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly iations. decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = −0.81, p < 0.0001), MTHFR protein expression (r = −0.8; p = 0.01), and Copyright: © 2021 by the authors. MTHFR enzyme activity (r = −0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in Licensee MDPI, Basel, Switzerland. WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion- This article is an open access article induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of distributed under the terms and nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent conditions of the Creative Commons de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear Attribution (CC BY) license (https:// MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA creativecommons.org/licenses/by/ uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell 4.0/). Int. J. Mol. Sci. 2021, 22, 9392. https://doi.org/10.3390/ijms22179392 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9392 2 of 27 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 into the 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. Keywords: folate depletion; MTHFR; hepatocellular carcinoma; nucleotide biosynthesis; uracil misincorporation; micronuclei 1. Introduction The genotoxic consequences of folate insufficiency [1], either through reduced folate consumption or functional folate deficiency due to genetic defects in folate-related genes, contribute to numerous pathological conditions in humans, including cancer [2,3]. 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 [2,4,5]. 1.1. Folate Depletion Causes Chromosome Instability Deoxynucleoside triphosphates (dNTPs) are essential for the replication and main- tenance of genomic stability. Regulation of cellular thymidylate synthesis is essential for DNA replication and genome stability in the nucleus [6]. Impaired de novo thymidylate synthesis due to folate deficiency results in deoxyuridine/uracil misincorporation into DNA and causes genome instability [7,8]. Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Human lymphocytes cultured in folate-deficient media exhibit DNA double strand breaks [9], re- duced DNA repair [9], and micronuclei formation [10–12]. High dietary folate consumption is associated with a lower micronucleus frequency in humans [13]. 1.2. Folate Metabolism, MTHFR and Hepatoma Different forms of folate cofactors are required in the reactions of DNA synthesis and methyl donor supply. Perturbations in folate-dependent methylation pathways have been associated with cancer occurrence [3]. Folate-mediated one-carbon metabolisms have been an important therapeutic target for numerous human diseases [14,15], including HCC [4]. However, the understanding of the repercussions of folate deficiency or supplementation on folate metabolic gene variations in the liver is limited. Methylenetetrahydrofolate reductase (MTHFR, EC 1.7.99.5) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate (methyleneTHF, 5,10-CH2- THF) to 5-methyltetrahydrofolate (methylTHF, 5-CH3THF) that can be inhibited by the universal methyl donor S-adenosylmethionine (SAM) [16]. By controlling folate cofactor distributions, MTHFR can modulate the partitioning of intracellular one-carbon moieties among purine synthesis, thymidine synthesis, and methyl donor supply [5]. Mildly reduced MTHFR is common in many populations due to a polymorphism at bp 677 known as C677T. This common mutation results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro [17] and an altered folate cofactor distribution in red blood cells [18]. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial [19]. In 2013, a meta-analysis paper reported that MTHFR A1298C polymorphism may play a protective role in the carcinogenesis of HCC [20]. Another meta-analysis paper reported in 2014 that MTHFR C677T polymorphism was significantly associated with an increased HCC risk in Asians but not in Caucasians [21], and in 2015 another meta-analysis also reported that MTHFR A1298C polymorphism might be related to an increased risk of HCC in Asians [22]. Int. J. Mol. Sci. 2021, 22, 9392 3 of 27 1.3. Folate Co-Factors vs. the Balance between Methyl Group and Nucleotide Supplies Previous studies indicated that folate cofactors are limited for cytoplasmic folate- dependent reactions and that the synthesis of DNA precursors competes with SAM synthe- sis [23]. Due to distinct expression patterns of tissue-specific metabolic enzymes, metabolic kinetics in response to medications [5,24,25] or under nutritional deprivation [26] may differ among cell types. We previously demonstrated that MTHFR C677T polymorphism increases methotrexate (MTX) sensitivity via the inhibition of SAM and de novo purine synthesis [5]. We also demonstrated that lymphoblast is more sensitive to antifolate drug MTX in cell proliferation, protein, and thymidine synthesis,