Downregulation of miR-150 Expression by DNA Hypermethylation Is

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Down-regulation of miR-150 expression by DNA hypermethylation is associated with high HMB-induced hepatic cholesterol accumulation in nursery piglets Yimin Jia, Mingfa Ling, Luchu Zhang, Shuxia Jiang, Yusheng Sha, and Ruqian Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03615 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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Journal of Agricultural and Food Chemistry

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Down-regulation of miR-150 expression by DNA hypermethylation is associated

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with high HMB-induced hepatic cholesterol accumulation in nursery piglets

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Running title: miRNA regulates hepatic cholesterol accumulation

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Yimin Jia†, Mingfa Ling†, Luchu Zhang†, Shuxia Jiang†, Yusheng Sha‡, Ruqian

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Zhao†,§,*

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†

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Medicine, Nanjing Agricultural University, Nanjing 210095, P. R. China.

Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary

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§

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and Safety Control, Nanjing 210095, P. R. China

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‡

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China.

Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality

China Feed Industry Association, Ministry of Agriculture, Peking 100125, P. R.

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*Corresponding author, email: [email protected] Tel. 00862584395047 Fax:

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00862584398669

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The authors declare no conflict of interest.


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Abstract：：

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Excess 2-hydroxy-(4-methylthio) butanoic acid (HMB) supplementation induces

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hyperhomocysteinemia which contributes to hepatic cholesterol accumulation.

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However, it is unclear whether and how high level of HMB breaks hepatic cholesterol

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homeostasis in nursery piglets. In this study, HMB over-supplementation suppressed

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the food intake and decreased the bodyweight in nursery piglets.

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Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in

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HMB groups. Accordantly, HMB significantly increased the protein content of

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3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine

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N-methyltransferase (GNMT), but decreased that of acyl-coenzyme A:cholesterol

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acyltransferase-1 (ACAT1). Significant down-regulation of miR-150, miR-181d-5p

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and miR-296-3p targeting the 3’UTRs of GNMT and HMGCR was detected in the

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liver of HMB-treated piglets and their functional validation was confirmed by

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dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter

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was detected in association with suppressed miR-150 expression in the livers of

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HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol

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un-homeostasis by dietary methyl donor supplementation.

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Key words: cholesterol, DNA methylation, HMB, microRNA, nursery piglets



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Introduction

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DL-2-hydroxy-(4-methylthio) butanoic acid (HMB) has been widely used in the

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animal feed industry as a substitute for methionine; HMB is easily absorbed in the

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intestinal tract and transported into the liver, where HMB is converted into

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L-methionine through two key enzymes - D-2-hydroxy acid dehydrogenase

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(D-HADH) 1 and L-2-hydroxy acid oxidase (L-HAOX) 2. It has been reported that

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excessive intake of methionine causes various toxic changes including suppression of

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feed intake and growth 3, 4, methemoglobin accumulation 5 and hemolytic anemia 6 in

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pigs, ducks, cats and rats. Also, excess HMB supplementation induces

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hyperhomocysteinemia which contributes to hepatic cholesterol accumulation 7, and is

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associated with non-alcoholic fatty liver disease 8, however, it is unclear whether and

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how a high level of HMB affects hepatic cholesterol homeostasis in nursery piglets.

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The liver is the critical organ for methionine metabolism, which is also called one

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carbon metabolism. In this biochemical process, methionine can be converted into

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S-Adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and homocysteine

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(Hcy) by three key enzymes - methionine adenosyltransferase II, beta (MAT2b),

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glycine N-methyltransferase (GNMT) and adenosylhomocysteinase-like 1

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(AHCYL1) ,respectively. Finally, HCY is catalyzed to generate methionine by

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betaine-homocysteine S-methyltransferase (BHMT). Much evidence indicates that

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hypermethioninemia appears to be responsible for elevated levels of plasma

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homocysteine in humans 9 and in mice 10. Plasma homocysteinemia is positively

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correlated with cholesterol in blood, which leads to cardiovascular and


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cerebrovascular disorders 11, 12.

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It was mentioned that homocysteine regulates gene expression at transcriptional levels.

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Both cystathionine -synthase deficiency 13 and high methionine supplementation 7, 14

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induce hyperhomocysteinemia (Hhcy) in mice, which causes hepatic cholesterol

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accumulation by enhancing 3-hydroxy-3-methylglutaryl coenzyme A reductase

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(HMGCR) gene expression. Also, sterol regulatory element binding proteins (SREBPs)

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have been found to be involved in the up-regulation of HMGCR expression 7, 13.

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Moreover, Hhcy increases DNA methylation level in the promoters of the

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ATP-binding cassette transporter A1 (ABCA1) and decreases acyl-coenzyme

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A:cholesterol acyltransferase-1 (ACAT1) gene expression, which causes cholesterol

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accumulation in monocyte-derived foam cells 15.

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Mature microRNAs (miRNAs) contain about 22 nucleotides and are involved in

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posttranscriptional regulation. The microRNA family miR33 is verified to target the

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3’UTR of SREBP and ABCA1 genes to repress cholesterol synthesis and efflux 16, 17,

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while miR122 and miR 21 inhibition cause a significant decrease of HMGCR

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expression involved in cholesterol synthesis 18, 19. Similar to RNA, miRNA locates in

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intergenic regions or embeds within introns of protein-coding genes. Each

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pre-miRNA has its own transcriptional start site and promoter. However, it is unclear

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whether Hhcy is involved in the regulation of hepatic cholesterol metabolism through

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DNA methylation in the promoter of miRNA.

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In this study, we have aimed to first clarify whether excess HMB supplementation

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was toxic to nursery piglets through hepatic cholesterol accumulation. We wanted to



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also identify which kinds of miRNAs were involved in hepatic cholesterol

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metabolism. Finally, we wished to explain how Hhcy impacts miRNA expression

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through DNA methylation.

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Materials and methods

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Materials

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Plasma total cholesterol (Tch, Osaka, Japan), LDL-cholesterol (LDL-c, Osaka, Japan),

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HDL-cholesterol (HDL-c, Osaka, Japan) assay kits were purchased from Wako Pure

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Chemical Industries, Ltd. and homocysteine (Hcy, Chengdu, China) assay kit was

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purchased from Sichuan Maker Biotechnology Co., Ltd. A Poly (A) tailing kit, a

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Pierce BCA Protein Assay kit and the SuperSignal West Dura Substrate Extended

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Duration Substrate were purchased from Thermo Fisher Scientific (Waltham, MA).

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S-Adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), NH4H2PO4 and

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1-heptanesulfonic acid sodium salt were obtained from Sigma (St. Louis, MO).

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Methanol was purchased from Fisher (South San Francisco, CA). TRIzol (Shanghai,

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China) was obtained from Pufei Biotech Co., Ltd. and the HiScript II 1st Strand

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cDNA Synthesis Kit (Nanjing, China) was purchased from Vazyme Biotech Co., Ltd.

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Protein A/G agarose beads were purchased from Santa Cruz (Santa Cruz, CA). The

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pmirGLO Dual-Luciferase miRNA Target Expression Vector was purchased from

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Promega (Madison, WI). The vacutainer tubes were purchased from Jiangsu Yuli

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Medical Instrument Co., Ltd (Taizhou, China). Antibodies were used as follows:

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BHMT, MAT2B, GNMT, AHCYL1 and LDLR (Proteintech, Wuhan, China);

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HMGCR, CYP27A1, ACAT1, DNMT3a and DNMT3b (Bioworld Technology,


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Nanjing, China) ; CYP7A1 and 5-methyl cytidine (Abcam, Cambridge, UK),

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SREBP2 (Santa Cruz, Santa Cruz, CA); β-actin-HRP (KangChen Bio-techs, Inc.,

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Shanghai, China). Secondary anti-mouse and anti-rabbit antibodies were obtained

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from Bioworld Technology.

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Animals and samples

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The animal experiment was performed at the Meilin NO. 21 farm, Xinghua, Jiangsu

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Province, China. Seventy-two Duroc×Landrace×Yorkshire crossbred weaned piglets

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aged from 30-35d were randomly divided into two groups. Each group had three

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replicates with twelve nursery piglets per pen. One group was fed a basal diet (Table 1)

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substituting 0.14% 2-hydroxy-4-methylthiobutyrate (HMB) with 0.12% methionine;

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the other group was fed the diet supplied with 2.05% HMB. The HMB (Rhodimet®

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AT88, Nanjing, China) was a gift from Bluestar Adisseo Company in Nanjing,

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Jiangsu, China. After one week of adaption, piglets were fed one-quarter of the daily

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meal at 08:00, 12:00, 16:00 and 20:00 h for one month, respectively; the daily feed

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intake was recorded as previously reported 20. Two piglets of the mean body weight of

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the replicate were selected from each pen and fasted overnight before slaughter; they

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received only water. Blood samples were collected from the jugular veins of the

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piglets by using vacutainer tubes containing heparin. After collection, the tubes were

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gently mixed by inverting 8-10 times. The tubes were centrifuged at 3,500 rpm for 20

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min, and then the separated plasma were stored at -20°C, and liver samples were

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snap-frozen in liquid nitrogen before being stored at -80°C. Livers were fixed in 4%

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paraformldehyde and embedded in paraffin. Histological analysis was determined by



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staining with hematoxylin and eosin (HE).

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The experimental protocol was approved by the Animal Ethics Committee of Nanjing

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Agricultural University, with the project number 2012CB124703. The slaughter and

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sampling procedures complied with the “Guidelines on Ethical Treatment of

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Experimental Animals” (2006) No. 398 set by the Ministry of Science and

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Technology, China.

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Determination of plasma concentrations of cholesterol, free amino acids and

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homocysteine and hepatic contents of total and free cholesterol

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Plasma total cholesterol, LDL-c and HDL-c were measured using the commercial kits

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by an automated chemiluminescence technique (Tokyo, Japan). One milliliter of each

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plasma sample was mixed with 1mL of 1.5 mol/L perchloric acid; the mixtures were

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vigorously shaken to remove protein. After resting for 10 min, the samples were

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centrifuged at 3000 g for 10 min, then the supernatants were transferred to other tubes

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by adding 0.5 mL 2 mol/L K2CO3 for neutralization. Free amino acids were

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determined in duplicate with an automatic amino acid analyzer (Tokyo, Japan).

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Hepatic total and free cholesterol concentration was determined by using commercial

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cholesterol assay kits purchased from Applygen Technologies Inc., China (Beijing,

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China) following the manufacturer’s instructions.

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Measurement of S-Adenosylmethionine and S-adenosylhomocysteinelevels in

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plasma and liver

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Plasma and hepatic SAM and SAH were determined according to previous published

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research with some modifications 21. Plasma and tissue extracts were prepared using


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0.4 mol/L perchloric acid (1:1, v/v), and then the extracts were centrifuged at 10,000

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rpm for 20 min at 4°C. The supernatants were neutralized with 2 mmol/L KOH and

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filtered through 0.22 µm membrane filters. Ultra performance liquid chromatography

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(UPLC) was performed with a reverse-phase column (ACQUITYUPLC BEH C18 1.7

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µm 2.1x150 mm, Waters, Milford, MA). The column temperature was set at 35°C and

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the wavelength UV monitor was set at 254 nm. A mobile phase (pH 3.0) was used

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that consisted of 40 mmol/L NH4H2PO4, 8 mmol/L 1-heptanesulfonic acid sodium salt

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and 18% methanol; the flow rate was maintained at 0.61 mL/min by an ACQUITY

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UPLC H-Class system (Waters, Milford, MA). Calibration curves were prepared by a

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six-point standard (1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 µmol/L) of SAM and SAH

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mixture in 0.4 mmol/L perchloric acid.

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Real-time RT-PCR for mRNA quantification

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Total RNA was isolated from liver samples using TRIzol Reagent according to the

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manufacturer’s instructions and reverse transcribed with the HiScript II 1st Strand

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cDNA Synthesis Kit. Two microliters of diluted cDNA (1:20) were used in each

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real-time PCR assay by Mx3000P (Stratagene, Santa Clara, CA). Peptidylprolyl

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isomerase A (PPIA) was chosen as a reference gene, because it is expressed in

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abundance comparable to the genes of interest and because its expression was not

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affected by the treatment. All primers were synthesized by Genewiz, Inc. (Suzhou,

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China) and listed in Table 2.

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Western Blotting for protein quantification

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Liver samples were homogenized at 4°C in a 50 mmol/L Tris-HCl buffer (pH 7.4)



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containing 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and

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protease inhibitor cocktail (Roche, San Francisco, CA) using a beads cell disrupter

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(TOMY, Tokyo, Japan). A Pierce BCA Protein Assay kit was used to determine the

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protein concentration. Forty micrograms of protein were used for electrophoresis on a

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10% or 7.5% SDS-PAGE gel. Western-blot analysis was carried out according to

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manual instructions provided by the primary antibody suppliers. Polyclonal antibodies

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against BHMT (diluted 1:1000), MAT2B (diluted 1:1000) , GNMT (diluted 1:1000),

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AHCYL1 (diluted 1:1000), HMGCR (diluted 1:1000), CYP7A1 (diluted 1:200),

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CYP27A1 (diluted 1:200), LDLR (diluted 1:1000), ACAT1 (diluted 1:1000),

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SREBP2 (diluted 1:200), DNMT3a (diluted 1:1000), DNMT3b (diluted 1:1000), were

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used in western blot analysis, and β-actin-HRP (diluted 1:10000) was selected as a

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loading control.

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MicroRNA expression assay

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miRNA analysis was performed according to our previous publication with some

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modifications 22. Six micrograms of total RNA were polyadenylated by poly (A)

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polymerase using a poly (A) tailing kit according to the manufacturer's instructions.

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Polyadenylated RNA was then dissolved and reverse transcribed using a poly (T)

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adapter. Real-time PCR was performed in duplicate with a miRNA-specific forward

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primer and a universal reverse primer complementary to part of the poly(T) adapter

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sequence. A random DNA oligonucleotide was added to total RNA samples before

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polyadenylation as an exogenous reference to normalize miRNA expression. The

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sequences of all the porcine miRNAs were acquired from miRBase


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(http://www.mirbase.org/). The miRNAs targeting the 3’UTR of GNMT, HMGCR and

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ACAT1 were predicted with an online miRNA prediction tool 23. All the predicted

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miRNAs were quantitated by real-time PCR. The primer sequences used for miRNA

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analysis are listed in Table 2.

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Functional assay for microRNA target identification and validation

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Genomic sequences of porcine miR-181d -5p, miR-150, miR-296-3p and miR-222

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precursors (Table 2) were synthesized and inserted into pSilencer 3.0-H1 small

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interfering RNA expression vectors according to the manual instructions. The 3’UTR

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sequences of porcine GNMT and HMGCR genes containing conserved motifs for

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miR-181d -5p, miR-150, miR-296-3p and miR-222 were amplified by LA Taq (Takara,

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Dalian, China). The PCR products were then inserted into the downstream fragment

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of the pmirGLO Dual-Luciferase miRNA Target Expression Vector.

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HEK293T cells were cultured in DMEM containing 10% fetal bovine serum at 37°C

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in 5% CO2 incubator and co-transfected with 180 ng pmir-GLO /GNMT plasmid and

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100 ng pSilencer 3.1-H1 neo miR-181d -5p, miR-150, miR-296-3p and mirR-222

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plasmid. The luciferase activity was detected by using the GloMax® 96 Microplate

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Luminometer (Promega, Madison, WI), according to the manufacturer’s instructions

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at 24h after transfection.

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Slot blot analysis for whole-genome DNA methylation

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Slot blot analysis for hepatic whole-genome DNA methylation was performed as

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previously described with some modifications 24. Forty micrograms of sonicated DNA

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samples (~1000 bp) were heated in order to separate into single-strained DNA and



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raised to 200 µL with distilled water; the unused slots were filled with 200 µL

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distilled water, and then drawn by vacuum and immobilized by heating at 80°C for 1h.

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The membrane was incubated in Tris–HCl buffered saline containing Tween-20

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(0.025 M Tris–HCl, 0.15 M NaCl, pH 7.6, 0.05% Tween-20, TBST) and 5% skim

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milk for 2h, then incubated overnight at 4°C with a TBST buffer containing 5% skim

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milk and 5-methyl cytidine (5mC, diluted 1:500) antibody. The membrane was

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washed three times with TBST and the blot was processed using the Supersignal West

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Dura Extended Duration Substrate according to the manufacturer’s instructions.

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Methylated DNA immunoprecipitation assay

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Methylated DNA immunoprecipitation (MeDIP) analysis was performed as

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previously described 22. DNA isolated from liver was sonicated into small fragments

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(~500 bp) by the sonicator (Sonics & Materials, Inc., Newtown, CT). Two

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micrograms of fragmented DNA were heated to separate into single-strained DNA.

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MeDIP was performed by using a mouse monoclonal antibody against 5mC and

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normal mouse IgG. The DNA-antibody complex was captured with protein A/G

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agarose beads, then digested by protein K. Finally, the immunoprecipitated DNA was

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diluted in 30 µL ddH2O. Normal IgG captured DNA was used as a negative control

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and a portion of the denatured DNA was used as input DNA. The primers were

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designed according to the sequences in the CpG islands of the miRNAs’ promoter

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(Table 2).

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Statistical analysis

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Data are presented as means ± SEM. Student’s t-test was used to compare the


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difference between two groups by SPSS 19.0. Results from relative quantifications of

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mRNA and protein were presented as the fold change relative to the mean value of the

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control group. The differences were considered statistically significant when P

Downregulation of miR-150 Expression by DNA Hypermethylation Is

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