| Objective:To investigate the role of voltage-gated chloride channel clc-2on non-small-cell lung cancer and the significance of the expression of it, we examine the gene expression of chloride channel clc-2in human non-small-cell lung cancer during occurrence, invasion and metastasis.Materials and Methods:Samples involved in this research were divided into four groups. They are:①Pulmonary adenocarcinoma;②squamous lung carcinomas;③Pulmonary tuberculosis;④ormal lung tissue besides lung cancer. The groups of non-small-cell lung cancer and benign pulmonary diseases are the experimental groups. The group of normal lung tissue beside lung cancer is the control group. In each experiment, group①and group②comply with the same principles of gender, pathological T staging and the degree of differentiation. The rest groups were selected at random for the comparison and analysis within those groups. From the results of semi-quantitative RT-PCR, Western blot and immunohistochemistry analysis, the expressions of clc-2mRNA and clc-2protein was determined, and difference in the the expression and distribution of clc-2among different kinds of cells and tissues were also assessed. Furthermore, we preliminary analysed the expression of clc-2in non-small-cell lung cancer cells. The48cases of pathological materials applied in this research were collected from freshly frozen tissues stored after surgical resection(2009.05-2010.08) from tissue library of Bethune no.3hospital in Jilin University. These cases have clearly postoperative pathologic diagnosis. The patients who provided specimens didn’t undergo preoperative systemic anticancer therapies such as radiotherapy and chemotherapy. Results:The mRNA level of clc-2in non-small-cell lung cancer cells:The result of RT-PCR detection showed that both referencing GAPDH gene band and clc-2gene were observed among all these tissue groups. The mRNA is reversely transcribed to cDNA with primers which have clc-2specific activity. Then PCR was carried out. The result indicates that an obvious specific fragment with length of250-500bp can be seen in group①and group②while the fragments shown in group③and group④is weak. Then we got the relative content by calculating the ratio of the content of the product of target gene in samples and GAPDH. It shows that the clc-2expression in non-small-cell lung cancer groups dramatically increases compared with other groups.The expression of clc-2protein in non-small-cell lung cancer cells:From Westernblot, a referencing P-Actin and an obviously specific clc-2protein band at98KD were both observed among all the tissue groups. The expression of clc-2protein in group①,②and③are higher than group④. Among the3groups, the expression of group②was highest and the difference between expression level in group①and in group③was statistically insignificant. Furthermore, the expression of clc-2protein in group④is fewer than experimental group. There is a certain degree of expression of chloride channel clc-2in each group.Immunohistochemistry analysis and the difference in the expression and distribution of clc-2protein:The immunohistochemistry analysis indicates that clc-2protein in group①and②were over-expressed, while that in group③and④were comparatively lower. The expression of clc-2in non-small-cell lung cancer cells of group②is distinctly higher than that in group①.The data mentioned above all confirm that clc-2is over-expressed in non-small-cell lung cancer cells. This reveals that clc-2plays an important role in the process of occurrence, invasion and metastasis of clinical lung cancer and the development of pulmonary disease as a voltage-gated ion channel through various mechanisms, clc-2may be a new molecular target for the clinical treatment of non-small-cell lung cancer, and it may provide the new approach to further study the association of clc-2with the pathogenesis and development issues that involved in lung cancer.Conclusion:The expression of clc-2mRNA, the genetic derivative of clc-2protein, in the non-small-cell lung cancer cells was significantly higher than that in normal lung tissues and tissues with benign pulmonary diseases.The expression of clc-2protein is higher in tissues with benign pulmonary diseases and malignancies than normal lung tissues. The results show that the expression of clc-2protein in squamous lung carcinomas is highest while difference between the pulmonary adenocarcinoma group and the group with benign pulmonary diseases was statistically insignificant.The immunohistochemistry analysis reveals that clc-2is widely distributed in non-small-cell lung cancer cells in a great amount. While only a few can be found in the group with benign pulmonary diseases and normal lung tissues. Furthermore, it is indicated that the expression of clc-2protein in squamous carcinomas group is higher than that in adenocarcinoma group.Taken together, these data suggest that chloride channel2(clc-2) plays a regulatory role as a voltage-gated ion channel in the process of occurrence, invasion and metastasis of non-small-cell lung cancer from gene and protein levels. |
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