Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS)
- Jian Huang†1,
- Pei Hao†2, 3,
- Yun-Li Zhang†1,
- Fu-Xing Deng1,
- Qing Deng1,
- Yi Hong5,
- Xiao-Wo Wang6,
- Yun Wang1,
- Ting-Ting Li6,
- Xue-Gong Zhang6,
- Yi-Xue Li2, 4,
- Peng-Yuan Yang1Email author,
- Hong-Yang Wang5Email author and
- Ze-Guang Han1Email author
© Huang et al; licensee BioMed Central Ltd. 2007
Received: 20 November 2006
Accepted: 02 July 2007
Published: 02 July 2007
The liver is the largest human internal organ – it is composed of multiple cell types and plays a vital role in fulfilling the body's metabolic needs and maintaining homeostasis. Of these cell types the hepatocytes, which account for three-quarters of the liver's volume, perform its main functions. To discover the molecular basis of hepatocyte function, we employed Massively Parallel Signature Sequencing (MPSS) to determine the transcriptomic profile of adult human hepatocytes obtained by laser capture microdissection (LCM).
10,279 UniGene clusters, representing 7,475 known genes, were detected in human hepatocytes. In addition, 1,819 unique MPSS signatures matching the antisense strand of 1,605 non-redundant UniGene clusters (such as APOC1, APOC2, APOB and APOH) were highly expressed in hepatocytes.
Apart from a large number of protein-coding genes, some of the antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via a cis-/trans-regulation mechanism. Our result provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases.
The liver – one of most important organs in the human body – performs the main digestive function in the metabolism of most substances. In addition, liver has a number of other functions, including the generation of red blood cells during embryonic development, production of various plasma proteins, detoxification of xenobiotics and phagocytosis of solid materials. It forms a protective barrier between the digestive tract and the rest of the body. The liver also plays a vital role in activation, catabolism and excretion of retinols that are essential to the vision, growth, reproduction, immunity, cell proliferation and differentiation of the body. Furthermore, as a major organ of drug elimination, the liver has a significant effect on drug metabolism. However, to date the molecular mechanisms of liver function have not been completely characterized.
Massively Parallel Signature Sequencing (MPSS) as a global view with no bias towards the transcriptome of certain tissues or cells will provide a profound understanding of organ and cell functions [1, 2]. It is well known that human liver is composed of many types of cells, such as hepatocytes, bile duct cells and kupffer cells, where hepatocytes account for three-quarters of the volume and perform the main functions of the liver. In this study, a powerful transcriptomic approach was employed to profile the gene expression of human hepatocytes obtained by laser capture micro dissection (LCM) for providing an available resource to address the molecular basis of hepatocyte biology.
Results and Discussion
Identification of UniGene clusters in hepatocytes by MPSS
Distribution of Unique MPSS signatures with expression levels
Classification of MPSS signatures expressed in hepatocytes
To evaluate whether those signatures with low abundance (≤ 3 TPMs) were also reliably expressed in the hepatocytes, RT-PCR was employed to detect the transcripts of the 16 UniGene clusters with less than 3 TPMs, where the UniGenes were randomly selected for the estimation. The interesting results showed that the transcripts of all UniGene clusters examined were indeed detected in the human hepatocytes at different levels (Figure 1B), suggested that transcripts detected at 3 TPMs or less by MPSS assay could in fact be proven to be expressed by RT-PCR.
Taken together, the resulting data suggested that 10,279 UniGene clusters could be detected in human hepatocytes using MPSS technique, implying that 7,475 known genes represented by the MPSS signatures could be expressed in the cell type of liver (see additional file 2).
Enriched genes in hepatocytes
Here S is the enrichment, E1 to En are the expression levels across all tissues and Eh is the expression value observed in hepatocytes for a given gene. S values > 2 would be considered as hepatocyte-enriched genes. The results indicated that 327 non-redundant UniGene clusters were enriched in hepatocytes (see additional file 3). Many of these were well-known to be secreted by hepatocytes and enriched in the liver. For example, ALB, APOA, APOB, and APOC are related to lipid metabolism and maintaining the balance of proteins in plasma. Interestingly, a number of genes with unknown functions, such as FLJ32745 and MGC40405, were also enriched in hepatocytes. Whether these genes are involved in hepatic functions should be further investigated. However, it should be pointed out that the distinct discrepancy of gene expression profiles between liver and other human tissues, based on hierarchical clustering of MPSS data (Figure 2), might result partially from the technical difference between the data collected in this study and the reference dataset.
Natural antisense transcripts (NATs) have been identified from plants  and animals , and these antisense RNAs are thought to be very important in the regulation of gene expression in such higher eukaryotes [11, 12]. Recently, approximately 5000 UniGene clusters of potential human NAT pairs had been identified by several groups [13–15]. The synchronous presence of both sense and antisense transcripts in the same cells or tissues may be an important indicator of antisense regulation [16, 17]. Interestingly, among 19,435 signatures uniquely matched with UniGene clusters, we here identified 1,819 unique signatures matching antisense strands according to the signature classification (see additional file 1 and 4), according to the previous description by Jongeneel et al . Herein, these antisense strands represented 1,605 non-redundant cis-antisense transcripts in hepatocytes, of which 1,127 (70.2%) of these antisense transcripts are co-expressed with their corresponding sense transcripts, whereas 478 (29.8%) of these antisense transcripts were found to be expressed alone. Compared with published NATs, 1,222 out of 1,605 UniGene clusters of the antisense transcripts were previously uncharacterized, implying that a large number of NATs could be found in given cells or tissues by the powerful transcriptomic approach.
To evaluate the evolutional conservation of these NATs, we identified their ortholog pairs between human and mouse through the reciprocal best matches between both genomes. 167 (10.40%) out of 1,605 human NATs were considered to have the corresponding antisense orthologs from mouse through comparing the data from the HomoloGene database with Mouse Genome Informatics, suggesting that some NATs could be evolutionarily conservative. Furthermore, these human NATs derived from hepatocytes were compared with 1,127 mouse NATs from the liver based on MPSS signatures deposited in NCBI GEO (GPL1010). The resulting data showed that 167 NATs, including 146 with both sense and antisense transcripts, were commonly expressed in human and mouse livers (see additional file 8). For example, the NATs of these genes, such as CYP4A2, HADH2, HSD17B4 and ACOX2 involved in fatty acid metabolism, CYP27A1 and ACADSB in bile acid biosynthesis, and ADH4 in alcohol metabolism, were conserved in both human and mouse livers, suggesting that these conservatively co-expressed sense-antisense pairs could play important roles in hepatic functions.
This study provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases. In addition, the data suggested that, like a large number of protein-coding genes, some antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via cis-/trans-regulation mechanisms.
Adult human livers
Normal human livers from ten patients were resected surgically because of hemangioma in liver in China. The samples were obtained from the portion unaffected by the hemangioma and frozen in liquid nitrogen immediately. All procedures and risks were explained verbally and in a written consent form. The samples were sectioned and confirmed to be normal histologically. All laboratory data assessing hepatic function were within normal ranges, including serum alanine aminotransferase, aspartate aminotransferase, g-glutamyl transpeptidase, alkaline phosphatase, total bilirubin, albumin, prothrombin activity, glucose, cholesterol, and triglycerides (data are not shown). Serological tests for hepatitis B surface antigen, Hepatitis C virus antibodies, and Human immunodeficiency virus antibodies also showed negative. Neither heavy alcohol consumption nor the intake of chemical drugs was observed before surgical resection.
Laser Capture Microdissection and RNA extraction
Four- micrometer sections of frozen liver tissues were not stained with any dye. The sections were immediately microdissected with a Leica AS LMD Laser Capture Microdissection System (Japan) using laser pulses of 7.5-μm diameters, 70 mW, and with 2–3 ms duration. Approximately 106 cells of human hepatocytes were microdissected and stored on microdissection caps with TRIZol reagent. Each cell population was determined to be 95% homogeneous by microscopic visualization of the captured cells. Laser-capture-microdissected hepatocytes were added to 1 ml TRIZol reagent (Invitrogen, Carlsbad, CA), and then total RNA was extracted according to the manufacturer's instructions and RNAse-free DNase I was used to remove DNA contamination. The nucleotide acid concentration and purity were assessed at 260 nm using a spectrophotometer (DU 530, Beckman-Coulter Inc., Fullerton, CA), and the quality was assessed by an Agilent 2100 Bioanalyzer. In addition, for the purposes of considering gene expression polymorphism, the total RNAs from ten livers were pooled equally.
MPSS was performed using RNA from the pooled livers and evaluated for the presence of LZP markers and absence of markers for AFP. The mRNA was converted to cDNA and digested with DpnII. The last DpnII site and the downstream 14 bases were cloned into Megaclone vectors and their sequences determined according to the MPSS protocol. This experiment was carried out by TaKaRa Co., Japan.
Semi-quantitative Reverse Transcription PCR
Reverse Transcription (RT) was performed in a 20 μl reaction system which contained 2 μg total RNA, 20 pmol oligo-dT, mixed up to 11 μl with DEPC-H2O and then incubated at 70°C for 5 minutes. After 5 mintues at 0°C, 4 μl 5 × buffer, 2 μl 0.1 M DTT, 2 μl dNTP (10 Mm) and 1 μl (200U) SurperScript II reverse transcriptase (Life Technologies), incubated at 42°C for 2 hours. In PCR, β-actin was used as a control to estimate the quality of cDNA (forward primer: 5'-TCACCCACACTGTGCCCATCTACGA-3' and reverse primer: 5'-CAGCGGAACCGCTCATTGCCAATGG-3'). To further avoid DNA contamination, all primers in this study were designed to span at least one exon. Each PCR was performed as follows: pre-denature at 94°C, 5 min; denature at 94°C, annealing at 55°C, extend at 72°C, 40 seconds, respectively, and finally at 72°C for 7 min. The PCR products were observed by electrophoresis on 2% agarose gel.
32 tissues for human transcriptomic analysis by MPSS were extracted from the NCBI GEO database (GPL1010). Genomic mapping data were taken from the human genome database version 17 at UCSC.
Availability and requirements
All data is available by download from our website http://184.108.40.206/hepatocytes/.
List of abbreviations
Massively Parallel Signature Sequencing
Laser Capture Microdissection
This work was supported by the Chinese Human Liver project (CNHLPP, 2004BA711A19), the National Natural Science Foundation for Outstanding Youth (30425019), the Chinese National Key Program on Basic Research (2006CB0D0802, 2004CB518605, 2006CB0D1205), the Chinese High-Tech Research and Development Program (863), the National Foundation for Excellence Doctoral Project, and the Shanghai Commission for Science and Technology (06ZR14069, 04XD14014 and 03DZ14024). We thank InforSense Ltd. for providing the KDE platform for the data analysis. We also thank Dr. Alex Michie in InforSense Ltd and Dr. Daixing Zhou in Solexa Company for the revision of this manuscript. In addition, we thank that TaKaRa Co. in Japan for performing MPSS experiment.
- Meyers BC, Tej SS, Vu TH, Haudenschild CD, Agrawal V, Edberg SB, Ghazal H, Decola S: The Use of MPSS for Whole-Genome Transcriptional Analysis in Arabidopsis. Genome Research. 2004, 14 (8): 1641-1653. 10.1101/gr.2275604.PubMed CentralPubMedView ArticleGoogle Scholar
- Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M: Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol. 2000, 18 (6): 630-634. 10.1038/76469.PubMedView ArticleGoogle Scholar
- Jongeneel CV, Iseli C, Stevenson BJ, Riggins GJ, Lal A, Mackay A, Harris RA, O'Hare MJ, Neville AM, Simpson AJ, Strausberg RL: Comprehensive sampling of gene expression in human cell lines with massively parallel signature sequencing. Proc Natl Acad Sci USA. 2003, 100 (8): 4702-4705. 10.1073/pnas.0831040100.PubMed CentralPubMedView ArticleGoogle Scholar
- Meyers BC, Vu TH, Tej SS, Ghazal H, Matvienko M, Agrawal V, Ning J, Haudenschild CD: Analysis of the transcriptional complexity of Arabidopsis thaliana by massively parallel signature sequencing. Nat Biotechnol. 2004, 22 (8): 1006-1011. 10.1038/nbt992.PubMedView ArticleGoogle Scholar
- Brandenberger R, Khrebtukova I, Thies RS, Miura T, Jingli C, Puri R, Vasicek T, Lebkowski J, Rao M: MPSS profiling of human embryonic stem cells. BMC Dev Biol. 2004, 4: 10-10. 10.1186/1471-213X-4-10.PubMed CentralPubMedView ArticleGoogle Scholar
- Jongeneel CV, Delorenzi M, Iseli C, Zhou D, Haudenschild CD, Khrebtukova I, Kuznetsov D, Stevenson BJ, Strausberg RL, Simpson AJ, Vasicek TJ: An atlas of human gene expression from massively parallel signature sequencing (MPSS). Genome Res. 2005, 15 (7): 1007-1014. 10.1101/gr.4041005.PubMed CentralPubMedView ArticleGoogle Scholar
- Smith AD, Sumazin P, Xuan Z, Zhang MQ: DNA motifs in human and mouse proximal promoters predict tissue-specific expression. Proc Natl Acad Sci USA. 2006, 103 (16): 6275-6280. 10.1073/pnas.0508169103.PubMed CentralPubMedView ArticleGoogle Scholar
- Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ, Murray HL, Volkert TL, Schreiber J, Rolfe PA, Gifford DK, Fraenkel E, Bell GI, Young RA: Control of pancreas and liver gene expression by HNF transcription factors. Science. 2004, 303 (5662): 1378-1381. 10.1126/science.1089769.PubMed CentralPubMedView ArticleGoogle Scholar
- Krivan W, Wasserman WW: A predictive model for regulatory sequences directing liver-specific transcription. Genome Res. 2001, 11 (9): 1559-1566. 10.1101/gr.180601.PubMed CentralPubMedView ArticleGoogle Scholar
- Terryn N, Rouze P: The sense of naturally transcribed antisense RNAs in plants. Trends Plant Sci. 2000, 5 (9): 394-396. 10.1016/S1360-1385(00)01696-4.PubMedView ArticleGoogle Scholar
- Vanhee-Brossollet C, Vaquero C: Do natural antisense transcripts make sense in eukaryotes?. Gene. 1998, 211 (1): 1-9. 10.1016/S0378-1119(98)00093-6.PubMedView ArticleGoogle Scholar
- Lehner B, Williams G, Campbell RD, Sanderson CM: Antisense transcripts in the human genome. Trends Genet. 2002, 18 (2): 63-65. 10.1016/S0168-9525(02)02598-2.PubMedView ArticleGoogle Scholar
- Li YY, Qin L, Guo ZM, Liu L, Xu H, Hao P, Su J, Shi Y, He WZ, Li YX: In silico discovery of human natural antisense transcripts. BMC Bioinformatics. 2006, 7: 18-26. 10.1186/1471-2105-7-18.PubMed CentralPubMedView ArticleGoogle Scholar
- Chen J, Sun M, Kent WJ, Huang X, Xie H, Wang W, Zhou G, Shi RZ, Rowley JD: Over 20% of human transcripts might form sense-antisense pairs. Nucleic Acids Res. 2004, 32 (16): 4812-4820. 10.1093/nar/gkh818.PubMed CentralPubMedView ArticleGoogle Scholar
- Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, Shoshan A, Diber A, Biton S, Tamir Y, Khosravi R, Nemzer S, Pinner E, Walach S, Bernstein J, Savitsky K, Rotman G: Widespread occurrence of antisense transcription in the human genome. Nat Biotechnol. 2003, 21 (14): 379-386. 10.1038/nbt808.PubMedView ArticleGoogle Scholar
- Kumar M, Carmichael GG: Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol Mol Biol Rev. 1998, 62 (4): 1415-1434.PubMed CentralPubMedGoogle Scholar
- Knee R, Murphy PR: Regulation of gene expression by natural antisense RNA transcripts. Neurochem Int. 1997, 31 (3): 379-392. 10.1016/S0197-0186(96)00108-8.PubMedView ArticleGoogle Scholar
- Hastings ML, Milcarek C, Martincic K, Peterson ML, Munroe SH: Expression of the thyroid hormone receptor gene, erbAalpha, in B lymphocytes: alternative mRNA processing is independent of differentiation but correlates with antisense RNA levels. Nucleic Acids Res. 1997, 25 (21): 4296-4300. 10.1093/nar/25.21.4296.PubMed CentralPubMedView ArticleGoogle Scholar
- Peters NT, Rohrbach JA, Zalewski BA, Byrkett CM, Vaughn JC: RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts. RNA. 2003, 9 (6): 698-710. 10.1261/rna.2120703.PubMed CentralPubMedView ArticleGoogle Scholar
- Tufarelli C, Stanley JA, Garrick D, Sharpe JA, Ayyub H, Wood WG, Higgs DR: Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat Genet. 2003, 34 (2): 157-165. 10.1038/ng1157.PubMedView ArticleGoogle Scholar
- Chen J, Sun M, Hurst LD, Carmichael GG, Rowley JD: Genome-wide analysis of coordinate expression and evolution of human cis-encoded sense-antisense transcripts. Trends Genet. 2005, 21 (6): 326-329. 10.1016/j.tig.2005.04.006.PubMedView ArticleGoogle Scholar
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