一个在肺腺癌细胞系中差异表达cDNA片段的定位及表达分析

Mapping and Expression Analysis of a Different Expression cDNA Fragment from Lung Adenocarcinoma Cell Line

为了研究和克隆肺癌转移相关候选基因 ,探讨肺癌发生及转移的分子基础 ,应用细胞培养、cDNA克隆、North ern印记杂交和生物信息学技术分析了在细胞来源相同 ,但转移能力不同的肺腺癌细胞系AGZY83 a和Anip973中差异表达片段OPB7 1在人不同组织中和不同人肺癌细胞系中的表达情况 ,并应用RH定位技术对该片段进行了基因定位。表明OPB7 1与已知基因同源性差 ,该基因在正常人多种组织中有表达 ,心肌和骨骼肌中高表达 ,转录本均为 3 0kb左右。在不同人肺癌细胞系中存在该基因的表达差异 ,高转移潜能、低分化及高浸润的细胞系中呈高表达趋势 ,且表达的片段大小略有差别。提示OPB7 1是一个具有广泛表达谱的基因 。

Lung cancer is one of the most common malignant tumors in humans. Metastasis is the basic biological feature of malignant tumors, which is the main cause of death.Molecular mechanism of metastasis is still unclear, although lots of studies have been done in tumor metastasis. To study and explore the molecular basis of metastasis in lung cancer, and isolate tumor metastasis related genes, two human lung adenocarcinoma cell lines AGZY 83 a and Anip 973 were chosen as research materials.The Anip973 was derived from AGZY83 a, but manifested much higher metastasis potential than the parent line.Using mRNA differential display technique, an unknown cDNA fragment, OPB 7 1,which is over expressive in Anip973 cell line, was obtained.It was used as a template to isolate its corresponding cDNA through dbEST searching and PCR.To search and clone lung adenocarcinoma metastasis related candidate gene, and to explore the molecular basis of development of lung carcinoma,differential expression of OPB 7 1 cDNA fragment among 9 human lung adenocarcinoma cell lines and 12 normal human tissues were detected using cell culture, cDNA clone, Northern blot analysis and bioinformation technology. Results showed that there were significant differences in OPB 7 1 expression among 9 human lung adenocarcinoma cell lines. High expression tendency was obserued in Anip973 cell line with high metastasis potential, TKB 18 cell line with high invasion potential and GLC 82 cell line with low differentiation potential. Besides, a bigger fragment can be found in Anip973 cell line on the Northern blot hybridization. The 3 0kb transcriptions were found in various tissues. Over expression in heart and skeletal muscle could be observed, whereas expression in spleen, liver, kidney, placental and lung could be found except colon, thyroid gland and small intestine. These manifests indicate that OPB 7 1 gene has a wide rage expression in human multiple tissues. A 1.0kb cDNA fragment was acquired by linking up EST fragments homologous match 5′ end and PCR. BLAST analysis revealed that OPB 7 1 gene has extremely low sequence identity with any known genes from GenBank and any sequences from EST database. The chromosomal localization of it was determined by RH location method. The OPB 7 1 fragment was localized to chromosome 1p31～34. That OPB 7 1 gene has an extensive expression pattern, may be a novel tumor gene related to lung carcinoma.Further research needs to be done to obtain the full length cDNA of OPB 7 1 gene. It will be helpful to investigate the expression in lung cancer cases and other tumor tissues for further determining the function of OPB 7 1 gene in development of tumor.