Expression of Connexin 43 and E-cadherin Protein and mRNA in Non-small Cell Lung Cancers in Chinese Patients

Lung cancer is the most common cancer in the world. Non-small cell lung cancer (NSCLC) is the most common bronchial tumor, which can be classified in two major histological subtypes, the adenocarcinoma (AC) and the squamous cell carcinoma (SCC). In 2008, among males, the highest lung cancer incidence rates are in Eastern and Southern Europe, North America, Micronesia and Polynesia, and Eastern Asia, and Chinese females have higher lung cancer rates than those in certain European countries such as Germany and Italy. Lung cancer rates are also increasing in China (Parkin, 2001; Jemal et al., 2011). Connexins was a group of homologous proteins which from the inter membrane channels of gap junctions (Proksch et al., 2008). The connexins are the products of an identified gene family which has both highly conserved and highly divergent regions. The variety contributes to the wide range of functional properties of gap junction (Sohl and Willecke, 2004; Laird, 2006; Solan and Lampe, 2009). The abnormal connexin expression and distribution are closely related to the tumor formation (Laird, 2006; Cronier et al, 2008).The wild-type connexin genes, which


Introduction
Lung cancer is the most common cancer in the world.Non-small cell lung cancer (NSCLC) is the most common bronchial tumor, which can be classified in two major histological subtypes, the adenocarcinoma (AC) and the squamous cell carcinoma (SCC).In 2008, among males, the highest lung cancer incidence rates are in Eastern and Southern Europe, North America, Micronesia and Polynesia, and Eastern Asia, and Chinese females have higher lung cancer rates than those in certain European countries such as Germany and Italy.Lung cancer rates are also increasing in China (Parkin, 2001;Jemal et al., 2011).
Connexins was a group of homologous proteins which from the inter membrane channels of gap junctions (Proksch et al., 2008).The connexins are the products of an identified gene family which has both highly conserved and highly divergent regions.The variety contributes to the wide range of functional properties of gap junction (Sohl and Willecke, 2004;Laird, 2006;Solan and Lampe, 2009).
The abnormal connexin expression and distribution are closely related to the tumor formation (Laird, 2006;Cronier et al, 2008).The wild-type connexin genes, which
Adhesion molecules play an important role in tumor metastasis by controlling the gap junction function (Fujimoto et al., 1997;Pertz et al., 1999;Kobayashi et al., 2007;Dittmar et al., 2007;Jeanes et al., 2008;Wheelock et al., 2008;Makrilia et al., 2009).Cell surface adhesion molecules, especially Ca 2+ -dependent adhesion molecule E-cadherin, can provide a stable structure that induces cell membrane move closer to another cell membrane and promote two connected bodies relative to each other, so as to form a gap junction (Jongen et al., 1991;Fujimoto et al., 1997;Wheelock et al., 2008).These calcium-dependent adhesion molecules on the regulation of gap junction function are affected by calcium ion concentration changes (Pertz et al., 1999).
Although so many studies have proven that connexin and E-cadherin are related with the tumor progress, it is not clear that how of connexin and E-cadherin protein and mRNA expression in Chinese patients with lung cancer.The present study tried to investigate Cx43 and E-cadherin protein and mRNA expression in Chinese patients with NSCLC, so as to understand possible Cx43 and E-cadherin protein and mRNA expression as biomarkers for judging the metastasis and prognosis of NSCLC.

Case information
Fifty Chinese patients with non-small cell lung cancer including 39 male and 11 female, aged 26-77 years, average aged 46.5 years, were studied.The experiments were approved by the Ethics Committee of 117 hospital of People's Liberation Army and all of the patients were signed the informed consents.The patients did not accept the treatments of chemotherapy and radiotherapy before the study.The diseases were diagnosed by the pathological methods after the surgery and biopsy, in which 32 cases had lymph node metastasis and 18 not, 25 cases suffered with adenocarcinoma, 19 with squamous carcinoma and 7 with large cell undifferentiated carcinoma.Tumor-nodemetastasis (TNM) stage designations were according to UICC standard, and all the tumors were classified as stage I, II, III and IV respectively (n=12,15,20 and 3).For the pathological grade, 10 cases were in welldifferentiation, 20 in moderate-differentiation and 20 in poor-differentiation.For 20 control samples, the lung tissues, which were diagnosed as normal by the pathological methods, were taken from the tumor tissue 3 cm or more away in the patients with NSCLC.

Materials
Rabbit anti-human monoclonal Cx43 antibody, Rabbit anti-human monoclonal E-cadherin antibody and ABC kit were purchased from Zymed Co., Ltd., USA.The specific steps of ABC immunohistochemical method were operated according to the manual instruction.

Immunohistochemical evaluation
C x 4 3 e x p r e s s i o n e v a l u a t i o n : A f t e r A B C immunohistochemical test, Cx43 protein showed brown stains that distributed on the adjacent membranes between lung cancer cells.The Cx43 expression evaluation standard was depended on the brown stain intensity and extent.It was considered as positive if over 10% lung cancer had brown stains, otherwise negative.
E-cadherin expression evaluation: After ABC immunohistochemical test, E-cadherin protein showed brown stains that distributed on the membrane of lung cancer cells.The E-cadherin expression evaluation standard was depended on the brown stain intensity and extent.It was considered as positive if over 60% lung cancer had brown stains, otherwise negative.

RT-PCR
During surgery, the lung tissues isolated were quickly semi-frozen.Using TRIzol reagent isolated the total RNA from each tissue sample.TRIzol reagent 0.3 ml maintains the integrity of the RNA during the sample lysis.Addition of 0.3 ml chloroform was followed by centrifugation and the aqueous phase was transferred.The RNA is recovered by precipitation with isopropyl alcohol.The extracted RNA was treated with DNase according to the manufacturer's protocol.Thereafter, to 1 μg of RNA, the sample was mixed with 1 μl of DNase (1 U/μl, Invitrogen), 10× DNase reaction buffer, and adjusted to 10 μl with DEPC-water.The mixed solutions were incubated at 25 °C for 15 min and stopped by adding 1 μl of 25mM EDTA at 65 °C for 10 min.
A review of GenBank using BLAST program showed that these primers were specific for Cx43 and (Gene Bank accession No. NC000006) and E-cadherin mRNA (Gene Bank accession No. NM001670.2) as shown in Table 1.

Statistical analysis
All values were expressed as the positive expression rate (%) and mean ± standard error of the mean (SEM).The data were analyzed by SPSS 17.0, which χ 2 test and two-way analysis of variance (ANOVA), followed by the Bonferroni test and one-way ANOVA followed by Dunnett test and Newmann-Keuls test.P < 0.05 was considered statistically significant.

The relationship between Cx43 and E-cadherin protein expression
In 50 Chinese patients with NSCLC, there were 21 cases (42%) showing the positive expression of both Cx43 protein and E-cadherin protein; there were 2 cases (4%) showing the negative expression of Cx43 protein and the positive expression of E-cadherin protein; there were 6 cases (12%) showing the positive expression of Cx43 protein and the negative expression of E-cadherin protein; and there were 21 cases (42%) showing the negative  There was very significant (χ 2 =23.86,P<0.01, γ=0.96) (Table 2).

Correlation of Cx43 or E-cadherin protein expression and clinical pathology
In 50 NSCLC patients, the Cx43 positive expression was correlated with the histological type, clinical stage and cancer cell differentiation, but without the lymph node metastasis; E-cadherin protein expression was associated with clinical stage, cancer cell differentiation and lymph node metastasis, but without histological type.

Correlation of Cx43 or E-cadherin mRNA expression and clinical pathology
In 50 NSCLC patients, the relative quantity of the Cx43 or E-cadherin mRNA expression was calculated by comparing with the neighboring health lung tissue of each lung cancer sample.As the results of the protein expressin above, the relative quantity of the Cx43 or E-cadherin mRNA expression was correlated with the the histological type, clinical stage, cancer cell differentiation and the lymph node metastasis (Table 4).

Discussion
Reduced connexin (Cx) 43 gene expression has been shown in most of lung tumors and cancer cell line (Chen et al., 2003;Kato et al., 2005;Brehm et al., 2006;Xu et al., 2008;Jinn et al., 2010;Losa et al., 2011).Jinn et al. detected connexin 32 (Cx32) and Cx43 expression levels by immunohistochemistry in human lung cancer tissue (Chen et al., 2005).They found that Cx43 expression level reduced, but Cx32 expression was not, and Cx43 expression level was correlation with the degree of differentiation of lung cancer.With Cx43 expression decreasing, the degree of differentiation was lower, and the prognosis was worse.In our research, 50 cases of primary lung cancer were studied by using Immunohistochemistry.The date showed that Cx43 expression positive rate was 54% (27/50), whose number and distribution were different and showed a significant heterogeneity.Alveolar cells, macrophages, epithelial cells, lymphocytes had a strong expression of Cx43, but have no significant relationship with Cx43 expression in lung cancer cells.
In the study of Cx43 expression and clinical stage, histological grade of lung cancer, we found that with tumor progression, Cx43 expression gradually decreased, and the difference of the positive rate between stageI~II and stage III~IV was statistically significant (P <0.05), and Cx43 positive expression rate of moderately, well differentiated lung cancer cases was significantly higher than that in poorly differentiated cancer (P <0.05).These results suggested that, Cx43 expression was corrected with histological grade and clinical stage of lung cancer.The tumor cells are easy to escape normal growth control and host immune surveillance because of decreased Cx43 expression.Therefore, decreased Cx43 expression may be one of the reasons that lung cancer cells in the middle, late stage and poorly differentiated are easy to spread and distant metastasis.These show that Cx43 plays an important role in judging the prognosis of patients with lung cancer.In Lung cancer with or without lymph node metastasis, the difference of Cx43 positive expression rate was not statistically significant (P>0.05).In addition, adenocarcinoma was prone to invasiveness and blood transfer.In this group, Cx43-positive expression rate in lung adenocarcinoma was significantly higher than squamous cell carcinoma and large cell undifferentiated carcinoma.It is not yet clear whether Cx43 positive expression of lung cancer cells are prone to hematogenous shift and the expression have no significant relationship with lymph node metastasis, the exact mechanism awaits further studies.
The study of 54 cases with NSCLC conducted by Smyth et al. showed that E-acdherin and its associated proteins were associated with the differentiation of lung cancer (Smythe et al., 1999), but not with its clinical stage and histological type.However, Chen XF et al. believed that E-cadherin and nm23 functions asputative metastasissuppressor genes in the progression of malignancies in NSCLC (Jinn et al., 1998).Aberrationsin mRNA expression were observed and associated with degrees of histological differentiation and increasing stage as well as lymph node metastases, although no change of genetic structure was detected in the E-cadherin and nm23 genes.Our study showed that (1) E-cadherin expression level of lung cancer without lymph node metastasis was higher than those with lymph node metastasis; (2) E-cadherin positive expression in stage I~II was significantly higher than that in stage III~IV; (3) E-cadherin positive expression rate of moderately, well differentiated lung cancer was significantly higher than the poorly differentiated level of lung cancer; (4) E-cadherin expression had no correction with the tissue type of NSCLC.These demonstrated that E-cadherin played a key role in lung cancer occurrence, development and metastasis, which could be used as an important prognostic indicator.Musil et al. reported that E-cadherin fens were transected into cell communication dysfunction of tumor cell lines, whose gap junctional intercellular communication increased (Musil et al., 1990).The E-cadherin expression showed high in the cell while connexin protein increased.The data indicated that E-cadherin might have control function for transcription and translation of connexin protein mRNA (Naus and Laird, 2010).
In our study, the results also showed that the relationship of E-cadherin expression lever with Cx43 protein expression was positive; indicating that in the development and metastasis of lung cancer, Cx43 and E-cadherin played a common control function.
In summary, the investigation demonstrated that dysfunction of Cx43 and E-cadherin had a role in progression of NSCLC, and that the examination of Cx43 and E-cadherin expression could provide experimental evidence for clinical treatment.In clinical work, detection of the expression of these two indicators had significance in the metastasis and prognosis of lung cancer.

Table 3 . Correlation of Cx43 or E-cadherin Protein Expression and Clinical Pathology
*P<0.05, **P<0.01compared with the other group in the same clinical pathology expression of both Cx43 protein and E-cadherin protein.