Department Of Thoracic Surgery, Union Hospital Of Huazhong University Of Science And Technology

Purpose Cancer stem cells show poor sensitivity to chemotherapeutics, which often become the root of tumor recurrence. So far, numerous studies have focused on development of novel drugs specific to tumor stem cells, which would be one of the most effective treatment of cancers. Of note, to separate and purify lung cancer stem cells by surface marker, study their gene expression patterns and then find out the essential functional protein for oncogenesis as new drug targets for lung cancer treatment, is a dynamic research direction and may provide important information for developing novel therapy. In this study, we detect the expression of Galectin-3 in CD133+ pulmonary adenocarcinoma cells and investigate its biological functions by synthesized galectin-3-specific siRNA.Methods In the first part, we sepatated CD133+ pulmonary adenocarcinoma cells from excised pulmonary adenocarcinoma specimens of 10 patients by magnetic activated cell sorting (MACS) and determined the percentage of CD133+ cells in different cell populations by flow cytometry(FCM). In the second part, we detected the expression of Galectin-3 in lung cancer cells on both transcriptional and translational level by FQRT-PCR and Western blot respectively, whereas extracellular expression by ELISA.In the third part, the studies were aimed to investigate the biological functions of Galectin-3 in CD133+ cells. CD133+ cells were transfected with galectin-3-specific siRNA to explore the affects of galectin-3 inhibition on cancer cell growth, metastasis and its ability of inducing CD8+T cell apoptosis. The interference efficiency of galectin-3 expression by siRNA was determined by FQRT-PCR and Western blot, and then the proliferation rate of CD133+ lung cancer cells post-transfected was detected by colony formation assay and MTT test. Boyden chamber model was used to observe the metastasis ability of CD133+ lung cancer cells. Finally, Annexin V and PI double staining flow cytometry was performed to determine CD8+T cell apoptosis induced by supernatant of CD133+ lung cancer cellsResults Cells expressing CD133 constituted 90% of the CD133+subpopulation after separation by MACS whereas only 1.2% in the unsorted cells. Expression of galectin-3 was 1.24 folds,1.5 folds and 2 folds higher in CD133+cells than in CD133-cells detected by FQRT-PCR, Western Blot and ELISA respectively(p<0.05). siRNA was able to interfere with galectin-3 and the interference efficiency was 80.3%, showed by FQRT-PCR and Western Blot. The MTT assay showed that proliferation rate of CD133+ cell treated with siRNA was decreased correlated with incubation time after transfection. At the point 96h after transfection, the viability rate of CD133+ cell treated with siRNA was significantly lower than CD133+ cell without treatment(p<0.05), the former 75 percents of the later, whereas there was no much difference between the viability rates of siRNA group and siRNA mutant group at the point 48h after transfection. The colony formation rate of CD133+ cells post-transfected (60.12%±4.32%) was lower than that of non-treated group (87.34%±4.89%). Boyden chamber model showed that siRNA transfected CD133+ lung cancer cells were less invasive than non-treated group,68±7 cells compared with 100±8 cells (p<0.05). Apoptosis detection by flow cytometry showed that the supernatants of CD133+ cells induced more efficiently apoptosis of CD8+T cells (18.6±3.5) compared with siRNA transfected cells(8.2%), which could be also inhibited by lactose, anti-galectin-3 polyclonal antibody.Conclusion Our data indicate that galectin-3 is expressed at a relatively higher level in CD133+ lung adenocarcinoma cells and could induce CD8+T cell apoptosis in vitro, both of which can be down-regulated by galectin-3 siRNA. Galectin-3-specific siRNA also reduce proliferation and metastasis abilities of CD133+ lung cancer cells, as well as their capacity to induce CD8+T cell apoptosis. These findings indicate Galectin-3 may play an important role during oncogenesis, implying a potential therapeutic target for pulmonary adenocarcinoma.