Study Of BRD4 Via PTEN/PI3K/AKT Pathway To Regulate Iodine Uptake Indicator In Human Papillary Thyroid Cancer Cells

Background:The most prevalent pathological type of thyroid cancer is papillary thyroid cancer（PTC）.The therapy for PTC is currently based on surgical resection combined with postoperative I¹³¹radiation therapy and thyroxine replacement therapy,with a good prognosis.Nevertheless,there are some patients who develop distant metastases and even develop radioiodine refractory thyroid cancers（RAIR-TC）.More recently,bromodomain-containing protein 4（BRD4）has been characterized as an effective therapeutic target for multiple hematologic malignancies and solid tumors,and it has also emerged as a potential target in thyroid cancer,especially in PTC.Simultaneously,JQ1,a selective inhibitor of BRD4,has been shown to inhibit a number of tumors and to affect the sodium iodide symporter（NIS）performance,which is an indicator of PTC iodine uptake,whereas the exact mechanism has not been fully elaborated.Therefore,to further probe the molecular mechanisms relevant to the alteration of iodine uptake rate in PTC by BRD4 and its inhibitors will be instrumental in both the enhancement of therapeutic efficacy and the amelioration of survival prognosis of PTC patients.Objective:To investigate the effect of BRD4 expression level in PTC and its inhibitor JQ1 on the proliferation and migration ability of PTC cells,as well as to further explore the implications and possible mechanisms on the expression of the iodine uptake indicator NIS,in order to improve the survival and enhance the prognosis of RAIR-TC patients.Methods:（1）Both real-time quantitative PCR（q RT-PCR）and Western blotting（WB）techniques were applied to assay the levels of BRD4 expressing in normal thyroid cell Nthy-ori 3-1 and human PTC cell lines TPC-1 and K1,respectively;（2）Expression of BRD4 in PTC tissues was detected by q RT-PCR;（3）Meanwhile,the clinical information of 45 PTC patients was collected and analyzed the relevance of BRD4 to the clinicopathological characteristics of PTC patients withX²test;（4）To investigate the impact of JQ1 on the proliferation ability of TPC-1 and K1 cells by Cell Counting Kit-8（CCK-8）technology,as well as its half-inhibitory concentration（IC50）was calculated;（5）To examine the influence of JQ1 on the migration ability of TPC-1 and K1 cells by scratch assay;（6）The morphological and numerical changes of cells after different periods of JQ1 interaction were observed using high-content imagining technology;（7）The expression of genes and proteins affecting PTC iodine uptake was detected by q RT-PCR and WB techniques;（8）Three-dimensional（3D）spherical cultures of Nthy-ori 3-1,TPC-1 and K1 cells were performed,while their aggregation patterns and morphology were observed using inverted fluorescence microscope,so as to initially investigate whether they correlated with iodine uptake.Results:（1）Both q RT-PCR and WB results showed that BRD4 was highly expressed in PTC cells compared to normal thyroid cells;（2）The q RT-PCR results indicate that BRD4 is highly expressed in PTC tissues compared to paraneoplastic tissues;（3）There was no statistically correlation between the expression of BRD4 and the clinicopathological characteristics of PTC patients,such as tumor size,presence of lymph node metastasis,and presence of distant metastasis;（4）The CCK-8 results demonstrated that JQ1 showed time and concentration dependent inhibition of proliferation ability of TPC-1 and K1 cells.The IC50 of TPC-1 cell and K1 cell for48h was（9.045±0.772）μM and（14.169±1.406）μM,respectively;（5）The results of scratch assay indicate that JQ1 can suppress the migratory of TPC-1 and K1 cells;（6）The results of high-content imaging demonstrated that JQ1 significantly altered the number and morphology of PTC cells and inhibited their growth and proliferation with time and concentration dependence;（7）The q RT-PCR and WB results have shown that the m RNA and protein expression of BRD4,phosphoinositide 3-kinase（PI3K）and protein kinase B（AKT）were decreased in the experimental group after JQ1 was applied to TPC-1 and K1 cells for 48h,separately.The m RNA and protein levels of phosphatase and tension homology deleted on chromosome ten（PTEN）and sodium iodine protein（NIS）were increased;（8）The 3D spherical culture showed that Nthy-ori 3-1 and TPC-1 cells could aggregate to form single cell spheres,whereas K1cells were cell number independent and could form concentrated single cell spheres or multiple cell spheres scattered in distribution.Conclusions:BRD4 is highly expressed in PTC.JQ1 effectively inhibits the expression of BRD4 and probably inhibits the proliferation and migration of PTC cells through the PTEN/PI3K/AKT pathway,while increasing the expression of the iodine uptake indicator NIS,which in turn increases the iodine uptake capacity of PTC cells.Treatment targeting BRD4 may be an effective measure to inhibit PTC progression and improve iodine uptake.