Hexavalent chromium Cr (VI) is one of the environmental carcinogens, which is considered as highly toxic metal to human health. According to epidemiologic studies, long term Cr (VI) exposure is highly associated with increased rate of lung cancer risks. Underlying mechanisms of Cr (VI) induced carcinogenicity and its induced malignant cellular transformation need to be explored.
Transcription factor Nrf2 is a redox sensitive and it is involved in regulating heavy metal induced oxidative stress balancing under cellular environment. Interestingly, Nrf2 plays in two different roles in metal induced carcinogenesis. At initiation process of metal induced cellular transformation Nrf2 protects cells from oxidative stress and acts as a tumor suppressor. However, at later stage of malignant cell transformation Nrf2 is constitutively activated and acts as an oncogenic by preventing malignant cells from excessive oxidative stress. Therefore, there is a need to investigate the underlying reason of Nrf2 activation in hexavalent chromium induced cellular transformation.
Nrf2 signaling pathway is regulated by many different cellular signaling pathways including autophagy dysregulation, ER stress dysregulation and epigenetic reprogramming. Among them, epigenetic alterations such as DNA methylation, histone modifications and microRNA dysregulation have been growing area to understand Cr (VI) carcinogenesis as well as abnormal activity of Nrf2 in metal induced malignancy.
The purpose of this study was to determine the underlying mechanisms of Nrf2 upregulation and microRNA dysregulation in Cr-T cells. Our microRNA differential expression studies among parental B2B and Cr-T cells revealed that oxidative stress sensitive miR-27a/b expression is downregulated in Cr-T cells compared with B2B. Additionally, miR-27a/b contains the seed sequences which could potentially target redox sensitive transcription factor Nrf2. Here, we aimed to determine underlying mechanisms of Nrf2 upregulation and loss of activity of miR-27a/b in Cr-T cells, and miR-27a/b functional role in Cr-T cells.
By exposing immortalized and nontumorigenic human lung epithelial BEAS-2B (B2B) cells to Cr (VI) at lower dose for longer time, we created in vitro model BEAS-Cr (Cr-T). This cell culture model of malignant chromium transformation allowed us to do research on Cr (VI) induced carcinogenicity. Throughout our studies, Cr (VI) free medium treated passaged control B2B and Cr-T cells were used.
Our results collectively demonstrate one of the possible mechanisms for constitutive activation of Nrf2 is through loss of miR-27a/b in Cr-T cells. We also highlighted the Cr (VI) induced oxidative stress and ROS generation could be one reason of dysregulation of Nrf2 and miR-27a/b by showing the restoration result of Nrf2 and miR-27a/b expression in ROS inhibitor treated Cr-T cells. Potentially, Nrf2 3’UTR is a direct target of both miR-27a and miR-27b. Additionally, miR-27a/b acts as a tumor suppressor in Cr-T cells by inhibiting cellular proliferation, migration, colony formation, angiogenesis and tumor growth rate of Cr-T cells. Those microRNAs may reflect its tumor suppressor function by targeting 3’UTR of Nrf2. Such findings enhance our current understanding of Cr (VI) induced carcinogenicity: this knowledge related with Cr (VI) induced molecular mechanism may increase the potential prevention of Cr (VI) carcinogenicity and further tumor development.