Analysis of AC3-33 gene expression in multiple organ cancer and adjacent normal tissue by RNA in situ hybridization

Introduction

AC3-33 (GenBank name: C3orf33, accession no. FLJ31139), also known as chromosome 3 open reading frame 33, encodes a classical secretory protein with a predicted molecular mass of 29.3 kDa (1). Transcription factor activator protein-1 (AP-1) is crucial in the regulation of cellular proliferation, transformation and death (2). Using a dual-luciferase reporter assay system, our previous study found that AC3-33 significantly inhibited AP-1 transcriptional activity. Further investigation indicated that AC3-33 significantly inhibited the transcriptional activity of Elk1 and c-jun, but not of c-fos; additionally, AC3-33 significantly inhibits Elk1 transcriptional activity via the extracellular-signal-regulated kinases 1/2/mitogen-activated protein kinases pathway. This occurs via disruption of ERK1/2 MAPK pathway (3). AC3-33 is highly expressed in a number of tissues, including the adrenal glands and cervix, and expression is comparatively significantly reduced in the human leukemia cell lines, K562 and KG1a (4). However, the expression of AC3-33 in multiple organ tumors and cancer-adjacent normal tissue remains to be elucidated.

In the present study, RNA in situ hybridization was used to detect the AC3-33 gene expression in multiple organ tumors and cancer-adjacent normal tissue. An improved understanding of the expression of AC3-33 may offer more information as to the role of AC3-33 in the pathological process of tumorigenesis, which may subsequently provide a new insight into AC3-33 and its potential applications in the treatment and diagnosis of human disease.

Materials and methods

Tissue microarray

Tissue microarray was purchased from Chaoying Biotechnology (Xian, China; MCN602). Specimens for microarray were obtained from a total of 56 cases of multiple organ tumors and adjacent normal tissue. This included 10 organ types (esophagus, stomach, colon, rectum, liver, lung, kidney, breast, uterine cervix, ovary), three tissue cores for cancerous tissue, three adjacent normal tissue cores for each organ and a single specimen per case. For all specimens, details of age, gender, organ, pathological diagnosis, clinical grade, TNM classification, clinical stage, specimen type and results were recorded. This study was approved by the ethics committee of Hebei United University (Tangshan, China).

Preparation of digoxigenin-labeled probes for RNA in situ hybridization

Sense and anti-sense probes that matched the AC3-33 corresponding sequence were: Anti-sense, TATAA*GTTCTCTGAACTTCAGTATTAAGGAGCAGTTGTTCATGTTGTCTTTC-DIG; and sense, GAAATG*TTAAACTACGTGGACGATTACGCCGAATAACTGAGAATGGTTTA-DIG. The asterisk indicates that the 3′ terminal was labeled with digoxigenin. All probes were synthesized by Sangon Biotech (Shanghai, China).

RNA in situ hybridization

Hybridization procedures were performed in this study as described. Hybridization conditions were as follows: Anti-sense or sense probe concentration, 20 ng/ul; anti-digoxigenin antibody (catalog no. ab76907; Abcam, Cambridge, UK) dilution, 1:500; was hing temperature, room temperature; dyeing temperature, 37°C; dyeing time, 2 h. Deparaffinized sections were mounted on Denhardt-coated glass slides (D2532; Sigma Aldrich, St. Louis, MO, USA) and treated with pepsin (0.25 mg/ml in diethylpyrocarbonate H2O-HCl) for 30 min in a 37°C water bath. The treated sections were then processed for in situ hybridization at 42–47°C for 24 h. The hybridization mixture contained the labeled oligonucleotide probe, 50% formamide, 10 mmol/l Tris-HCl, 1 mmol/l vanadyl-ribonucleoside complex (Sigma-Aldrich; catalog no. 94740), 1 mmol/l cetrimonium bromide (Sigma-Aldrich; catalog no. 855820, pH 7.0), 0.15 mol/l NaCl, 1 mmol/l EDTA (pH 7.0), 1×Denhardt's mixture and 10% dextran sulfate. Following hybridization, the slides were was hed three times, 30 min each time, in 0.1 mol/l Tris buffered saline (TBS) at 47°C, and subsequently treated with TBS (100 mmol/l Tris, pH 7.5, 150 mmol/l NaCl) containing 1% blocking reagent (Roche Diagnostics, Shanghai, China) and 0.03% Triton X-100 for 30 min at room temperature and incubated for 30 min with antidioxigenin alkaline phosphatase-conjugated antibodies (Roche Diagnostics) diluted at 1:4000 in TBS containing 0.03% Triton X-100 and a 1% blocking reagent. After was hing three times in TBS and 0.05% Tween, 15 min each time, the slides were rinsed in a diammonimum phosphate (DAP) buffer (100 mmol/l Tris, pH 9.5, 100 mmol/l NaCl, 50 mmol/l MgCl2) and subsequently hybridization signals were visualized using nitroblue tetrazolium and 5-brom-4-chlor-3-indolyl phosphate as substrates [DAP in 10% polyvinyl alcohol (Sigma-Aldrich; catalog no. 341584)]. Positive expression was determined to be 1+, 2+ and 3+ staining, and negative expression was observed as no staining.

Results

The association between AC3-33 expression and multiple pathological cell types

The AC3-33 gene expression in multiple organ and cancer-adjacent normal tissue was detected by RNA in situ hybridization. As shown in Table I and Fig. 1, the expression level of AC3-33 varies between the different tissues. The expression of AC3-33 is positive in squamous cell carcinoma (SCC) of the esophagus and adenocarcinoma of the rectum, but is negative in cancer-adjacent normal esophageal tissue and cancer-adjacent normal rectal tissue. AC3-33 exhibits positive expression in hepatocellular carcinoma and cancer-adjacent normal hepatic tissue, clear cell carcinoma of the kidney and cancer-adjacent normal kidney tissue. Negative AC3-33 expression was observed for adenocarcinoma of the stomach and cancer-adjacent normal gastric tissue, adenocarcinoma of the colon and cancer-adjacent normal colon tissue, serous adenocarcinoma of the ovary and cancer-adjacent normal ovarian tissue. AC3-33 exhibited positive expression in squamous cell carcinoma of the lung, invasive ductal carcinoma of the breast and squamous cell carcinoma of the uterine cervix. However, the expression of AC3-33 in cancer-adjacent normal breast tissue is partially positive.

Figure 1. Reflectance in situ hybridization showing expression of AC3-33 in different tumor tissue samples. AC3-33 in cancer adjacent normal hepatic tissue, clear cell carcinoma of the kidney and squamous cell carcinoma of uterine cervix.

Table I. The association between AC3-33 expression and multiple organ tumors and cancer adjacent normal tissue.

Table I.

The association between AC3-33 expression and multiple organ tumors and cancer adjacent normal tissue.

Organ	Pathological diagnosis	Tumors, n	AC3-33 mRNA-positive tumors (+/+/++)	AC3-33 mRNA-negative tumors
Esophagus	Squamous cell carcinoma	3	3 (1/2/0)	0
	Cancer adjacent normal esophageal tissue	3	0	3
Stomach	Adenocarcinoma	3	0	3
	Cancer adjacent normal gastric tissue	3	0	3
Colon	Adenocarcinoma	3	0	3
	Cancer adjacent normal colon tissue	3	0	3
Rectum	Adenocarcinoma	3	3 (1/2/0)	0
	Cancer adjacent normal rectal tissue	3	0	3
Liver	Hepatocellular carcinoma	3	3 (0/3/0)	0
	Cancer adjacent normal hepatic tissue	3	3 (0/0/3)	0
Lung	Squamous cell carcinoma	3	3 (1/2/0)	0
	Cancer adjacent normal lung tissue	1	1 (0/1/0)	0
Kidney	Clear cell carcinoma	3	3 (0/2/1)	0
	Cancer adjacent normal kidney tissue	3	3 (0/3/1)	0
Breast	Invasive ductal carcinoma	3	3 (0/2/1)	0
	Cancer adjacent normal breast tissue	4	3 (1/2/0)	1
Uterine cervix	Squamous cell carcinoma	3	3 (0/2/1)	0
Ovary	Serous adenocarcinoma	3	0	3
	Cancer adjacent normal ovarian tissue	3	0	3

[i] Staining was scored using a 0–3+ scale. 0, no staining; 1+, 2+ and 3+ indicate increased intensity of the staining. Sub-regions excluding necrosis, macrophages and infiltrated neutrophils and lymphocytes were selected and scored. The intensity score for an array spot is the average of all its sub-regions.

Discussion

With >300,000 new cases per year, cancer of the esophagus, predominantly SCC, is one of the 10 most frequently diagnosed tumor types (5). Esophageal cancer often occurs in developing countries, and the incidence is greatly different between different regions (5). The development of molecular oncology in the last decade has provided much information with regard to genetic abnormalities in cancer, and the clinical characteristics of cancer patients can now be predicted on the basis of these genetic abnormalities. Expression of N-myc (6), int-2 (7), cyclin D1 (8) and p53 (9) may be useful markers for predicting the outcome and distant organ metastasis in patients with SCC of the esophagus. The current study also found that AC3-33 exhibits positive expression in esophagal SCC, but negative expression in cancer-adjacent normal esophageal tissue. These results indicate that AC3-33 may be a novel prognostic factor.

Colorectal cancer (also known as colon cancer, rectal cancer, bowel cancer or colorectal adenocarcinoma) is a cancer due to uncontrolled cell growth in the colon or rectum, or in the appendix (10). Genetic analysis has shown that colon and rectal tumors are genetically the same cancer (11). Although the prognosis of rectal adenocarcinoma is associated with histopathological features, including invasion of the rectal wall or perirectal fat and lymph node involvement, a number of patients experience recurrence despite undergoing potentially curative procedures and early pathological staging (12–13). It has been proposed that genetic alterations acquired during tumor development may predict prognosis (14). For example, the expression of the p53 protein has been found to predict a worse prognosis in rectal adenocarcinoma (14). Similarly to SCC of the esophagus, the current study identified positive AC3-33 expression in rectal adenocarcinoma, but negative expression in cancer-adjacent normal rectal tissue.

In conclusion, the differential expression of AC3-33 may be significant in the development and progression of rectal adenocarcinoma and esophagal SCC, and may be used as a prognostic indicator. However, the mechanism of AC3-33 function appears to be complex and further investigations are required to elucidate the role and molecular mechanisms of AC3-33 in the development and progression of rectal adenocarcinoma and esophagal SCC.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (grant nos. 81072093; 30671092; 81302323), the Natural Science Foundation of Hebei Province (grant nos. C2009001260; C2013209024; C2014209140), the General Higher Education Young Talents Program of Hebei Province (BJ2014027), and the Science and Technology Support Program of Tangshan City (grant no. 14120208a).

References