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Expression profiling suggests a regulatory role of gallbladder in lipid homeostasis.

AIM
To examine expression profile of gallbladder using microarray and to investigate the role of gallbladder in lipid homeostasis.


METHODS
33P-labelled cDNA derived from total RNA of gallbladder tissue was hybridized to a cDNA array representing 17,000 cDNA clusters. Genes with intensities > or =2 and variation <0.33 between two samples were considered as positive signals with subtraction of background chosen from an area where no cDNA was spotted. The average gray level of two gallbladders was adopted to analyze its bioinformatics. Identified target genes were confirmed by touch-down polymerase chain reaction and sequencing.


RESULTS
A total of 11 047 genes expressed in normal gallbladder, which was more than that predicted by another author, and the first 10 genes highly expressed (high gray level in hybridization image), e.g. ARPC5 (2 225.88+/-90.46), LOC55972 (2 220.32+/-446.51) and SLC20A2 (1 865.21+/-98.02), were related to the function of smooth muscle contraction and material transport. Meanwhile, 149 lipid-related genes were expressed in the gallbladder, 89 of which were first identified (with gray level in hybridization image), e.g. FASN (11.42+/-2.62), APOD (92.61+/-8.90) and CYP21A2 (246.11+/-42.36), and they were involved in each step of lipid metabolism pathway. In addition, 19 of those 149 genes were gallstone candidate susceptibility genes (with gray level in hybridization image), e.g. HMGCR (10.98+/-0.31), NPC1 (34.88+/-12.12) and NR1H4 (16.8+/-0.65), which were previously thought to be expressed in the liver and/or intestine tissue only.


CONCLUSION
Gallbladder expresses 11 047 genes and takes part in lipid homeostasis.


INTRODUCTION
Cholesterol cholelithiasis is an extremely common, economically significant digestive disease that affects some 10-15% of the global population [1] . Gallstone is also the main cause of gallbladder carcinoma, biliary pancreatitis and iatrogenic lesions of the biliary tract. It has been reported that the USA spends 8-10 billion dollars on gallstone disease annually [2] . It was suggested from the data in the late 1980s that about 5.6% of the population in China was affected with gallstones [3] , and the incidence may be increased in recent years. The patients with gallstone-related diseases hospitalized in the surgical department of our hospital accounted for 47% in 2001. Clearly, it is an important disease that deserves more attention.
Cholesterol saturated bile secreted by the liver is the prerequisite of gallstone formation, so liver is the place of lipid metabolism and becomes the focus of study. However, gallbladder is the place of stone formation and its relationship with lipid metabolism has been seldom investigated. Furthermore, the molecular mechanisms of gallstone formation in gallbladder-related with lipid metabolism are far from clear. Only about 40 genes (including gallstone susceptible gene loci) have been identified presently, and most of the previous studies were based on the changes in a single gene. Obviously, a total list of genes expressed in the gallbladder should be identified. The relatively new advent of cDNA array technology has provided a powerful method for largescale expression profiling [4] , and has led to the elucidation of a number of regulatory pathways involved in complex biological processes especially in tumor-related areas [5] . In this study, we used a powerful tool to examine expression profile of gallbladder and to investigate the role of gallbladder in lipid homeostasis. This would build a basis for understanding the physiological function of gallbladder, especially the mechanism of gallstone formation. MATERIALS AND METHODS cDNA array construction cDNA clones were derived from liver and hepatocarcinoma cell lines, isolated from hypothalamus-pituitary-adrenal libraries [6] or purchased from Research Genetics (Huntsville, AL, USA). The assembled cDNA array contained 17 000 cDNA clones (representing the same number of independent cDNA clusters), of which 7 565 clusters were homologous to those found in the UniGene Database. All cDNA fragments were amplified and verified by electrophoresis. The average length of the cDNA fragments was -1 kb. PCR products were precipitated in isopropanol, redissolved in 10 L of denaturing buffer (1.5 mol/L NaCl, 0.5 mol/L NaOH), and spotted on two 8×12-cm Hybond-N nylon membranes (Amersham Pharmacia, Buckinghamshire, UK) with an arrayer (BioRobotics, Cambridge, UK). Each spot carried -100 nL in volume and was 0.4 mm in diameter each cDNA fragment was placed in two different spots (double-offset). Lambda phage and pUC18 vector DNAs were spotted as negative controls.

Clinical samples
Normal gallbladders were removed within 4 h postmortem from two adult males (aged 25 and 20 years, respectively) who died in traffic accidents. The Institute of Biomedical Science, Shanghai Second Medical University, approved the study, and all samples were obtained with informed consent. Tissues were frozen in liquid nitrogen immediately after separation, and kept frozen until used.

RNA extraction and probe preparation
Total RNA was extracted using a standard TRIzol RNA isolation protocol (Life Technologies, Inc., Grand Island, NY, USA). Poly (A)+ mRNA was then isolated from total RNA using a poly (dT) resin (Qiagen, Hilden, Germany

Hybridization and image processing
Prehybridization was carried out in 20 mL of prehybridization solution (6× SSC, 0.5% SDS, 5× Denhardt's, and 100 µg/mL denatured salmon sperm DNA) at 68 ℃ for 3 h. Overnight hybridization with the 33 P-labeled cDNA in 6 mL of hybridization solution (6× SSC, 0.5% SDS, and 100 µg/mL salmon sperm DNA) was followed by stringent washing (0.1× SSC and 0.5% SDS at 65 ℃ for 1 h). Membranes were exposed to phosphor screens overnight and scanned using an FLA-3000A Plate/Fluorescent Image Analyzer (Fuji Photo Film, Tokyo, Japan). The radioactive intensity of each spot was linearly digitalized to 65 536 gray-grade in a pixel size of 50 µm in an Image Reader, and recorded with Array Gauge software (Fuji).

Data collection and analysis
After subtraction of background values (3±3) measured in an area where no cDNA was spotted, genes with intensities ≥2 were considered positive signals; this ensured that positives were distinguished from the background with a statistical confidence of >99.9%. Normalization among arrays was based on the sum of background-subtracted signals from all genes on the membrane [7] . The average hybridization intensities of two gallbladders were adopted to analyze the bioinformatics.

Touchdown reverse transcription polymerase chain reaction
For most of the genes, mRNA levels in the gallbladder samples were too low to be detected by standard dot blot and hybridization in situ, or even by conventional PCR, we used touch-down PCR to confirm the array hybridization results. All PCR products were verified by sequencing to avoid false positives.

Establishment of cDNA array system
Human cDNA clones randomly picked from cDNA libraries were terminally sequenced and compared with the Unigene database prior to their use in creating a cDNA array representing 17 000 genes or cDNA clusters ( Figure 1). The reproducibility of the cDNA array analysis was evaluated in multiple replicated tests in which cDNA probes independently generated from the same mRNA sample were hybridized to different replicates of the cDNA arrays. The results from these experiments were almost perfectly concordant with a scatterplot R 2 (the square of the Pearson correlation coefficient, which measures similarity in gene expression patterns) of 0.97-0.98 ( Figure 2). Of the 17 000 genes, only 0.2% showed >two-fold differences in their expression levels across different measurements. This showed that the cDNA array system was highly reproducible.

Gene list expressed in the gallbladder
In our work, a catalog of genes expressed in the human gallbladder was identified by cDNA array hybridization. The radio-intensities of corresponding spots on two parallel arrays were averaged. If the value was >2 and the variation <0.33 between the two samples, the signal was considered efficient.
Of the 17 000 genes tested, a total of 11 047 genes were expressed in human normal gallbladder tissue, which is more than the number of 3 754 predicted by Lewis [8] .

Top 10 genes expressed in the gallbladder
The 10 most highly expressed genes are listed in Table 1, and the top 3 breakpoint cluster region protein, uterine leiomyoma, 2; actin-related protein 2/3 complex, subunit 5 (16 ku) and eukaryotic translation initiation factor 4A, isoform 1, respectively.

Lipid metabolism-related genes and gallstone candidate genes in gallbladder
Totally 149 lipid metabolism-related genes were expressed in the gallbladder ( Table 2). Eighty-three of them were identified for the first time in gallbladder (results after searching Unigene and Pubmed). Lammert et al have listed 45 possible gallstone candidate genes based on previous knowledge, 24 of which were assembled in our array, and 19 of these 24 genes were lipid-related genes and expressed in the gallbladder. We selected four lipid-related genes randomly, and by touchdown reverse transcription polymerase chain reaction (RT-PCR) and sequencing, confirmed the results in cDNA array ( Figure 3).

DISCUSSION
A cDNA array representing 17 000 human genes or cDNA clusters was established. Compared with the cDNA array without hybridization intra membrane controls, the HIC in the cDNA array system significantly contributed to the evenness of hybridization among different parts of the array membrane and therefore improved the reliability of the array analysis (data not shown). Replicated examinations of the same sample indicated that only 0.2-0.3% of the genes spotted or 0.5% informative genes, might be false-positive signals. Because we chose the genes that were lower than 0.33 in the variation of average on two samples, the possibilities of false-positives were remote. Totally 11 047 genes were expressed in the gallbladder, which is almost thrice the number of 3 754 predicted by Lewis. The results remind us the importance of the role of gallbladder in the human body.
The human body contains both exogenous and endogenous cholesterol. Exogenous cholesterol comes from diet while the endogenous is synthesized inside the body by liver cells. Excess total cholesterol in the plasma will eventually become deposited on the arterial walls, leading to atherosclerosis [17] . High-density lipoprotein is the only lipoprotein that can transport cholesterol to the liver for degradation; this is called reverse cholesterol transport [18] . Liver cells catalyze cholesterol into bile acid, which is secreted into the biliary tract. Supersaturated cholesterol remains in the gallbladder bile, and can result in the formation of cholesterol monohydrate crystals, and finally gallstones [19] . Normally, the gallbladder epithelium absorbs high amounts of biliary cholesterol and phosphatidylcholine in a proportion determined by their molar ratio in the bile entering the lumen. This physiological lipid absorption continuously reduces biliary cholesterol molar percentage in the gallbladder, thus avoiding crystal precipitation. In contrast, gallbladder epithelium in patients affected with cholesterol gallstone disease may be hyperplastic and/or hypertrophic, and consistently absorbs cholesterol and phosphaticylcholine less efficiently, leading to a higher likelihood of cholesterol crystal precipitation [20] . A number of lipid metabolism genes or proteins are reportedly expressed in gallbladder tissue, including scavenger receptor class B type I(SR-BI) [21] , Apo B [22] , acyl-coA: cholesterol acyltransferase (Soat1) [23] , sodium-dependent bile acid transporter and organic anion transporting polypeptide(Oatp1) [24] , and the multidrug resistance protein (MRP) [25] . However, most of the lipid metabolism-related genes we identified in this study (Table 2) have not previously been reported in the gallbladder. From our results, we hypothesize that supersaturated lipids in bile lead to impaired regulation of the buffering ability of the gallbladder, resulting in the formation of gallstones. In our previous work, we observed that high cholesterol diet decreased the expression of cholecystokinin-A receptor in guinea pig, which led to the formation of gallstone [26] . Usually, it is nucleation factors or impaired motility of gallbladder that results in the formation of gallstone [27] . However, we found 19 lipid metabolism-related genes expressed in the gallbladder in our previous research [28,29] , and currently a total of 149 lipid metabolism-related genes were expressed in the gallbladder, and most of them were firstly identified (83 genes), thus, we think it is necessary to consider the regulation ability of gallbladder in lipid homeostasis while we study the mechanism of gallstone formation.
Lammert et al [2] collected 45 possible candidate genes in gastroenterology. Twenty-four of the 45 genes were included in our array, and 21 of them were expressed in the gallbladder. Among the 21 genes, 19 genes were lipidrelated genes, and they can be divided into five groups: lipid regulatory enzymes, lipoprotein receptors and related proteins, intracellular lipid transporters, membrane lipid transporters and lipid regulatory transcription factors. Previously those genes were believed to be expressed in the liver and/or intestinal cells only, not in gallbladder, and they could not be searched in Unigene and Pubmed for their association with gallbladder except LRPAP1 [30] , FABP1 [31] , SCP2 and ABCC2 [32] . It is well known that lipid metabolism has a close relationship with liver, but we found those genes were expressed in the gallbladder, too, suggesting gallbladder takes part in both digestion and lipid homeostasis. Those genes of patients with gallstones expressed in the gallbladder are involved in each step of gallstone formation, thus, if we study the differentially expressed genes between normal and gallstone affected gallbladder by cDNA hybridization, we may find some valuable gallstone-related genes.
In summary, we established a cDNA array and identified a catalog of genes expressed in normal gallbladder tissue, the number is greater than previous predictions. In addition, we identified the expression of 139 lipid metabolism-related genes, eighty-three of them were first discovered, suggesting that the gallbladder takes part in lipid homeostasis.