Downregulation of miR-150 Expression by DNA ... - ACS Publications

Sep 20, 2016 - Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p ... Furthermore, hypermethylation of miR-150 promoter was detected i...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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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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