Study On T3 Regulating KISS-1 Gene Expression In The Hypothalamic GT1-7 Neuron Cells

Objective: To investigate the effect of T3 on KISS-1 gene expression in GT1-7 neuron cells of hypothalamus.Methods: 1.Expression of KISS-1 gene m RNA in GT1-7 neuron cells was measured by RT-PCR. 2.After GT1-7 neuron cells were treated with 200ng/ml T3 for different times(12、24、48、72h),realtime PCR、western blotting were used to observe the expression of KISS-1 gene respectively from m RNA and protein levels. 3.Western blotting detected the effect of inteference after treating GT1-7 neuron cells with TRα、TRβsi RNA. Inteferencing GT1-7 cell with effective si RNA sequences,and then treated it with 200ng/ml T3 for 24 h to observe the expression of KISS-1 gene m RNA with realtime PCR.4. The 2-kb PGL3-Kiss1 promoter constructs were transfected into GT1-7 cells for 18 h prior. After GT1-7 cells were treated with T3 for 24 h, luciferase dual reporter gene detection system was used to observe the change of KISS-1 gene promoter activity.Results: 1.The results from RT-PCR showed that 140 bp KISS-1 band existed. 2.The results from realtime PCR showed that after GT1-7 neuron cells were treated with 200ng/ml T3 for different times, compared with the control group(0h), the expression of KISS-1 m RNA increased in 12 h group,but which has no statistical significance(p>0.05). The expression of KISS-1m RNA were significantly increased in 24, 48, 72 h groups(p < 0.01), that of 48 h group increased was most obvious. 3.The results from western blot showed that after GT1-7 neuron cells were treated with 200ng/ml T3 for different times(12-72h), compared with the control group(0h), the expression of KISS-1 protein was began to increase in 12 h group, but which has no statistical significance(p>0.05). The expression of KISS-1 protein were significantly increased in 24, 48, 72 h groups(p < 0.01), that of 24 h group increased was most obvious. 4.The results from si RNA interference showed that after GT1-7 cells were treated with TRα、TRβsi RNA for 48 h,the expression of TR protein decresaed in si RNA664 and si RNA1259 group. After inteferencing GT1-7 cells with effective si RNA sequence for 18 h,200ng/ml T3 treated for 24 h follewed.Compared with control group, the expression of KISS-1 m RNA decreased were most obvious in si RNA664 group(p < 0.01), that of si RNA1259 group decreased(p < 0.05). 5.The results from dual luciferase reporter gene detection system showed that the activity of KISS-1 gene promoter were increased in 20,200,2000ng/ml groups, compared with the control group,that of 200ng/ml group increased was most obvious. The increased kiss1 promoter activity effect diminished when the concentration of T3 exceeds 200 ng/ml.Conclusion: T3 promoted the expression of KISS- 1 gene in GT1-7 neurons of hypothalamus.