Expression And Function Of GSTA1 On Lung Cancer Cells

Currently, lung cancer is one of the most common cancers in the world with the highest incidence and mortality, as well the first major cancer in china, the incidence and mortality of which is growing rapidly. However, because of its insidious onset, prognostic 、three-year and five-year survival rate of it is far from satisfactory, the main reason is the lack of early diagnosis and lung cancer markers. In addition to reduce the incidence on etiology prevention, it is particularly important to find the lung cancer marker for the enhancement of the efficacy of early diagnosis.The diagnosis and screening of lung cancer is still depend on the diagnostic imaging(X-ray, CT, MRI and PET-CT), blood enzyme,biochemical diagnosis(including serum cancer marker, biochenmical- enzymic and immunology index) and histological diagnosis at present. However, the golden stander of diagnosis of malignant tumor is molecular pathology method such as tissue section and immunohistochemistry. The diagnostic imaging has great limitations in the diagnosis in subclinical stage.It can detect cancer earlier with the tumor marker as observed indicators than diagnostic imaging.Therefore, the screening and identification of highly specific tumor markers of lung cancer has been the focus and difficulty of the study in the early diagnosis of lung cancer. In the early stage of the study, we selected GSTA1 tumor antigens of lung cancer with dozens of lung cancer-specific binding polypeptides by Phage display technology, which has not been reported in literature.On this basis, this paper was performed the effects of GSTA1 to the proliferation and apoptosis of lung cancer, and to clarify the mechanism.The first part: The location and the protein expression of GSTA1 in different tumor cells and normal cell were detected by confocal laser analysis. Results show GSTA1 localization in the cell membranes and the relative GSTA1 protein levels in lung cancer cells are much higher than the control group.The expression of m RNA and protein of GSTA1 of different cancer cells(A549、H460、SPCA1、LTEP-A2、Hep G2) and the lung normal cells(MRC-5) were validated by Western blotting and Real-time PCR.Results shown that m RNA and protein expression of cancer cells lines, especially the A549 cells were higher than MRC-5 by Real-time PCR and Western blotting.On the result, A549 cells were selected as the further study subjects.The second part:Screening optimal GSTA1 small interference RNA by Western blotting and Real-time PCR for the sake of building virus vector(GSTA1 lower expression group and GSTA1 over-expression group).After infecting A549 cells, the effect of GSTA1 in A549 cells growth was observed by MTT, Hoechst33258 and flow cytometry. Results show that the over-expression of GSTA1 will accelerate the proliferation of A549 lung cancer cells, on the contrary, the proliferation of A549 cells will be inhibited by the silence of GSTA1 expression which will promote the cell apotosis.The third part:The nude mouse transplantation model of A549 lung cancer was build to study the effect of GSTA1 on A549 cell lines in vivo. Respectively, the A549 lung cancer cells transfected by Si-GSTA1 virus vector and GSTA1 vector were inoculated to the left axillary of nude mice. The curves of tumors formation were drew by measuring the tumor volume and weight every week in three weeks. Respectively GSTA1 protein expression level and m RNA expression level of the tumor tissues were detected by using Western blotting methods and Real – time PCR method. Results show that tumor capacity, growth rate,tumor volume and weight of GSTA1 group are significantly higher than that of the control group(p < 0.05),On the contrary,that of Si-GSTA1 group are much lower than that of the control group(p < 0.05).The protein levels and the m RNA levels of the GSTA1 group are significantly higher than that of the control group,with statistical significance(p < 0.05),that of the Si-GSTA1 group are significantly lower than the group(p<0.05) by Western blot and Real-time PCR respectively.It confirms that the inhibitory proliferation of lung cancer cells in vivo by the under- expression of GSTA1.We explored the effects of GSTA1 on lung cancer cell proliferation and apoptosis in vitro and in vivo,and found that the over-expression of GSTA1 can promote the growth of cancer, suggesting the degree of malignancy of lung cancer tissue by detecting the expression of GSTA1 in the tissues.The fourth part:The effect of apigenin on the proliferation of the lung cancer cells was detected by MTT, the change of lung cancer cell nucleus morphology was detected by flow cytometry method, relative protein levels of Bax and Bcl-2 were detected by western blotting in order to evaluate the apotosis of lung cancer cells caused by apigenin.The protein levels and m RNA of GSTA1 of different lung cancer cells were detected by western blotting and Real-time PCR in order to study what a role can GSTA1 play in the cell proliferation and apotosis.Results show that apigenin can inhibit lung cancer cell proliferation and promote apoptosis on concentration- and time- dependent way.The GSTA1 protein levels and m RNA levels of the apigenin groups are much lower than that of the control group(p<0.05),suggesting that the expression of GSTA1 can be inhibited by apigenin.In summary, GSTA1 may play an impotant role in the occurrence and development of human lung cancer, not only as a valuable human lung cancer biomarker, but also as a potential lung cancer candidate targets. Apigenin is expected to be a new kind of traditional Chinese medicine fighting against lung cancer which can inhibit the expression of GSTA1 and promote the apoptosis of lung cancer with its low toxicity, abundant source and the attractive in price and quality.