Comparative Analysis of Long Noncoding RNAs Expressed During

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Omics Technologies Applied to Agriculture and Food

Comparative Analysis of Long Noncoding RNAs Expressed During Intramuscular Adipocytes Adipogenesis in Fat-type and Lean-type Pigs Yunmei Sun, Xiaochang Chen, Jin Qin, Shuge Liu, Rui Zhao, Taiyong Yu, Guiyan Chu, Gongshe Yang, and Pang Wei-jun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04243 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Journal of Agricultural and Food Chemistry

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Comparative Analysis of Long Noncoding RNAs Expressed During Intramuscular

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Adipocytes Adipogenesis in Fat-type and Lean-type Pigs

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Yunmei Sun#, Xiaochang Chen#, Jin Qin, Shuge Liu, Rui Zhao, Taiyong Yu, Guiyan Chu,

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Gongshe Yang*, Weijun Pang*

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Laboratory of Animal Fat Deposition & Muscle Development, College of Animal Science

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and Technology, Northwest A&F University, Yangling Shaanxi 712100, China.

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#These

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* Correspondence:

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Weijun Pang, Ph.D.

authors contributed equally to this work.

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Address: No. 22 Xinong Road, Yangling, Shaanxi Province 712100, China

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Tel: 86-29-87091017

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Fax: 86-29-87092430

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E-mail: [email protected]

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Gongshe Yang, Ph.D.

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Address: No. 22 Xinong Road, Yangling, Shaanxi Province 712100, China

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Tel: 86-29-87091017

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Fax: 86-29-87092312

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: The meat quality of local breed pigs is more tender and juicier than the

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imported varieties. The important reason is that the intramuscular fat content is high. Even

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though modest sequence conservation and evolution, the expression pattern and function

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of long non-coding RNAs (lncRNAs) seem to be conserved. In spite of that, analysis of

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lncRNAs associated with intramuscular fat development remains unknown to us in porcine.

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Here, we systematically investigated lncRNAs of intramuscular adipocytes of fat local

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Bamei pig and lean Large White pig to consider the function of lncRNAs on intramuscular

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fat development. We selected three piglets of both breeds separately to isolate

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intramuscular preadipocytes and performed RNA sequencing across four stages (0, 2, 4

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and 8 d) during the intramuscular preadipocytes differentiation, and identified 1932

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lncRNAs (760 novel). In addition, we have screened lnc_000414 closely related to fat

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synthesis. This lncRNA function as an inhibitor in the proliferation of porcine

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intramuscular adipocytes. These novel findings will provide new targets for improving

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pork quality and making pig breeding better.

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KEYWORDS: pig, lncRNA-seq, intramuscular adipocyte, adipogenesis, lnc_000414

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INTRODUCTION

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Now, with the improvement of living standards, people pay more attention to meat

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quality. Pork, as a major source of meat, is closely related to human health. Its quality is

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strongly influenced by fat distribution and deposition1. Therefore, how to improve meat

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production and quality becomes an important issue for porcine breeding scientists.

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Coincidently, more and more studies demonstrated that intramuscular fat (IMF) was

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closely connected with the quality of meat, affecting properties such as flavor, water-

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holding capacity and tenderness2. Thus, in the past few years, researches on IMF have

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increased frequently in the field of porcine fat deposition3, suggesting it is essential to study

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candidate genes controlling IMF deposition.

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The regulation processes of proliferation, differentiation and tissue development in

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porcine IMF are actually a result of the interaction between inheritance, epigenetics and

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signaling molecules4-6. And most importantly, long noncoding RNA (lncRNA) plays a

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major role in these processes. LncRNAs is a type of non-coding RNA longer than 200 nt7,

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and previous studies have shown that it could involve in many biological processes,

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including transcription level and post-transcription level, although it has only weak ability

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to encode proteins. They were associated with many life processes, including cell

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proliferation, differentiation and apoptosis by regulating the expression of their target

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genes8-10. There were some evidences demonstrated that lncRNAs play critical roles during

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adipogenesis in vitro as well as in vivo. As a matter of fact, more and more studies have

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demonstrated that lncRNAs could regulate adipogenesis. For instance, PU.1 AS lncRNA



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elevates adipogenic differentiation of preadipocyte through inhibiting the translation of

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PU.1 mRNA in different animal models

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are thought to be potential therapy targets for weight loss, suggesting that the associated

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lncRNAs 11may have an effect on the formation of brown and beige adipocytes, and

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contribute to the treatment of obesity13.

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11,12.

Nevertheless, brown and beige adipocytes

Bamei pig, a Chinese native fat-type pig breed, is known for good meat quality and 14.

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relative high lean meat percentage

Thus, it is a useful model for studying muscle

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development and meat yield of indigenous pig breed. Although, there are also some reports

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on differentially expressed lncRNAs between fat-type pig and lean-type pig breeds15. There

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are also more new lncRNAs to consider in other breeds especially indigenous fat-type pig

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breed. In this study, we obtained the lncRNAs expression variation in preadipocytes

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between Bamei pig and Large White pig at different differentiation stages by lncRNA high-

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throughput sequencing, and aimed to explore the lncRNAs involved in intramuscular fat

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development of Chinese indigenous pigs. Our findings will be useful to expand the

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knowledge of regulating meat quality by lncRNAs and be beneficial for the genetic

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improvement of porcine meat quality traits.

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MATERIALS AND METHODS

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Animals and samples

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The male Chinese native Bamei piglets (three-day-old, 1.2 kg) were purchased from

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Bamei original breeding filed of Huzhu, Gansu, and the male Large White piglets (three-

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day-old, 1.5 kg) were from the experimental station of Northwest A&F University. These


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piglets lived in similar rearing conditions.

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Intramuscular preadipocytes were isolated from longissimus dorsi muscle (LDM) of

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3-day-old piglets as described previously16. In briefly, we digested the muscle tissues by

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Dulbeccos modiﬁed Eagles medium (DMEM; Gibco, Carlsbad, CA) with 0.1%

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collagenase I (270 U/mg; Gibco, Carlsbad, CA) at 37°C for 1.5h. The digest samples were

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sequentially filtered through 70 and 200 mesh filters to separate the cells. Next, we used

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DMEM/F12 medium to wash the cells thrice. Cells were seeded in dishes containing

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DMEM/F12 medium added 10% fetal bovine serum (Gibco, Australia). After 1 h, we

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rinsed off unattached cells with DMEM/F12 medium. When density of cells reaches to

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approximately100 %, they were induced to differentiate with DMEM/F12 containing 10%

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FBS, 5μg/ml (872 nM) insulin, 1 μM dexamethasone and 0.5 mM isobutyl methylxanthine

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(IBMX, Sigma–Aldrich, St. Louis, MO) for 2 days. The medium with 10% FBS and

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5μg/ml insulin was used to retain differentiation. Finally, we harvested the cells at0, 2, 4

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and 8 d for subsequent research.

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In our study, all the experimental procedures with pigs are based on the Experimental

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Animal Manage Committee of Northwest A&F University (2011-31101684).

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RNA isolation, library preparation, and sequencing

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We used Trizol reagent (Invitrogen, USA) to extract the total RNA from all cell

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samples, and the method was followed manufacturer’s instructions. The quality of RNA

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and concentration was detected by the NanoDrop 2000 (Thermo Scientific, Waltham, MA,

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USA). After that, the qualified RNA was stored at -80°C for later use.



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Each cell sample was extracted with 3μg of RNA for preparation of sequencing

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samples. Firstly, we used Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA) and

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ethanol to remove ribosomal RNA and free residue separately Subsequently, sequencing

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libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional

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RNA Library Prep Kit for Illumina® (NEB, USA). Briefly, fragmentation Buffer was

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added to the obtained mRNA to make the fragment into a short fragment. Then, using the

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fragmented mRNA as a template, the first strand of cDNA was synthesized with random

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hexamers and buffer, dNTPs, RNase H and DNA polymerase I synthesizes the second

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strand of cDNA, which is purified by QiaQuick PCR kit and eluted with EB buffer. The

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end-repair, base A is added, the sequencing linker is added, and the fragment of interest is

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recovered by agarose gel electrophoresis, and PCR is performed. Amplification was

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performed to complete the entire library preparation work, and the constructed library was

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sequenced using Illumina HiSeq2000.

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The remaining overhangs were converted to blunt ends by exonuclease/polymerase

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activity. After adenylation at the 3' end of the DNA fragment, a NEBNext adaptor with a

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hairpin loop structure was ligated to prepare for hybridization. The cDNA fragment of 150

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to 200 bp was preferentially selected, and a cDNA fragment library was purified using an

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AMPure XP system (Beckman Coulter, Beverly, USA). The appropriately sized cDNA

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was then ligated at 37 °C for 15 minutes following 95 °C for 5 minutes by USER Enzyme

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(NEB, USA). Subsequently, PCR was performed by using Phusion High-Fidelity DNA

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polymerase, Universal PCR primers and Index (X) Primer.


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Quality control

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Firstly, raw data is measured by each internal script in the fastq format. Moreover,

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with the strict screening criteria, the clean data from raw data were collected.

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Synchronously, the related indicators of clean data were calculated, including Q20, Q30

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and GC content17. And the high quality clean data was used for further downstream

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analyzes.

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Transcriptome assembly

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The Cufflinks (v2.1.1) and Scripture (beta2) were used for assembling the mapped

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reads for samples. In order to determine exons connectivity, two different approaches were

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used to splice reads in these two methods.

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quantify the expression level of the expression data by using a probabilistic model in a

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given locus. And Scripture could distinguish expressed loci and used spliced reads to

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assemble expressed segments though a statistical segmentation model. We used a variety

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of methods to predict the coding ability of transcripts including PFAM database19, Coding-

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Non-Coding Index (CNCI) software20 , and Coding Potential Calculator (CPC)18, if the

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transcript cannot pass the test, it will be excluded. On one hand, all parameters were set as

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default in Scripture analysis, and on another hand, Cufflinks analysis was based on the

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program named ‘min-frags-per-transfrag=0’ and ‘--library-type’. Besides these two

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parameters, other parameters were run with default.

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Quantification of gene expression level

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In briefly, Cufflinks could assemble and

Cuffdiff (v2.1.1) was used to calculate FPKMs of both lncRNAs and coding genes in



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each sample. Gene FPKMs were counted by summing the FPKMs of transcripts in each

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group. FPKM computed the number of fragments in each exon isoform. This was based on

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the length of the fragments and reads count mapped to this fragment.

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Differential expression analysis

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Cuffdiff provided statistical routines for determining differential expression in digital

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transcript or gene expression data using a model which was based on the negative binomial

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distribution. Transcripts were considered to be differentially expressed, if P-adjust

Comparative Analysis of Long Noncoding RNAs Expressed During

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