Inactivation Of MiR-100 Combined With Arsenic Treatment Enhances The Malignant Transformation Of BEAS-2B Cells And Its Molecular Mechanism

[BACKGROUND]The occurrence of tumor is a complex process.We have been committed to the research of tumor pathogenesis and intervention.The role of miRNAs in cancer is of great importance.The abnormal expression of miRNAs might promote the carcinogenesis of lung cancer.MiR-100 has been shown to be involved in this biologic process.The research about the relationship between miR-100 and tumor has made significant progresses,but the data so far are still controversial.Some investigators found that human bronchial epithelial cells(BEAS-2B cells)long-term contacted with sodium arsenic increased proliferation,migration and gained anchorage-independent growth,increasing the risk of lung cancer..There has been a lot of research on the mechanism of arsenic carcinogenesis,and some achievements have been made.Long-term exposure of arsenic induces EMT and promotes cell malignant transformation.In this study,we hypothesize that inactivation of miR-100 combined with low concentration of arsenic exposure could promote the malignant transformation of BEAS-2B cells by promoting EMT.To test this hypothesis,lentiviral vectors were used to mediate the inhibition of miR-100 expression,low-dose of As2O3 treated chronically BEAS-2B cells.Thus we intend to study the effect on the cellular behavior of BEAS-2B after being treated with low-dose of As2O3 chronically and silencing the expression of miR-100 by infecting with miR-100 inhibitor lentivirus vector.The mouse xenograft model was used to investigate the cell malignant growth in vivo,and western blot was used to detect EMT related marker expressions.Our results showed that,the inactivation of miR-100 combined with arsenic treatment significantly promoted the proliferation,migration and anchorage-independent growth of BEAS-2B cells in vitro,and tumorigenesis in vivo.Consistently,we found that as the epithelial marker,the expression of E-cadherin declined.Instead,the expressions of the mesenchymal markers increased,such as ZEBl,vimentin,and so on.Our data indicate that inactivation of miR-100 combined with chronic arsenic treatment promotes tumorigenicity of BEAS-2B cells via activation of EMT.This novel insight may help us to better understand the pathogenesis of lung cancer and facilitate the development of miRNA-directed diagnostics and cancer prevention.[METHODS]1.Construct the miR-100 inhibitor lentivirus vector and stable infect BEAS-2B cells with it.2.Detect the expression of miR-100 after infecting with miR-100 inhibitor lentivirus vector by RT-PCR.3.Low-dose of As2O3 chronically treated BEAS-2B(miR-100-inhibitor)and BEAS-2B(miR-NC)cells,take the 0,20 and 40 passages of cells for experiments.4.Detect the proliferation of BEAS-2B cells treated with arsenic after miR-100 inactivation by MTT assay,Flow cytometry,plate colony assay;observe the anchored-independent growth capacity of BEAS-2B cells by soft agar assay;evaluate the adhesion and migration of BEAS-2B(miR-100-inhibitor)and BEAS-2B(miR-NC)cell treated with arsenic by Transwell migration assay and adhesion test.5.The mouse xenograft model was used to investigate the cell malignant growth in vivo.6.Western blotting was used to detect EMT related marker expressions.Meanwhile,comparison of the effects of acute and chronic arsenic treatment on EMT marker proteins in BEAS-2B cells[RESULTS]1.We have established BEAS-2B cell lines infecting with stable expression of miR-100 inhibitor.The expression of miR-100 in BEAS-2B cells infecting with miR-100 inhibitor lentivirus vector was down-regulated obviously.Furthermore,BEAS-2B cells were contacted with sodium arsenic.2.The results of MTT and plate colony assay showed that the proliferation ability of BEAS-2B cells increased when the expression of miR-100 was down-regulated;Furthermore,chronic arsenic treatment promotes the proliferation3.The cell cycle was detected by flow cytometry,and chronic arsenic treatment led to the increase of the cell proportion in the s-phase of the BEAS-2B cells.However,in the low expression of miR-100 cell group,the S stage accounted for 62.47%.4.Soft agar assay showed that inactivation of miR-100 combined with chronic arsenic treatment promotes anchorage-independent growth of BEAS-2B cells5.Adhesion and transwell migration assay showed that inhibition of miR-100 promoted the ability of migration of BEAS-2B cells,while chronic arsenic treatment increased migration synergistically.6.Tumor xenograft model showed that the miR-100 inactivation combined with chronic arsenic treatment significantly increased the growth of the mammary tumors.7.Western blotting showed that treatment of BEAS-2B(miR-100-inhibitor)and BEAS-2B(miR-NC)cells with arsenic,induced EMT,as evidenced by reduction of E-cadherin,and induction of ZEB1,vimentin,and the matrix metalloproteinases MMP-3,MMP-9,and nuclear p-catenin.The similar results was observed in BEAS-2B(Inh-miR-100)cells treated with acute arsenic.[Conclusion]1.Inactivation of miR-100 combined with chronic arsenic exposure could promoted the cell malignant transformation of human bronchial epithelial cells(BEAS-2B cells).2.Inactivation of miR-100 combined with arsenic treatment enhances the malignant transformation of BEAS-2B cells via stimulating epithelial-mesenchymal transition...