Analysis Of SOCS3 Gene Expression In Human Lung Cancer And Study On Its Biological Effects In Lung Cancer Cells

The STAT family of transcription factors plays a critical role in regulating physiological and pathological responses to cytokine stimulation. Recently aberrant STAT activation was found in many malignanacies, and recently one of STAT members, STAT3, was identified being an oncogene. Suppressors of cytokine signaling(SOCSs) comprise a recently identified family of negative regulators of STAT mediated signaling pathway, and SOCSs family consists of 8 members(CIS and SOCS1 to SOCS7), among which the action of SOCS3 was becoming clearer. The finding of STAT3 binding sites in SOCS3 promoters indicates STAT3 as transcription factors play an role in expression of SOCS3 gene, and SOCS3 expression inhibits action of STAT3. Aberrant STAT3 expression and activation has been found in lung cancer specimens, however, only little is known about the expression of SOCS3 and its possible function in lung cancer. In this study, we addressed whether abnormal STAT3 mediates SOCS3 expression. Moreover, by transfecting SOCS3 expression vector into A549, we examine effects of SOCS3 gene on proliferation and expression of proto-genes(c-onc) in A549, which might provide a novel target for treating cancer.Methods1. Expressions of SOCS3 and STAT3 mRNA in human lung cancer were performed by RT-PCR.2. SOCS3 expression vectors and screening vectors were cotransfected into the A549 with lipofectmine2000 in vitro. Positive cell clones were screened with G418. RT-PCR and immunocytochemistry were used to analyze the expression of SOCS3 mRNA and protein in A549 before and after transfection.3. Expression of STAT3 mRNA and activated protein in A549 were detected by semi quantitive RT-PCR (sqRT-PCR) and western blot before and after transfection.4. The changes of cell proliferation were observed by flow cytometric DNA analysis, ~3H-TdR incorporation and MTT assay.5. Expression of c-Myc, c-Fos, c-Jun mRNA before and after transfection weredetected by sqRT-PCR respectively. Results1. Expressions of SOCS3 mRNA in human lung cancer and normal lung tissue were not detected by RT-PCR. Expressions of STAT3 mRNA in human lung cancer were significantly higher than normal lung tissue.2. The positive cell clones cotransfected with SOCS3 gene expression vectors and screening vectors were obtained after being screened with 500 ug/ml G418. The strong expression of SOCS3 mRNA and protein was respectively confirmed by RT-PCR and immunocytochemistry in cells transfected with SOCS3 gene.3. There were no significant difference in the expression of STAT3 mRNA between control cells and SOCS3 gene-transfected cells. The level of tyrosine-phosphorylated STAT3 in SOCS3 gene-transfected cells was lower significantly than that in control cells.4. Compared with control cells, cells transfected with SOCS3 gene decreased in growth by MTT; H-TdR incorporation was significantly lower. In addition, cells transfected with SOCS3 gene at Go/Gi increased, cells in S+G2/M decreased significantly.5. Compared with control cells, the level of c-Myc, c-Fos and c-Jun mRNA decreased significantly in SOCS3 gene-transfected cells respectively.Conclusions1. SOCS3 gene was not detected in human lung cancer and human lung adenocarcinoma cell strain A549. Expression of STAT3 mRNA in human lung cancer was significantly higher than normal lung tissue.2. The SOCS3 expression vectors and the screening vectors were cotransfected into A549 by lipofectmine 2000. RT-PCR and immuncytochemisty revealed strong expression of SOCS3 gene after transfection.3. SOCS3 gene inhibited tyrosine-phosphorylation of STAT3 protein, but had no effects on expression of STAT3 mRNA in A549 cells.4. SOCS3 might regulate c-Fos, c-Jun and c-Myc gene expression negatively by suppressing STAT3 tyrosine-phosphorylation, which inhibited A549 cells proliferation.