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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 740-746 |
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doi:10.1111/j.1745-7270.2008.00454.x |
Identification of differentially expressed genes in rat silicosis model by suppression subtractive hybridization analysis
Zhongyuan Jin#, Baoan Liu#, Deyun Feng, Chen Chen, Xiang Li, Yongbin Hu, Jinwu Peng, Yu Liu, Jing Du, Chunyan Fu, and Jifang Wen*
Department of Pathology, Xiangya Medical
School, Central South University, Changsha 410013, China
Received: April 9,
2008�������
Accepted: June 15,
2008
This work was
supported partly by a grant from the Natural Science Foundation of Hunan Province
(No. 04JJ3094)
#These authors
contributed equally to this work
*Corresponding
author: Tel, 86-731-2355098; Fax, 86-731-2650400; E-mail, [email protected]
The critical molecular mechanism in the development
of the pulmonary fibrosis remains unknown, leaving diagnosed patients with a
poor prognosis. To isolate the genes specifically up-regulated in pulmonary
fibrosis, we established a rat silicosis model 360 d after treatment with
crystalline silica suspension. Radiographs of chests showed that some scattered
high-density shadows appeared in the lung field. Typical microscopic fibrosing
silicotic nodules formed in the lung, alveolar epithelial cells and bronchial
epithelial cells, particularly around the partial fibrosing silicotic nodules;
some of them showed atypical hyperplasia that suggested a correlation between
silicosis and lung cancer. Suppression subtractive hybridization analysis was
performed to compare gene expression in lung tissue with silicosis and normal
lung tissue. Reverse transcription-polymerase chain reaction showed that the
expressions of seven novel cDNA sequences identified by suppression subtractive
hybridization in lung tissue with silicosis differed from normal lung tissue. Bioinformatics
analysis showed that 47 positive clones represented 35 genes containing two
putative proteins and four predicted similar proteins. The analysis also showed
that some screened genes in silicosis, such as prolyl 4-hydroxylases,
actin-related protein-2/3 complex and acidic mammalian chitinase, have not been
previously reported. These genes may provide new clues for investigating the
molecular mechanisms in the development of pulmonary fibrosis.
Keywords������� pulmonary fibrosis; silicosis; suppression subtractive hybridization; cDNA subtracted library; differentially expressed gene
Pulmonary fibrosis is a destructive lung disease induced by multiple agents, and patients with the disease have a poor prognosis. The pathological processes of pulmonary fibrosis include inflammatory injury, tissue damage and repair. Many kinds of cells, such as alveolar macrophages [1], epithelial cells and fibroblasts [2,3], play important roles in the progress of pulmonary fibrosis. These cells function directly or indirectly by secreting bioactive compounds, such as cytokines and mediators of inflammation, and constitute a complicated cellular network interacting with each other and promoting the development of pulmonary fibrosis. However, the critical molecular mechanism in the development of the pulmonary fibrosis remains unknown. Based on the understanding of the pathogenesis of this disease, it is vital to search for better treatment plan of pulmonary fibrosis. Suppression subtractive hybridization (SSH) has recently been reported as a differential display technique that is the basis of suppressive polymerase chain reaction (PCR). SSH is a powerful technique that is capable of isolating differentially expressed genes without knowing their identity [4-7], and it has been successfully applied in studies on tumors, developing biology and immune regulations. For example, Luo et al successfully cloned six novel genes involved in the biosynthesis of ginsenoside in Panax ginseng plant using SSH [8].
In this study, we aim to use SSH to screen and identify differentially expressed genes in pulmonary fibrosis.
Materials and Methods
Construction of the rat silicosis model
Female Sprague-Dawley (SD) rats (8-week old; 180-200 g body weight) were purchased from Central South University (Changsha, China). The animals were housed individually in cages containing a bedding of wood shavings and had easy access to food and water. Fifty female SD rats were randomly divided into five experimental groups and five control groups; each group had five rats. Each rat from the experimental groups was treated with crystalline silica suspension (200 mg/kg body weight) poured through a trachea cannula. In the control group, rats were treated with sterile physiological saline. Radiographs of the chest were taken of rats in both the experimental and control groups, and five rats from both the groups were randomly selected and killed at 1, 30, 90, 180 and 360 d after treatment. The lung tissue was collected; some lung tissues were formalin-fixed, paraffin-embedded and sliced, and the rest were stored at -80 �C.
Total RNA extraction
Total RNA was extracted from individual lung tissue samples from the silicosis and control groups using TRIzol Reagent (Invitrogen, Carlsbad, USA). Total RNA concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany) by measuring absorbance at 260 nm and 280 nm (A260:A280). All pulmonary total RNA samples had an A260:A280�2.0.
Isolation of polyA+ RNA
PolyA+ RNA was isolated from previously purified total RNA using a PolyATtract mRNA isolation system II kit (Promega, Madison, USA) according to the manufacturer�s instructions. The absorbance was determined at 260 nm. From each of the 10 sample groups (five silicosis and five control), 180 mg total RNA was harvested and pooled together for a final volume of 900 mg per group. The concentration of mRNA was determined spectro�photo�metri�cally. RNA was stored at -80 �C until used.
Suppression subtractive hybridization (SSH)
SSH was performed using the Clontech PCR-Select cDNA subtraction kit (Clontech, Palo Alto, USA). Two different forward and reverse subtractions were performed to compare the gene expression between lung tissue with and without silicosis. We constructed the forward subtraction (SNA) using the cDNA of the silicosis as the tester and the cDNA of the control as the driver. We also constructed the reverse subtraction (SNB) using the cDNA of the control as the tester and the cDNA of the silicosis as the driver. cDNA was synthesized with 2 mg pooled mRNA according to the recommended methods by the manufacturer. The cDNA was then processed by restriction digestion of RsaI; adaptors were then added, hybridized in two rounds, and followed by suppression and nested PCR amplification for 27 cycles at 94 �C for 30 s, 66 �C for 30 s and 72 �C for 1.5 min and for 12 cycles at 94 �C for 30 s, 66 �C for 30 s and 72 �C for 1.5 min.
Cloning and DNA sequencing analysis
The products of the nested PCR were cloned into pMD20-T vector (TaKaRa, Shiga, Japan), transformed into JM109 competent cells (TaKaRa) and screened on Luria broth plates containing ampicillin/X-gal/IPTG. About 400 white colonies were obtained. For each library, over 100 white colonies were isolated and incubated overnight in Luria broth-ampicillin broth at 37 �C. Clones were then chosen for PCR identification using nested PCR primers; 52 clones containing inserted fragments from forward and reverse libraries were randomly chosen for sequence analysis by internal universal M13 primer in the vector.
Reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was carried out for additional putative gene products identified by SSH to confirm the validity of targets detected in our data set. For RT-PCR, total RNA was treated with DNase I (Promega) at 1 U/2 ml total RNA in 10 ml reaction volume and incubated for 30 min at 37 �C; 1 ml 20 mM EDTA was then added for enzyme inactivation and incubated for 15 min at 65 �C. cDNA synthesis was performed using 2 mg total RNA in 20 ml reaction volume with Superscript II Reverse Transcriptase (Invitrogen) according to the manufacture�s instructions using 4 ml 5�first-strand buffer, 1 ml 10 mM dNTP, 200 U Superscript II enzyme, 2 ml 0.1 M dithiothreitol, and 250 ng oligo(dT)18 primer (Invitrogen). For PCR reactions, 1 ml each synthesized cDNA was used as template in a 50 ml reaction volume containing 200 mM dNTPs, 1.5 mM MgCl2, 0.25 mM each primer, and 1 U Taq DNA polymerase in the buffer, as recommended by the manufacturer (Invitrogen). The reaction was allowed to denature for 5 min at 95 �C, followed by amplification (25, 28, 30 and 32 cycles each at 94 �C for 30 s, 58 �C for 30 s, and 72 �C for 1.5 min). At indicated cycles, a 5 ml sample was colleted from each reaction. The sequences of the primers and product length are listed in Table 1. PCR products were loaded onto a 1% agarose gel and electrophoresed in Tris-acetate-EDTA. Gels were subjected to ethidium bromide staining and were imaged in an ultraviolet transilluminator using a digital Kodak camera (Kodak, Shanghai, China).
Bioinfomatics analyses
Each sequence output was identified using Basic Local Alignment Search Tool (BLAST). Sequences were first searched for vector contamination using VecScreen, and portions of inserts with contamination were not used for BLAST. Similar sequences and percent identities were determined by BLASTn search in GenBank (http://www.ncbi.nlm.nih.gov/).
Results
Animal model of silicosis
In the present study, the SD rats were treated with crystalline silica suspension poured through trachea cannula. At 30 d, X-rays of the silicosis group showed thickened pulmonary markings. Morphologic alveolitis and macrophage-like cellular nodules appeared in lung tissue. At 180 d, scattered high-density shadows appeared in lung fields. Histopathologic features showed some scattered small, gray nodules in lung tissue, alveolar epithelial cells proliferation and typical fibrosing silicotic nodules. After 360 d, pulmonary markings were getting thicker, and the high-density shadows of varying sizes appeared in lung field [Fig. 1(A)]. Many different sized nodules were distributed on the surface and cross section of the lung [Fig. 1(B)]. Typical fibrosing silicotic nodules were formed in lung. Alveolar epithelial cells and bronchial epithelial cells proliferated around partial fibrosing silicotic nodules [Fig. 2(A)], and some cells showed atypical hyperplasia [Fig. 2(B)]. Diffused pulmonary interstitial fibrosis was also observed. The X-rays showed that the animal model of silicosis was successful and that this model could be used for later stage.�
Analysis of ligation
At least 25% of the cDNA should have adaptors on both ends to meet the requirements of the experiment. The fragments that spanned the adaptor/cDNA junctions were amplified by primer adaptor l or adaptor 2 and G3PDH 3�- or 5�-primers. After 25 cycles, the products were identified by 2.0% agarose gel electrophoresis. The results showed that the adaptors were efficiently ligated with double-stranded cDNA.
PCR analysis of subtraction efficiency
The efficiency of subtraction was estimated by comparing the abundance of G3PDH cDNA before and after subtraction. PCR was performed using G3PDH primer from the subtracted or unsubtracted cDNA. After 18, 23, 28 and 33 cycles, 5 ml amplified products were removed from each reaction and examined electrophoretically on a 2.0% agarose/ethidium bromide gel. The G3PDH product was observed after 23 cycles in the unsubtracted group and after 28 cycles in the subtracted sample, which indicated that the subtraction efficiency was satisfactory.
Amplification and TA clones of differentially expressed cDNA
After the second PCR amplification, the differentially expressed cDNAs were enriched. The PCR products were cloned into T-vectors. Nested PCR primers were used to determine approximate inserted fragment size, which varied from 200 to 600 bp, possibly representing differentially expressed genes (Fig. 3).
RT-PCR confirmation
In order to confirm the differentially expression of cDNA fragments in the lung tissue of the rats with and without silicosis, seven novel cDNA sequences (SNA-11, SNA-28, SNA-43, SNA-48, SNB-13, SNB-30, SNB-42) were examined by RT-PCR. The results showed that the expressions of these sequences in the lung tissue with silicosis were different from those in the lung tissue without silicosis (Fig. 4). According to the quantitative analysis of the scanned images, the difference of the gene expression quantity between two groups of lung tissues was 2-5 times. The results showed that two reliable high qualitative subtracted cDNA libraries were constructed.
�
Bioinfomatics analyses of gene fragments
Fifty-two clones from forward and reverse libraries were randomly chosen for sequence analysis and submitted to GenBank for homology analysis. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing two putative proteins and four predicted similar proteins (Tables 2, 3). Most genes were functional as including the structure and movement of cell or extracellular matrix, the defense of cell and body, material transportation, and the regulation of expression and metabolism.
Discussion
The pathological processes of pulmonary fibrosis include inflammatory injury, tissue damage and repair. In this study, 360 d after treatment with silica, the animal model of silicosis was successfully constructed as evidenced by the formation of fibrosing silicotic nodules and diffuse pulmonary interstitial fibrosis in lung tissue. Proliferated alveolar epithelial cells and bronchial epithelial cells surrounded the partial silicotic nodules, and some cells showed atypical hyperplasia. These results revealed that fibrotic reaction of recurrent inflammation and repair may cause repeat cellular injury, genetic damage to local epithelial cells, and a predisposition to the development of cancer through sequential cellular morphologic alterations of atypia [9,10].
The critical molecular mechanism in the development of the pulmonary fibrosis is still unknown [11]. To get a better understanding of this mechanism, we used SSH to compare the differentially expressed genes in normal lung tissue and in lung tissue with silicosis. We also constructed cDNA libraries from rat lung tissues with and without silicosis as tester and driver populations for SSH procedures. Positive clones from the libraries were randomly selected and a total of 52 clones were sequenced and compared to sequences in the GenBank. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing two putative proteins and four predicted similar proteins. Most genes were functional and played an important role in cell or extracellular matrix structure and movement, cell defense, material transportation, and the regulation of expression and metabolism. Some genes, such as prolyl 4-hydroxylase (P4H), actin-related protein-2/3 (ARP2/3) complex and acidic mammalian chitinase (AMCase), had previously not been reported in silicosis and were cloned. These isolated fragments provide the basis for the future cloning of full-length genes and functional analysis.
P4H catalyze the formation of 4-hydroxyproline by the hydroxylation of prolines in -X-Pro-Gly-sequences in collagens and more than 15 other proteins that have collagen-like domains. P4H plays a central role in the biosynthesis of collagens, as 4-hydroxyproline residues are essential for the formation of the collagen triple helix [12]. It has recently been shown that the hypoxia-inducible transcription factor a (HIFa) is regulated by a novel cytoplasmic HIF P4H family [13]. Interestingly, the gene for the a(I) subunit of human type I collagen P4H has been shown to be a hypoxia-inducible target gene of HIFa [14].
The eukaryotic actin cytoskeleton plays an important role in a remarkable number of diverse processes essential for cellular survival, including cell migration, endocytosis, vesicle trafficking and cytokinesis. The core constituent of the actin cytoskeleton is monomeric globular (G)-actin, a 43 kDa ATPase that can self-assemble into filamentous (F)-actin. ATP hydrolysis in the filament is tightly coupled to polymerization and regulates the kinetics of assembly and disassembly, as well as the association of interacting proteins. The ARP2/3 complex is a central player in this regulation [15-17]. ARP2/3 complex dysfunction might be associated with diseases, such as cancer metastasis, as well as with Wiskott-Aldrich Syndrome, a rare X-linked recessive genetic disorder [18]. The function of ARP2/3 complex in silicosis is unclear.
AMCase is a 50 kDa protein that contains a 30 kDa N-terminal catalytic domain. It can hydrolyse chitin, a polymer of N-acetylglucosamine [19]. AMCase is a member of the glycosyl hydrolase 18 family and is structurally similar to other members of this family, including the lectins Ym1 and Ym2, which are expressed in macrophages [20], human cartilage glycoprotein-39 (HC-gp39) and chitotriosidase [19]. Recently, it was reported that AMCase is expressed in T helper 2-mediated inflammation and can induce airway hyperresponsiveness in allergic asthma patients. However, pan-chitinase inhibitor can significantly ameliorate T helper 2-mediated inflammation and airway hypersensitivity. Therefore, chitinases have been proposed as a potential therapeutic target in T helper 2-mediated inflammation. In addition, rats exposed to silica exhibit increased expression of this protein in bronchoalveolar lavage fluid and alveolar macrophages in comparison to untreated rats, suggesting a role of this enzyme in lung defence mechanisms [21].
In conclusion, we have revealed that silicosis may be related to
lung cancer. In the course of this study, we also discovered some genes that
were not reported in silicosis previously and cloned them. Further studies on
these genes may provide a better understanding of the mechanisms underlying
pulmonary fibrosis.
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