HMGA2 Mediates The Atg12-Atg5 Conjugation In Cr(Ⅵ)-induced Autophagy In A549 Cells

Chromium is the silver metal.Divalent chromium(Cr(II)),Trivalent chromium(Cr(III))and Hexavalent chromium(Cr(VI))are three stable chromium oxidation states.Cr(VI)is serious toxic.International Agency for Research on Cancer(IARC)had affirmed Cr(VI)as a group I carcinogen since 1990.A large number of epidemiological studies and animal studies have shown that exposured to the environment of Cr(VI)are closely related with cancers and tumors.Some studies reported that low-dose Cr(VI)could induce autophagy.Autophagy promoted tumor by phagocytosis of damaged organelles or cytosolic components in tumor cells,maintenance the activity of tumor cells,and enhancement of tumor cell proliferation.Autophagy was associated with cancer,which could promote cancer,and provided a low-oxygen and nutrient-limiting condition.Autophagy is in progress by autophagy-related(ATG)proteins.HMGA2 is a structural transcription factors,involves in gene transcription regulation,chromatin condensation,DNA damage repair and a series of nuclear events.HMGA2 proteins are abundant in embryogenesis and many malignant neoplasms.In our previous study,we found that cadmium(Cd)-induced ROS formation provoked HMGA2 up-regulation,caused cell cycle changes and led to cell proliferation in MRC-5 cells.So,in this study,we aimed to know the function of HMGA2 in autophagy in Cr(VI)-treated A549 cells and BALB/c mice.Objective: To demonstrate the effects of low-dose Cr(VI)on autophagy,investigated the involvement of HMGA2 in Cr(VI)-induced autophagy.Methods: In this study,A549 cells and HEK 293 cells were treated as in vitro models and BALB/c mice were treated as in vivo models.The lung tissues of Cr(VI)-treated BALB/c mice were used for Western blot to observe the expression of HMGA1,HMGA2 and autophagy-related proteins.Transmission electron microscopy was used to investigate the effects on formation of autophagosomes in Cr(VI)-treated BALB/c mice.Cr(VI)-treated A549 cells were determined the proliferated changes by MTT assay on different concentrations of chromium.And examined the effect of 3-MA on Cr(VI)-induced proliferation.Acridine orange(AO)and Transmission electron microscopy were used to measure the number of autophagolysosomes(AVOs)and autophagosomes(AVs)in Cr(VI)-treated A549 cells.Western blot was used to observe the expression of autophagy-related proteins in A549 cells.To investigate the function of HMGA2 in Cr(VI)-induced autophagy,A549 cells were transfected by si RNA.The effect of HMGA2 on the autophagosomes and autophagolysosomes numbers of A549 cells was investigated using Transmission electron microscopy and AO assay.To elucidate the involvement of HMGA2 in Cr(VI)-induced autophagy in A549 cells,expressions of autophagy-related proteins were further investigated with Western blot analysis.And q PCR was performed to determine the gene expression levels of Atg5 and Atg12 in HMGA2-si RNA A549 cells.Plasmid pc DNA 3.1-HMGA2 was transfected in HEK 293 cells.Cells were used for Western blot to observe the expression of autophagy-related proteins.Results: After the treatment the lung tissues of BALB/c mice were used for Western blot,the expressions of autophagy-related proteins(LC3-II,Atg12-Atg5,Atg4,Atg5,Atg7,Atg12 and Beclin 1),HMGA1 and HMGA2 were all increased significantly in Cr(VI)-treated mice compared with the control mice,and the protein of p62 was decreased significantly compared with the control(P<0.05 and P<0.01).And the analysis of Transmission electron microscopy showed that the quantification of the AVs numbers per viable cell in lung tissues increased significantly compared to the control.These results demonstrated that Cr(VI)induced autophagy in lung tissues of BALB/c mice.In in vitro studies,using A549 cells,we found that the expressions of autophagy-related proteins(LC3-II,Atg12-Atg5 and Atg4),HMGA1 and HMGA2 were all increased significantly,while p62 level decreased significantly in Cr(VI)-treated A549 cells compared to the control.The effect of Cr(VI)-induced autophagy was further proven by the appearance of autophagosomes and autophagolysosomes in Cr(VI)-treated A549 cells examined by Transmission electron microscopy and AO assay.In Cr(VI)-treated HMGA2-si RNA transfected A549 cells,the number of autophagolysosomes and autophagosomes was decreased,the protein level of LC3-II and Atg12-Atg5 wasdepleted,the level of p62 was accumulated,while the level of Atg5 and Atg12 was not change.These results demonstrated that HMGA2 might mediate the Atg12-Atg5 conjugation in Cr(VI)-induced autophagy in A549 cells.To determine this effect,q PCR was used to observe the m RNA level of Atg5 and Atg12.The results showed that the m RNA levels of Atg5 and Atg12 did not changed significantly in Cr(VI)-treated HMGA2-si RNA A549 cells compared with the only Cr(VI)-treated group.The expression of HMGA2 was up-regulated in HEK 293 cells by transfection of plasmid pc DNA 3.1-HMGA2.The results of Western blot showed that the expression of LC3-II and Atg12-Atg5 proteins were significantly increased,while the level of p62 was depleted in the pc DNA 3.1-HMGA2 cells.And the expression of Atg5 and Atg12 did not change either.This result confirmed that HMGA2 played an important role in autophagy according to commanding the conjugation of Atg12-Atg5.Conclusion: HMGA2 might mediate the Atg12-Atg5 conjugation in Cr(VI)-induced autophagy.