Isolation And Functional Identification Of Two Novel Metastasis Associated Genes Of Human Lung Cancer

Tumor metastasis, a little understanding process, is still a great challenge faced by researchers and doctors of oncology, partly because the process consists of a series of steps, including invasion and mobilization, survived in the circulation, arrested in a distant capillary bed, extravasations and multiplies in organ parenchyma. All those steps must be completed by tumor cells if a metastasis was to be developed, so it is difficult to model the entire course using experimental approaches in vivo. Metastasis is a highly selective process that is influenced by both the intrinsic properties of tumor cells and host factors. In the 1970's Fidler pointed out that primary tumors contain cells with heterogeneous metastatic properties and the characteristic of metastases was acquired through multiple genetic alterations, so that, metastasis could even originate from the proliferation of single malignant cells. It was now widely accepted that cancer is attributed to the accumulation of genetic alteration in cells, but how to screen those genes related to metastasis from thousands of genes expressing in cancer cells had been another challenge until the emergence of technologies based on phenotype cloning theory. Using these methods, people could isolate genes by virtue of their effect alone and without requiring prior knowledge of their biochemical function or map position, further more, phenotype cloning has the potential to save a great deal of repetitive work involving analysis of multiple markers. There were many phenotype cloning methods such as mRNA Differential Display, Representation Difference Analysis and Suppression Subtractive Hybridization, the down to date phenotype cloning techniques, had also been used in our research.In the test of SSH, a pair of high and low-metastatic monoclonal cell strains, termed PLA801 D and PLA801 C, were derived from the same parental lung giant cell carcinoma cell line PLA801 by monoclonal technology were used as model cells. The highly metastatic cell subpopulation, PLA801D acted as tester in SSH and the poorly metastatic cell subpopulation PLA801C was driver. Microarray, a high throughput gene-analysis technology, was used to screen library generated by SSH efficiently. Sequences of the library were spotted on glass slides respectively and hybridized with two-colored fluorescence probes acquired from cDNA of model cells by reverse transcription. Then, some clones with high expressing level in the results of microarray had been confirmed tohave high expressing by RT-PCR and Northern blot.Finally, 79 ESTs were found to be expressed two times or more in D than in C. After sequencing, many were found to be identified with known genes encoding proteins of below: Cytokines and proteins correlated to receptor; kinases and related proteins; other proteins including enzymes, heat shock protein, receptors, proteins of cell skeleton and mitochondria, products of onco-genes etc; some hypothetical proteins, which might impact tumor metastasis in some ways that were still uncharted. Further bioinformatics analysis led to the discovery of two novel metastasis related genes, pmag-1 and 2, their homologous sequences in SSH library were NO.3 and 83. The pmag-1 gene was identical with BC006236 (the ID number of GenBank) except for "TG" insertion after the 144th base pair and pmag-2 had the same sequence as BC002420. The 8.5kb long pmag-1 gene composes with four exons and locates on the chromosome of 4q21. The gene of pmag-2, which is made up by 9 exons, has a full length of 5.2kb on genome and its chromosome site is 2q35. Both cDNA sequences have open reading frames, prognosticated promoters upstream the first exon of each genes and polyA signal "AATAAA" in their 3' ends. And the theories product of pmag-1 was proved to be located in cytoplasm by the PMAG1 -RFP (red fluorescent protein) fusion protein expressing plasmid pDsRed1-N1M1Both sense and anti-sense orientation of each gene were inserted into the multiply cloning sites of pcDNA3. The constructs were transfected into different ce...