The Mechanism Of HnRNP A2/B1 In Malignant Transformation Of BEAS-2B Cells By Cr(?)

Objective: Heavy metal chromium has become the main cause of lung cancer in occupational population in recent years.The proportion of lung cancer caused by chromium pollution in non-professionals has increased in lockstep with air pollution.It is extra-urgent to explore the mechanism of lung cancer caused by chromium.HnRNP A2/B1 is an RNA-binding protein and involved in the RNA transcriptional regulation,pre-m RNA splicing,and m RNA degradation.HnRNP A2/B1 is found highly expressed in lung cancer tissues and used as an auxiliary measure for lung cancer detection and prognosis.We also found that the hnRNP A2/B1 high expression is also a great probability of chromium induced cell malignant transformation in vitro.Therefore,it is valuable for both theoretical and clinical studies of lung cancer to explore the mechanism of hnRNP A2/B1 in chromium-induced malignant transformation cells.In this thesis,we will explore 1.BEAS-2B cell(Human normal lung epithelial cell)was used to simulate environmental exposure of Cr(?)and establish an vitro model of Cr(?)-induced malignant transformation,and screen hnRNP A2/B1 high expression cell line,named BEAS-2B-Cr-HN cells.2.Whether hnRNP A2/B1 affects downstream MMP-9 through AKT in BEAS-2B-Cr-HN cells.3.Whether hnRNP A2/B1 affects cell migration and invasion in the BEAS-2B-Cr-HN cells through AKT.4.Whether apigenin inhibits hnRNP A2/B1 and has chemoprevention effects on BEAS-2B-Cr-HN cells.5.Whether apigenin inhibits migration and invasion of BEAS-2B-Cr-HN cells through hnRNP A2/B1.To sum up,we hope to clarify the role of hnRNP A2/B1 in lung cancer development and its specific mechanism.It is expected to provide theory for clinical lung cancer prevention,therapy and prognosis.Methods:1.Transwell and Western Blot transwell assay were used to identify the migration and invasion ability and related proteins of BEAS-2B cells which have been induced by Cr(?)for 6 months;2.Soft agar colony formation assay was used to identify the malignant transformation ability of BEAS-2B-Cr cells;3.Xenograft tumor assay was performed to validate the tumorigenicity of BEAS-2B-Cr cells;4.Western Blot assay was done to detect AKT,downstream MMP-9 and migration and invasion-related proteins expression under the conditions of hnRNP A2/B1 knockdown or overexpression in BEAS-2B-Cr-HN cells;5.Transwell assay was performed to detect the changes of the ability of migration and invasion under the conditions of hnRNP A2/B1 knockdown or overexpression in BEAS-2B-Cr-HN cells;6.Co-transfection of hnRNP A2/B1 overexpression plasmid and AKT knockdown plasmid in BEAS-2B-Cr-HN cells and explored the possibility of hnRNP A2/B1/AKT/MMP-9 signaling pathways;7.Western Blot assay was performed to detcet the effects of apigenin on hnRNP A2/B1 in BEAS-2B-Cr-HN cells;8.Transwell assay was done to detect the effects of apigenin on the ability of migration and invasion in BEAS-2B-Cr-HN cells.Results: 1.HnRNP A2/B1,migration and invasion-associated proteins elevated with the increase of Cr(?)in BEAS-2B-Cr cells in a certain concentration manner;2.The ability of migration and invasion increased in BEAS-2B-Cr cells compared to normal BEAS-2B cells;3.The colonies formed by BEAS-2B-Cr cells in soft agar increased in a Cr(?)concentration dependent manner;4.The BEAS-2B-Cr cell colonies in soft agar were picked and subcultured,colony propagation could form tumors in nude mice in xenograft tumor assay;5.AKT,MMP-9 and migration and invasion-associated proteins such as FAK,c-Src were significantly changed with the upregulation/downregulation of hnRNP A2/B1;6.HnRNP A2/B1 regulated MMP-9 through AKT;7.Apigenin can inhibit the expression of hnRNP A2/B1 in BEAS-2B-Cr-HN cells;8.Apigenin can inhibit the ability of migration and invasion in BEAS-2B-Cr-HN cells..Conclusion:1.The malignant transformation model of BEAS-2B cells induced by Cr(?)was successfully established and can be used as a model of long-term exposure to Cr(?)in vitro study;2.HnRNP A2/B1 regulates MMP-9 through AKT,thereby regulating the migration and invasion ability of BEAS-2B-Cr-HN cells;3.Apigenin can inhibit the expression of hnRNP A2/B1,thereby declining the ability of migration and invasion in BEAS-2B-Cr-HN cells.We hope what we found will provide new ideas for the prevention of lung cancer.