| Summary: | 碩士 === 中山醫學大學 === 毒理學研究所 === 92 === To identify genes that may play roles in lung carcinogenesis, we established two subtracted cDNA libraries from a patient's normal lung tissue and lung adenocarcinoma. Among genes that showed differential expression patterns between normal lung and lung cancer, we identified a clone named N6. The N6 message is highly expressed in normal lung tissues and greatly repressed in lung cancers. Our previous studies suggested that N6 might exist as a stable 3'-processed RNA of Slit2 message. We thus assumed that the expression pattern of Slit2 would be similar to N6, which is repressed in lung cancers. Indeed, RT-realtime PCR showed that the relative expression level of Slit2 is repressed to 3-to 192- fold in lung cancers compared to that of their normal counterpart tissues. Similar expression patterns were shown via microarray analyses with 25 lung adenocarcinoma patients. Slit2 is a secreted protein and is known to function as a chemorepellent in axon guidance and neuronal migration. Recent studies showed that the function of Slit2 is related to human cancers. Thus, Slit2 may play different roles in lung carcinogenesis from that in carcinogenesis of other cancers'. An alternative spliced form of Slit2 with deleted exon15 was present in all lung cancer cell lines and lung tissues tested. To study the role of Slit2 in lung cancer, full-length cDNA clones with or without of Slit2 was constructed for overexpression assay in the future. The expression of Slit2 in lung cancer cell lines is very low except in CL1 series. CL1 cell lines were selected from an in vitro invasion assay (kindly provided by Dr. Yang Pan-Chyr), and their invasion ability is CL1-0 < CL1-1 < CL1-5. The expression of Slit2 is very high in CL1-0 and CL1-1 but low in CL1-5. Although Slit2 expressions in CL1 series are different from other lung cancer cell lines, the high expression level of Slit2 is suitable for RNA interference (RNAi) experiment. Stable clones expressed shRNA (short hairpin RNA) that mimic siRNA for RNAi showed longer doubling time. Flow cytometry analysis showed that Slit2 siRNA decreased the population of S phase cells. Slit2 RNAi does not affect cell death nor migration ability. High level of Slit2 favors cell growth however cannot explain why other cancer cell lines have low level of Slit2. Thus, other experiments ane needed to address the possible roles of Slit2 in lung cancers.
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