Expression and localization of apolipoprotein M in human colorectal tissues
© Luo et al; licensee BioMed Central Ltd. 2010
Received: 31 August 2010
Accepted: 16 September 2010
Published: 16 September 2010
It has been well documented that apolipoprotein M (apoM) is principally expressed in the liver and kidney. However we found that there was weak apoM expression in other tissues or organs too, which could not be ignored. In the present study, we therefore examined apoM expression in human colorectal tissues including cancer tissues, cancer adjacent normal tissues, polyp tissues and normal mucosa as well as inflammatory mucosa.
Tissue samples were collected from patients who underwent surgical resection or endoscopic examination. ApoM mRNA levels were determined by the real-time RT-PCR and apoM protein mass were examined by the immunohistochemistry.
ApoM protein can be detected in all colorectal tissues. However, apoM protein mass were significantly lower in the cancer tissues than its matched adjacent normal tissues, polyp tissues, normal mucosa and inflammatory mucosa. In parallel, apoM mRNA levels in the colorectal cancer tissues (0.0536 ± 0.0131) were also significantly lower than those in their adjacent normal tissues (0.1907 ± 0.0563) (P = 0.033). Interestingly, apoM mRNA levels in colorectal cancer tissues were statistic significant higher in the patients with lymph node metastasis than the patients without lymph node metastasis (P = 0.008). Patients under Dukes' C and D stages had much higher apoM mRNA levels than patients under Dukes' A and B stages (P = 0.034).
It is concluded that apoM could also be expressed in human colorectal tissues besides liver and kidney. ApoM mRNA levels in the colorectal cancer tissues were significantly increased in the patients with lymph node metastasis. Whether increased apoM expression in the patients with lymph node metastasis being related to patients' prognosis and the physiopathological importance of apoM expression in colorectal tissues need further investigation.
Apolipoprotein M (apoM) was first identified and characterized in 1999 . Human apoM gene is located in the major histocompatibility complex (MHC) class III locus on chromosome 6p21.31 (chromosome 17 in mouse) and contains six exons . It has been well documented that human apoM is exclusively expressed in the hepatocytes in liver and tubular epithelial cell in kidney, and small amounts were also found in fetal liver and fetal kidney in most investigations . However, in our previous study, during the human embryogenesis, relative high apoM mRNA could also be found in small intestine, stomach and skeletal muscle besides kidney and liver in the early stages of embryogenesis . More recently, Calayir et al., reported that apoM could be expressed in human colorectal adenocarcinoma cell line, Caco-2 cells , which indicates that apoM might be expressed in human colorectal tissues too. In the present study we investigated whether apoM presented in human colorectal tissues and further investigated the difference of apoM expression pattern in colorectal tissues from patients with colorectal cancer and benign diseases.
Materials and methods
Patients and samples
Both colorectal cancer tissues and their adjacent normal tissues were collected from 20 patients (13 men and 7 women, aged from 38 to 82 years old, median age was 60 years old) who underwent surgical resection in the hospital. All patients' colon or rectal tumors were histologically classified as the adenocarcinoma and none patient had chemotherapy or radiotherapy before operation. Immediately after resection, small pieces of both tumor and its adjacent normal tissues were collected with snap-frozen and stored in liquid nitrogen before further experiments. Parts of samples were fixed in 10% (v/v) formalin and embedded in paraffin until use. Twenty three colorectal biopsy samples (7 normal mucosa, 6 inflammatory mucosa and 10 polyp tissues), during endoscopic examinations, were collected for the immunohistochemistry. The present study was approved by the local ethics committee and all patients gave informed consent.
Total RNA extraction and real-time RT-PCR
Sequences of primers and probes
Gene (GenBank ID)
Sequence (5' to 3')
Normal mucosa, inflammatory mucosa, polyp tissues and both colorectal cancer tissues as well as their adjacent normal tissues from all patients were cut into sections under 3-μm thick for the immunohistochemical analyses. In brief, formalin-fixed, paraffin wax embedded tissues were dewaxed in xylene and rehydrated in graded ethanol solutions. Antigen retrieval was performed by incubating the slides at 100°C for 30 min in a 10 mmol/L citrate buffer (pH 6.0). Slides were cooled and immersed in a 0.3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity, and rinsed in PBS for 5 min, incubated with 5% bovine serum albumin at room temperature for 15 min to eliminate non-specific binding of primary antibody. Sections were then incubated with primary monoclonal antibodies against apoM (1:50 dilutions in PBS) (BD Biosciences) at 4°C overnight. PBS was used as negative control instead of the primary antibody. Then sections were incubated with HRP-labeled goat anti-mouse/rabbit secondary antibody (Ready to use, Maixin Biotechnology Limited Corporation, Fuzhou, China) for 30 min at room temperature. Diaminobenzidene (DAB) was used as the chromogen and hematoxylin as the nuclear counterstain. The sections were dehydrated, cleared and mounted.
Evaluation of apoM immunohistochemical staining
Two pathologists were invited to examine the slides. In brief, five high-power fields (×200) were randomly selected. Intensity of apoM immunochemical staining and percentage of positive cells were assessed. The extent of the staining was categorized into five semi-quantitative classes based on the percentage of positive cells: 0 (< 5% positive cells), 1 (6-25% positive cells), 2 (26-50% positive cells), 3 (51-75% positive cells) and 4 (> 75% positive cells) . The intensity of cytoplasmic and membrane staining was also semi-quantitatively determined on a scale of 0-3 as follow: 0 (negative), 1 (weak positive), 2 (moderate positive) and 3 (strong positive). Final staining score were obtained by multiplication of the intensity and the percentage scores and were represented as: 0 (negative), weak positive (1-4), moderate positive (5-8) and strong positive (9-12) . In the present study we practically divided patients into two groups: low apoM expression group (score 0-8) and high apoM expression group (score 9-12).
Results are expressed as means ± SE. Data were analyzed with the GraphPad Prism 4.0 software package (GraphPad Software, Inc., San Diego, California, USA). The paired t test, χ2-test and Mann Whitney test were applied for the statistical analyses. A P value less than 0.05 was considered to be significant.
Immunohistochemical staining of apoM in normal mucosa, inflammatory mucosa, polyp tissues and cancer tissues
Intensity of apoM immunochemical staining in the colorectal cancer tissues and their adjacent normal tissues
Colorectal cancer tissue
Adjacent normal tissue
Low apoM HIS (score: 0-8)
High apoM HIS (score: 9-12)
Scores of apoM immunohistochemical staining in different colorectal tissues
ApoM mRNA level and protein concentration in cancer tissue and its adjacent normal tissue
Correlation of apoM mRNA levels and protein mass to patients' clinical characteristics
Correlation of apoM mRNA levels and protein levels with patients' clinical characteristics
ApoM mRNA levels
ApoM protein levels
Ratio of ApoM/GAPDH (Median)
Staining score (Median)
Tumor size (diameter)
< 6 cm
≥ 6 cm
Lymph node metastasis
Tumor (T) stage†
The tissue distribution, location of cellular expression, structure feature [1–3, 7, 8], and potential roles [9–15] as well as regulation of apoM [9, 16–28] have been gradually elucidated since it was found and initially identified from human chylomicrons in 1999. According to the literature, apoM is exclusively expressed in the hepatocytes in liver and in the tubular epithelial cells in kidney [2, 3], and small amounts apoM expressed in the fetal liver and kidney too . More recently, Calayir et al. reported that apoM expression could be up-regulated by the LXR agonist in human colorectal adenocarcinoma cell line, Caco-2 cells , which indicates that apoM might be also expressed in human colorectal tissues. However, our previous study  and Calayir's report demonstrated that LXR agonist could significantly inhibit apoM expression in HepG2 cells , which indicates that apoM in liver and in colon may have different physiological functions. We have previously reported that plasma apoM levels in the hepatocellular carcinoma (HCC) patients were significantly increased than those in the normal subjects . However, both apoM mRNA levels and apoM protein mass in the HCC tissues were significantly lower than those in their adjacent tissues .
In the present study, we demonstrated that apoM was abundantly expressed in normal colorectal tissues and with inhibited levels found in the colorectal cancer tissues. Interestingly, apoM mRNA levels were significantly increased in the colorectal cancer tissues of patients with lymph node metastasis than the patients without lymph node metastasis. Moreover we demonstrated that apoM mRNA levels were much higher in the patients with Dukes' stages 3 and 4 than the Dukes' stages 1 and 2, which indicates that apoM mRNA levels in cancer tissues may have a potential positive correlation to the tumor progress, although apoM protein mass were not parallel increased in the patients with lymph node metastasis. This may suggest, although apoM expression is obviously decreased in the colorectal cancer tissues than in normal tissues, there are certain factors or mechanism could up-regulate apoM expression during the progress of the cancers. Perhaps apoM mRNA levels in colorectal cancer tissues can be determined for evaluating the metastasis, and further for patient's prognosis. Certainly it needs a standardized method for determining apoM mRNA level in the cancer tissues. In addition, it is still need to elucidate weather production of apoM in intestine influences plasma apoM pool in human.
Some proteins, such as alpha-fetoprotein (AFP), are normally produced in the developing embryo and fetus by hepatocytes, gastrointestinal cells and yolk sac cells. Synthesis of AFP stops at birth and its increased level is associated with pathological conditions . In our previous study, apoM expression could also be found in small intestine, stomach and skeletal muscle in the early stages of embryogenesis . We suspected whether apoM expression in adult colorectal tissues was influenced under cancer condition. In the present study, we examined apoM expression in normal mucosa, inflammatory mucosa, polyp and cancer tissues, which shows clearly that these tissues do express apoM, although apoM expression in the cancer tissues were significantly inhibited.
In conclusion, the present study revealed that apoM could also be abundantly expressed in human colorectal tissues, although the physiopathological importance of this expression and if it could influence plasma apoM pool are unknown yet. Moreover we demonstrated that apoM mRNA levels were significantly increased in the colorectal cancer tissues of patients with lymph node metastasis, although apoM expression in cancer tissues was generally much lower than in normal colorectal tissues. Determination of apoM mRNA in the surgical resected colorectal cancer tissues may have potential benefit for evaluating patient's prognosis.
We thank senior pathologists Changqing Lu, Qing Li, Bo Tian, Yuandong Chen and Prof. Tongyu Chen for their expert suggestions and technical assistances. We are also thankful to Dr. Haitao Wang for providing samples of patients with colorectal cancer and his critical suggestions. This work was supported by the National Nature Science Foundation of China (Project No. 30972955).
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