Liver fibrosis is a reparative reaction of the liver to various chronic disease states. It is also a common pathological change that occurs in all chronic hepatic diseases. The characteristics of liver fibrosis include misregulation of the synthesis and degradation of collagen in the extracellular matrix (ECM). Abnormal hyperplasia of fibrous connective tissue in the liver further destroys normal hepatic structures, which is followed by development of hepatic cirrhosis.1 Hepatic stellate cells (HSCs) are a major source of ECM and activation of HSCs is one of the key steps in the development of liver fibrosis. Complex interactions amongst cytokines and the ECM regulate the activation of HSCs, which directly influences synthesis of the ECM. Among the many cytokines that regulate HSC activation, leptin is a strong autocrine regulating cytokine that can activate HSCs and it plays a key role in liver fibrosis development. In this study, microarray analysis was used to screen for differentially expressed genes in leptin activated HSCs. We expect that our results will help illustrate the molecular mechanisms involved in the pathogenesis of liver fibrosis and highlight potential targets for gene therapy for the treatment of this disease.
Rat HSCs (cell line HSC-T6) were obtained from our lab and cultivated using standard techniques in 25-cm 2 culture dishes in DMEM medium (Invitrogen, USA) containing 10% fetal bovine serum (FBS, Invitrogen). Cell counting was carried out during logarithmic growth phase. A total of 1×106 /ml cells were cultivated in two 25-cm2 culture dishes, representing the experimental and control groups. After cultivation for 24 hours, the culture medium was replaced with new DMEM medium with 10% FBS. Leptin (PeproTech, UK) was added to the experimental group with the concentration of 100 ng/ml, while the control group without leptin. Cells were harvested after another 26 hours of cultivation. A total of 1 ml TRIzol (Invitrogen) was added to 5×106 cells followed immediately by liquid nitrogen preservation.
Total RNA extraction
TRIzol was used to extract total RNA from HSCs according to the manufacture's instructions. Total RNA was purified using an RNeasy® kit (Qiagen, Germany) and the quality of RNA was determined.
Fluorescent probe preparation
First strand cDNA was labeled. Nucleotides labeled with CyDye were inserted directly into strands of cDNA using reverse transcriptase to generate the fluorescent probes. mRNA (1 mmol/L) from the control group was labeled with Cy3-dCTP (Invitrogen, USA) and mRNA (1 mmol/L) from the experimental group was labeled with Cy5-dCTP (Invitrogen) under the following conditions: 42°C for 2 hours, 70°C for 5 minutes, and 37°C for 15 minutes, followed by addition of 10 μl 2 mol/L HEPES (Invitrogen) for neutralization. Labeled fluorescent probes were purified using a QIAquick Nucleotide Removal kit (Qiagen). Purified probes were transferred to an enzyme label plate and quantitative determination was carried out at A260, A550, and A650 using the following formula: Cy3 probe (pmol)=A550×elution volume/0.15, Cy5 probe (pmol)=A650×elution volume/0.25.
Probes were preserved in PCR tubes and dried by vacuum heating after quantitative determination. Dried probes were stored under photophobic conditions at -20°C prior to hybridization.
About 30 pmol of Cy3 and Cy5 labeled and quantitated probes were dissolved and denatured at 94°C for 3 minutes in a PCR machine. Then, 1-2 μg human Cot-1 DNA was added to the probes, followed by incubation at 70°C for 45 minutes. After the reaction, 10 μl of 4× hybridization buffer and 20 μl formamide were added. One drop of hybridization solution was added to the microarray (Shanghai Biochip, China) and then covered with a cover slip. The microarray was incubated in a photophobic hybridization chamber containing PBS and hybridized at 42°C for 16-18 hours.
After hybridization, the microarray was immerged sequentially in solution I (1×SSC/0.2% SDS) and solution II (0.1×SSC/0.2% SDS) at 55°C for 10 minutes each, and this sequence was repeated three times. The microarray was then immerged in solution III (0.1×SSC) at room temperature for 5 minutes, and this was repeated three times followed by washing in de-ionized water for 2 minutes at room temperature. The washed microarray was then transferred into a 50 ml centrifuge tube and spun at 1500 r/min for 5 minutes to dry. The dry microarray was immediately scanned or kept in a microarray chamber and preserved in a desiccator under photophobic conditions.
Statistical analysis and standardization
Images were obtained using an Agilent scanner (Santa Clara, USA) and introduced into Imagene image analysis software (BioDiscovery Inc., USA). Hybridization spots were identified by automatic and manual positioning and localization. Background noise was filtered to acquire fluorescent signal intensity values. Data were output as a list to complete the transformation between scanned image and quantified values. Because of sample variance, different fluorescence-label efficacies, and imbalanced detection rates, equalization and modification of the original Cy3 and Cy5 signals were necessary for further analysis of the experimental data. After standardization of these data using Genespring data analysis software (Agilent, Santa Clara, CA, USA), the ratio of Cy5 to Cy3 was calculated. The standard for determining differentially upregulated genes was a Cy5 to Cy3 ratio ≥2, while a ratio ≤0.5 was considered a downregulated gene.
Verification by microarray hybridization and analysis of results
There were 4096 cDNAs on the microarray. In order to monitor accuracy of the microarray hybridization there were eight mice genes at eight spots on the microarray that functioned as negative controls. Very low hybridization signals from these control spots indicated the reliability of the data. The Cy5 labeled pcDNA3.1 (-) ALR probe groups were displayed as red and the Cy3 labeled pcDNA3.1 (-) probe groups displayed as green, the difference between red and green signals indicated differential expression between the two groups, while yellow indicated there was no difference in expression levels. Scatter and hybridization plots are shown in Figures 1 and 2.
Analysis of differentially expressed genes
Following the analysis of the microarray, expression levels of six genes were found to be downregulated and six genes were upregulated. The ratios of Cy5 to Cy3 of these 12 genes are shown in Tables 1 and 2.
Researches on liver fibrosis have made great progress over the last decade. We understand that extraordinary complicated pathological processes are involved in the development of liver fibrosis and it is the result of the interaction of numerous factors including cells, cytokines, and the ECM. The leptin gene (ob) was identified in 1994 and codes for a protein that is mostly secreted by adipose cells. Considerable research indicates that leptin can activate HSCs and functions as a strong autocrine regulating cytokine, playing an important role in the formation of liver fibrosis. Activated HSCs secrete leptin and produce a large quantity of collagen, which is the primary cause of liver fibrosis. The mechanism by which leptin activates HSCs is complicated. There are a variety of positive and negative feedback pathways involving cytokines that initiate this process. However, little is known about the exact regulatory mechanisms involved. This research studied the changes in gene expression via microarray analysis following leptin treatment of HSCs and explored the molecular mechanisms by which leptin activates HSCs. These results provide new clues for possible future treatments of liver fibrosis.
In this study, we began to categorize the transactivated target genes of leptin. We identified 12 differentially expressed genes among 4096. These 12 genes are involved in cell differentiation, signal transduction, cell structure, cell composition, gene and protein encoding, cell cycle, cell apoptosis, immunity, energy metabolism, and other biological processes. Hence, as mentioned above, we can surmise that leptin induced liver fibrosis is the result of multifactor and multilevel regulation.
One representative downregulated gene encodes hepatic stearoyl coenzyme A desaturase (SCD-1). SCD-1 is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids (MUFAs) and plays a key role in regulation of fatty acid metabolism. The processing of precursor saturated fatty acids to MUFA involves anaerobic oxidation catalyzed by an enzyme system consisting of nicotinamide adenine dinucleotide (NADH) dependent flavoprotein cytochrome b5 reductase, cytochrome b5, and SCD. Stearoyl and palmityl CoA are catalyzed by SCD to oleoyl and palmitoleoyl CoA, which are the main precursors for the synthesis of triglycerides, cholesterol esters, and membrane phospholipids in cells. In the liver, oleic acid is a necessary fatty acid for the synthesis of triglycerides and cholesterol esters, which are key components for generating and excreting very-low-density lipoproteins. Therefore, SCD is a key control point for regulating the level of very-low-density lipoproteins. There are three isoenzymes of SCD: SCD-1, SCD-2 and SCD-3. SCD-1 is found primarily in the liver, kidney, lung, heart, and spleen. SCD-2 is mostly found in the kidney, lung, spleen, and adipose tissues. SCD-3 is only found in dermal sebaceous glands.2,3 In a previous report, the SCD-1 mRNA level was shown to decline in the livers of rats fed a fat-rich diet. The downregulation of the SCD-1 mRNA in our experiment may represent a similar response to high blood leptin levels that are induced by a fat-rich diet.4 Cohen et al5 suggested that the SCD-1 gene was a target of the leptin signaling, and leptin could suppress the expression of SCD-1.6 The total body fat of ob/ob rats without SCD-1 is 40% lower than ob/ob control rats. In normal rats, leptin controls the fat regulation pathway, including regulation of SCD-1 in the liver. Very-low-density lipoprotein excretion levels decline with the downregulation of SCD-1 mRNA levels, so that extra fat cannot be excreted from the liver and is deposited. This may result in a fatty liver and further the development of liver fibrosis.
One representative protein of the upregulated genes is the pulmonary-associated protein A1, SP-A. SP is a specific protein for binding dipalmitoyph-opatidylcholine (DPPC), which is a pulmonary surfactant. SP is a lung specific protein that is mainly synthesized in alveolar type II cells and is expressed in the lungs at high concentrations.7 The SP family contains hydrophilic macromolecules like SP-A and SP-D and hydrophobic small molecules like SP-B and SP-C. SP-A is the most abundant among the family members and about 50% of the total SP is SP-A. SP-A is involved in regulating the synthesis and metabolism of pulmonary surfactants, natural immunity, and inflammatory responses in the lung, as well as other important physiological functions.8-10 Serum SP-A levels are increased in idiopathic interstitial pneumonia (IIP) and idiopathic pulmonary fibrosis (IPF). Honda et al11 found SP-A levels reflected disease activity, as shown by the dynamic monitoring of serum SP-A levels in two IPF patients. The more severe the patients' condition, the higher the serum SP-A level and vice versa. Serum SP-A levels are a serological marker not only for auxiliary diagnosis for IIP and IPF and for monitoring disease development, but it also function as a predictive marker of patients' survival rate.12 IPF patients who died within three years had higher serum levels of SP-A relative to IPF patients that survived more than three years. Leptin can upregulate the expression of the SP-A gene. Therefore, we hypothesize that leptin can accelerate the development of liver fibrosis and may also play some role in the development of lung fibrosis.
Another interesting upregulated gene encodes short chain dehydrogenase, which is also known as retinol dehydrogenase. Cheng et al13 screened for interacting partners of the hepatic C virus (HCV) core protein-binding protein via the yeast two-hybrid technique and showed that the core protein of HCV can bind to retinol dehydrogenase 11 (RDH11). Huang et al 14 established a 293 cell line stably transfected with the gene encoding 11-cis-retinol dehydrogenase. Once accessory factor was given to the cells, 11-cis-retinol dehydrogenase could catalyze a series of transformations of sex hormones. This pathway is considered a non-classical pathway for generating active androgen in peripheral tissues. Therefore, 11-cis-retinol dehydrogenase can be considered a factor in the metabolism of steroids. The upregulated expression of retinol dehydrogenase may change enzyme catalysis and disturb the metabolism of sex and steroid hormones, which may result in disturbance of normal metabolic pathways in the liver. This provides a new avenue for future study of the biological functions of leptin.
Through this analysis of leptin-regulated genes in HSCs, we identify genes highly related to cell signal transduction, HSC apoptosis, synthesis of collagen, and regulation of the cell cycle, and each plays different roles in the development of liver fibrosis. This study presents new ideas for future clinical therapies for the treatment of liver fibrosis.
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