An Experimental Study On The Screening Of Lung Cancer Metastatic-related Genes By Suppression Subtractive Hybridization

Background and object Invasion and metastasis were not only the malignant markers and characteristics of lung cancer, but also the leading cause of failure to treatment and patient's death. Lung cancer metastasis was a complicated process in which multiple stages and multiple genes were involved. Although the modification of gene expression had been studied in many researches, lung cancer metastasis remained the major basic and clinical problem which needed to be clarified and solved nowadays and even in the future. One of the hotspots to solve this problem is cloning and screening the new metastatic-related genes with the new molecular biology techniques. Suppression subtractive hybridization (SSH) was a new method of phenotypic cloning which had the characteristics of convenience, good sensitivity and reduplication. For this reason, it was an ideal tool for particular phenotypic cloning of aim genes. With the development of SSH, it had been applying more and more in cancer metastasis research field.The object of this study was to construct the subtracted cDNA libraries of different metastastic potential cell lines NL9980 and L9981 by SSH method, and screened the cDNA segments which might be related to lung cancer metastasis. The results of this study could provide a valuable resource for cloning the new lung cancer metastatic-related genes and even set up the foundation of understanding the molecular biology mechanism of lung cancer metastasis.Method (1) The forward and reverse subtracted cDNA library were constructed in the different metastastic potential large cell lung cancer cell lines NL9980 and L9981 by SSH; (2) The positive clones were preliminarily screened by blue-white colony which was based on theα-complementary principal, and precisely identified by PCR; (3) The forward and reverse subtracted libraries were screened and identified by dot blot so as to obtain the clones corresponding to differential expression segments; (4) The sequences of differential expression segments were analyzed by DNA sequence; (5) Searching and comparing the sequences of different expressed segments by NCBI BLAST tools. Searching database included Human build 36 RNA alternate and reference assemblies, Database of GenBank+EMBL+DDBJ sequences from EST Divisions and All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, environmental samples or phase 0, 1 or 2 HTGS sequences).Results (1) The forward and reverse subtracted cDNA libraries of different metastastic potential large cell lung cancer cell lines were successfully constructed; (2) With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained; (3) 55 clones were successfully sequenced in the forward subtracted library. Homolog analysis confirmed 23 differential expression segments, which were similar to the human genes already known, including NME2, NPM1, MT 2A, HSPE1, TAFIA, EPRS, PX19 and EIF3S9 et al; (4) 31 clones were successfully sequenced in the reverse subtracted library. Homolog analysis confirmed 16 differentially expressed segments. 15 of them were similar to the human genes already known, including ANXA2, TUBB, PKN2, GNAS, EEF1A1, SSR2 and RPLPO et al. Only one segment had partial homology to known human genes. This segment was supposed to be the new EST segments which might not have been cloned. The segment was successfully submitted to GenBank and embodied by GenBank.The GenBank accession number was ES452735.Conclusion (1) The forward and reverse subtracted cDNA libraries of different metastastic potential large cell lung cancer cell lines were successfully constructed by SSH; (2) NME2, NPM1, MT 2A, HSPE1, TAF1A, EPRS, PX19 and EIF3S9 were differentially expressed in human low metastasis large cell lung cancer cell line NL9980. The function of these genes included cell apoptosis, transcription, assembling of ribosome, antioxidation, clearance of free radical and negative regulation of Rho activity, which might be related to inhibit the metastasis of lung cancer; (3) ANXA2, TUBB, PKN2, GNAS, EEF1A1, SSR2 and RPLPO were differentially expressed in human high metastasis large cell lung cancer cell line L9981. The function of these genes involved transcription, cytoskeleton, protein translation and cell signaling, which might be related to promoting the metastasis of lung cancer; (4) One new EST segment was found in the reverse subtracted cDNA library. This segment was supposed to be a novel lung cancer metastatic-related gene.