Promoter Activation Regions And Transcription Factors Regulating HMOX1 Expression In Cadmium Exposed HeLa Cells

Objective:Metal cadmium can enter human body through food chain and accumulate in human body.Metal cadmium can also combine with sulfhydryl protein in vivo to cause protein function loss,oxidative stress,apoptosis and necrosis.As a protective factor,HMOX1 gene plays an important role in anti-inflammatory,antioxidative stress and immune regulation.In this paper,we studied the promoter region of HMOX1 in Cd²⁺treated HeLa cells,and identified transcription factors.In order to understand the mechanism of HMOX1 transcription regulation in the mechanism of anti Cd²⁺toxicity in female reproductive system.At the same time,this study also provides a basis for the treatment of Cd²⁺induced oxidative stress-related diseases in the future.Methods:The changes of HMOX1 in HeLa cells treated with 0mmol/L、1mmol/L、2mmol/L、5mmol/L、10mmol/L、20mmol/L、50mmol/L Cd²⁺were detected by PCR and Western Blot;HeLa cells were transfected with PGL6-HMOX1-846 for 24 hours,then treated with different concentrations of cadmium chloride for 12 hours.The optimal concentration of Cd²⁺was determined by the reporter gene;PGL6 was used as the vector to construct a series of promoter reporter genes,and the activation regions of HMOX1 promoter was identified by a series of reporter gene detection;The transcription factors of HMOX1 were predicted according to the activation region,and the corresponding primers were designed.Results:（1）Compared with the control group,the level of HMOX1 mRNA and protein in HeLa cell model treated with 10mM、20mM、50mM Cd²⁺was significantly different（P<0.05）.（2）The concentration of cadmium chloride was determined to be 10mM by reporter gene test.（3）The activity of HMOX1-587⁺92 and HMOX1-567⁺92was significantly higher than that of HMOX1-577⁺92（P<0.05）,the results showed that HMOX1 promoter-577^-567 and-587^-577 had inhibitory and active transcription factor binding sites,respectively.（4）Compared with pGL6,the activity of HMOX1 promoter in-272^-28 was significantly increased（P<0.05）.There was no significant change in-14⁺29 activity（P>0.05）.Those results indicat that HMOX1promoter had some active transcription factors binding site in-28^-14.（5）Compared with pGL6-HMOX1-272⁺92,the promoter activity of HMOX1-28^-26 and-19^-11decreased significantly（P<0.05）,indicating that there may be a HMOX1 transcription factor binding site at this site.（6）According to the base sequence of promoter activation region-30^-10,the possible transcription factors were predicted which was MYC.Conclusions:After treated with a certain concentration of Cd²⁺,the expression of HMOX1 gene in protein and mRNA levels was significantly changed;HMOX1promoter-587^-577 and-577^-567 have active and inhibitory transcription factor binding sites,respectively;The HMOX1 promoter has an active transcription factor binding site in the range of-30^-10 and the transcription factor was MYC.