Significance and Expression of Aquaporin 1, 3, 8 in Cervical Carcinoma in Xinjiang Uygur Women of China

The aquaporins (AQPs) are a family of hydrophobic, small, and integrated transmembrane glucoproteins (30kDa monomer) that distribute widespreadly in the bodies of animals and plants, closely related with transmembrane water transport, and so far the discovery of AQPs, 13 mammalian homologs (AQPs 0~12) have been identified (Verkman et al., 2008). All vital movements of cancer cells depend on water microenvironment, especially transmembrane transport. Recent studies alluded to the key role of AQPs in human carcinogenesis. For example, AQPs abnormaly expressed in the tumor cells and stroma of the female malignant tumors such as breast carcinoma and endometrial carcinoma (Pan et al., 2008; Otterbach et al., 2010). The research on correlation of AQPs with cervical cancer is uncommon in document reports. In this study, we probed the effects of AQP1, AQP3, AQP8 on cervical carcinogenesis, invasion, metastasis and their clinical significance for Xinjinag Uygur women of China.


Introduction
The aquaporins (AQPs) are a family of hydrophobic, small, and integrated transmembrane glucoproteins (30kDa monomer) that distribute widespreadly in the bodies of animals and plants, closely related with Gynecology, Third Affiliated Hospital of Xinjiang Medical University. These samples were obtained from 11th month of 2008 to 8 th month of 2010, and included 10 cases of mild cervicitis, 10 cases of early cervical cancer and 10 cases of advanced cervical cancer. Paraformaldehydefixed and paraffin-embedded tissues were used to immunohistochemistry, including 36 cases of CIN2-3, 98 cases of cervical carcinoma and 42 cases of mild cervicitis. Tumor clinical stage was according to FIGO (federation international of gynecology and obstetrics).

Real-time PCR
Total RNA from human cervical lesion tissues was extracted using Trizol, cDNA synthesis was performed according to the manufacturer's instructions. Real-time PCR procedure: fore-denaturalization 95 ℃ 5 min, denaturalization 95 ℃ 30 s, renaturation 60 ℃ 30 s, amplification 72 ℃ 30 s, for 35 cycles, extension 72 ℃ 5 min. PCR products were identificated by 1.5% agarose gel electrophoresis. Expressing abundance of AQP genes was calculated according to AQP/actin. Primer sequences are listed in (Table 1), actin being considered as a house keeping gene.

Immunofluorescence analysis
Tissues were embedded by OCT, frozen sections were made and fixed for 10 min in cold acetone, washed 3 times with PBS, 5 min /time and stained with primary antibody of anti-AQP1, AQP3, AQP8 overnight at 4 ℃, followed by fluorescein isocyanate-labeled secondary antibody for 45 min. After washing, mounting media including buffering glycerine was added, and coverslips were mounted. PBS instead of primary antibody was used as negative control, AQP positive normal cervical tissue was used as control. Repeated experiments were performed for doubtful cases.

Statistical analysis
Data were expressed as the mean ± standard deviation. Statistical differences among groups were compared using the one-ANOVA for measurement data and Χ 2 test for numeration data. P<0.05 was considered significant.

Localization and abnormally high expression of AQP1, AQP3, AQP8 in cervical carcinoma tissues
Immunofluorescence and immunohistochemistry assays were performed. AQP1, AQP3, AQP8 proteins expressed in mild cervicitis, CIN and cervical carcinoma. AQP1 was observed to be predominantly localized in the microvascular endothelial cell in the stroma of mild cervicitis and cervical carcinoma (Figure 2A), and AQP1 expression in cervical carcinoma was significantly higher than CIN and mild cervicitis (Table 3). AQP3, AQP8 localized in the membrane of normal squamous epithelium and carcinoma cells, local positive signals were more common than diffuse positive signals ( Figure 2B, Figure  2C, Figure 3, Figure 4). AQP3 expression gradually increased in mild cervicitis, CIN2-3, and cervical cancer groups (P<0.05). AQP8 expression was highest in CIN2-3 group which was 91.67%, AQP8 expression in cervical carcinoma was higher than in mild cervicitis (P<0.05) (Table 4).

Discussion
Cervical cancer is generally the most common cancer in developing countries and the second most common in women globally (Farivar et al., 2012). In Xinjiang of China, especially South of Xinjiang, incidence of cervical carcinoma for Uygur women is very high, and its prevalence is 527/100,000, and about 80% patients are in the advanced stage while visiting doctors' office (Peng et al., 2003).Studies confirmed that AQPs overexpressed in many kinds of tumor tissues, their vascular endothelial cell and tumor cell lines, and this hinted that AQPs were close to tumorigenesis and tumor progress. For example, AQP5 overexpression was related with in cell growth and metastasis of human breast cancer (Jung et al., 2011). Human AQP1 expression was close to brain glioma (Hayashi et al., 2007). AQP3 and AQP5 expression increased in the gastric carcinoma tissues, both AQP3 and AQP5 expression were associated with lymph node metastasis and lymphovascular invasion in patients (Shen et al., 2010). But there is few reports concerning the expression and role of AQPs in human cervical carcinogenesis so far.
In this study, we detected AQP1, AQP3, AQP8 expression in cervical lesion tissues of Uygur women by real-time PCR, immunofluorescence, and immunohistochemistry, respectively, and analyzed correlation of their expression with clinicopathologic characteristics of patients. We found that AQP1, AQP3, AQP8 exhibited differential expression among cervical carcinoma, corresponding CIN and normal tissues at the levels of mRNA and protein. Immunohistochemical and immunofluorescence assays showed that AQP1 protein was expressed mainly in vascular endothelial cell, and AQP3, AQP8 was localized in the epithelial membrane of mild cervicitis, CIN and cervical carcinoma tissues. AQP1, AQP3 expression were detected remarkably stronger in carcinoma tissues than corresponding CIN and mild cervicitis, AQP8 expression was highest in CIN, and AQP1, AQP3, AQP8 expression increased markedly in advanced stage, metastatic lymph node, larger tumor size, deeper infiltration. MVD is used to represent tumor angiogenesis, and it is an accepted reliable indicator to evaluate tumor angiogenesis. In this study, MVD is used to represent AQP1 protein expression. Our results showed that AQP1 was clos to tumor vascularization and played an important role in the invasion and metastasis of cervical carcinoma. There are no blood vessels in the initial phase of tumor growth, tumor tissues mainly acquire nutrition by interstitial fluid diffusion. When a tumor grows to 1-2 mm, the formation of neovessels will occur. Formation of neoplasm metastasis focus demands neovessels provide nutrition too. Some animal experiment confirmed that in AQP1-knockout mice, tumor growth slowed down, angiogenesis decreased and general necrosis emerged from tumor tissues (Saadoun et al., 2005). Our results indicated that AQP3, AQP8 may play a certain role in advancement, infiltration and metastasis of cervical carcinomas. Similar reports were also seen in relative documents. For example, Kusayama et al. (2011) reported that a high level expression of AQP3 was observed in tumor areas of primary squamous cell carcinomas such as esophageal and lingual cancers, and lymph node metastasis. Galamb et al. (2007) pointed that AQP8 overexpressed in colorectal cancer by using gene chips. In this study, AQP8 expression rate in CIN was 91.67%, higher than in cervical cancer and normal tissues, we proposed that AQP8 played an important role in the process of precancerous lesion CIN transforming into cervical cancer. But Jablonski et al. (2007) reported that AQP8 expression down-regulated in mouse hepatocellular carcinoma model. Our results do not coincide with theirs. It is necessary to deeply probe AQP8 effects in tumorigenesis.
AQPs are a family of small transmembrane proteins that facilitate osmotically driven water transport, we propose that AQPs overexpression in cervical cancer may increase tumor cells permeability to water to alter tumor cells volume and shape, accordingly facilitate advancement, infiltration and metastasis of cervical carcinoma.
In summary, AQP1, AQP3, AQP8 expression abnormally increase in cervical carcinoma of Uygur women of China, and facilitate the advancement, invasion and metastasis, suggesting AQPs as targets potential for curing cervical carcinoma in future.