Luciferase Reporter Gene Assay Of Progesterone Levels In Women With Thyroid Tumors

Thyroid tumorsare common cervical tumor,the incidence of which in women is significantly higher than that in men,many scholars believe that this difference may be related to the different sex hormone level of male and female.Progesterone,a kind of female hormone,plays a major role in the reproductive and developmental system.However,as progesterone drugs are widely used in medical,animal husbandry and agriculture,progesteronesmake great contribution to the society,whilethey also caused serious hormone pollution to the environment,and have begun to affect organisms' endocrine systems.Progesterone receptor(PROG)belongs to the nuclear receptor,and only activated by PROGcan PR bind to the DNA region of progesterone receptor response element(PRE)to initiate the expression of the receptor response element downstream target genes.Based on this mechanism,a luciferase reporter gene was cloned to the downstream of PRE,and a stably transfected cell line with high-sensitivity and high-throughput bioassays for progesterone was established.This cell line was used for the bioassay of serum progesterone in thyroid tumor and cancer patients to explore the relevance of progesterone level with the occurrence of tumors and cancers.Firstly,three different expression vectors containing the human PR(hPR)gene and three kinds of vectors containing different series fragments of PRE were constructed in this study.Based on the comparison results of luciferase activity,the recombinant plasmids of pEGFP-N2-hPR/pGL4-PRE4 which showed the highest luciferase activitywere co-transfected into HeLa cells.And a total of ten stable clones were isolated and cultivated through antibiotics screening and limited dilution method.The positive stable clones were selected and identified via molecular cloning and subcellular localization technology,thenthe stability was further confirmed by the results of luciferase expression analysis from the fifth to twenty-thrid inconsecutive passages.Results showed that the stable clonespresented a very high sensitivity for progesterone with detection limit reaching 10 pM.Meanwhile,it was also observed that the luciferase activity increased as the concentration of PROG ascended in extracellular environment.But higher concentration of progesterone(more than 106 pM)could reduce the luciferase activity and even cause cells death.This also indicated that long-term progesterone pollution in the environment could cause harm to animals and human health,so human beings should pay more attention to environmental hormone pollution.In this study,the serum progesterone levels of female patients with thyroid diseases were detected and analyzed using the bioassay system of cell lines stably transfected with PR.Based on the biostatistics analysis,it was found that when compared to the normal group,the serum progesterone level of thyroid cancer and malignant tumor patients' was lower.And the average progesterone concentrationof 26.7% thyroid patients was less than 1 nM.However,the progesterone concentration was higher than 1 nM in all of normal group with 22.7% samples exceeded 50 nM,which was not observed in thyroid diseasegroup.The difference analysis revealed that there was a significant difference in progesterone levels between thyroiddisease groups and the normal groups.Although the progesterone levels in women are fluctuant,this study discovered that the progesterone levels in the serum of thyroid tumor or cancer patients were significantly lower than normal group,which suggested a negative correlation between the pathogenicity rate of thyroid cancer or tumors and progesterone levels within a certain range.Consequently,further study of progesterone in women blood may provide some reference value for thyroid tumor early clinical diagnosis.