Study Of The Relationship Between The Dna Methylation Of CLCN5/ANO1 And Sporadic Nodular Goiter

Objective: To study the difference between DNA methylation status of promoter of CLCN5 and ANO1 in nodule tissues of patients with sporadic nodular goiter and that in paranodal normal thyroid tissues,and to analyze its relationship with the occurrence and development of nodular goiter.Methods: Patients with nodular goiter who were operated at the Department of Thyroid Surgery of Yantai Yuhuangding Hospital Affiliated to Qingdao University.The methylation of promoter of CLCN5 and ANO1 genes in fresh thyroid nodule tissues and paranodal normal thyroid tissues was detected by Bisulfite sequencing PCR(BSP).The expression of mRNA and protein of above genes was respectively detected by quantitative real time polymerase chain reaction(qRT-PCR)and Western blot.The siRNAs of CLCN5 and ANO1 and the plasmids of them were separately transfected to the human normal thyroid cell line NTHY-ORI-3-1 by liposome transfection.Then low-expressed and over-expressed human thyroid cells were construted separately.The expression of protein of above genes was detected by Western Blot.The proliferation of them were detected by CCK-8.Results: We found that the DNA methylation level of CLCN5 gene in thyroid nodule tissues was significantly higher than that in paranodal normal thyroid tissues(50.38±5.95%VS 24.59±5.21%).The methylation level of ANO1 gene in thyroid nodule tissues was significantly higher than that in paranodal normal thyroid tissues(44.80±4.60% VS27.37±4.60%).And the differences were both statistically significant(P < 0.01).The quantity of mRNA expression of CLCN5 in thyroid nodule tissues was significantly higher than that in paranodal normal thyroid tissues(2.625±0.525 VS 1±1.074).The quantity of mRNA expression of ANO1 in thyroid nodule tissues were significantly higher than that in paranodal normal thyroid tissues(2.885±0.71 VS 1±0.102).And the differences were both statistically significant(P< 0.01).The protein expression of CLCN5 and ANO1 in thyroid nodule tissues of patients with sporadic nodular goiter was both statistically different comparing with the protein expression of CLCN5 and ANO1 in paranodal normal thyroid tissues.After siRNAs of CLCN5 and ANO1 and the plasmids of them were transfected to the human normal thyroid cell line NTHY-ORI-3-1 by liposome transfection,the lowexpressed and over-expressed human thyroid cells of CLCN5/ANO1 gene were constructed.Then the transfection was verified by Western blot.Finally,we measure the optical density of the cells to know their proliferation capacity on 12h/24h/36h/48 h by CCK-8.We found that comparing with the untreated normal thyroid cells,the proliferation capacity of the over-expressed cells of CLCN5/ANO1 gene was improved,the proliferation capacity of the low-expressed cells of CLCN5/ANO1 gene was reduced.Conclusion: Our results suggested that the level of the methylation of the promoter of CLCN5/ANO1 gene,the mRNA expression and the protein expression was all higher in thyroid nodule tissues of patients with sporadic nodular goiter than that in paranodal normal thyroid tissues.We proved that the level of the protein expression was positively correlated with the proliferation capacity of thyroid cells.In conclusion,Our results suggest that methylation of CLCN5 and ANO1 gene promoter regions of thyroid cells may be involved in the occurrence and development of nodular goiter.