| BackgroundAs the most common endocrine malignancy,the incidence of papillary thyroid carcinoma（PTC）has increased sharply in recent decades worldwide[1].An increasing number of clinical evidence suggests that serum thyroid-stimulating hormone（TSH）is an independent predictor of thyroid malignancy in patients with nodular thyroid disease.The level of TSH in papillary thyroid carcinoma（PTC）is significantly higher than that in benign thyroid nodules,and higher TSH values,even within the normal range,are associated with a greater risk of thyroid malignancy[2-4].The correlation between elevated serum TSH and papillary thyroid cancer has been widely studied,and some scholars have suggested it as a method to evaluate the risk of thyroid cancer in thyroid nodules with diameter less than 1cm.However,not all studies are consistent.Some studies have shown that there is no significant correlation between thyroid papillary microcarcinoma and TSH level[6,7].In fact,the causal role of TSH in causing thyroid cancer has not been fully confirmed[8].A common defect of these current clinical studies is that the regression method used can only prove the correlation between TSH and thyroid cancer,but no appropriate methods,such as Mendelian genetic studies,have been used to prove whether there is a causal relationship between TSH and thyroid cancer,so it cannot be proved that TSH is the inducement of thyroid cancer[9].Tumor environment is a key factor in determining the biological characteristics of a tumor.Cytokines,as an important part of the tumor environment,play a unique role in the occurrence and development of tumors through autocrine or paracrine mode.The retinal determinant gene network（RDGN）,composed of the Dach,SIX and Eya families,is essential for organ development in mammals.DACH1 is an important component of RDGN,which can regulate the expression of target genes through direct combination or interaction with other factors[10-13].In general,DACH1 acts as a tumor suppressor to inhibit the growth and metastasis of lung cancer,kidney cancer,breast cancer[14,15].Unlike its role in a variety of solid cancers,DACH1 appears to act as an oncogene in hematological malignancies[16].The different roles of DACH1 in different cancers may reflect the unique role of RDGN in specific cell lines and highlight the importance of cell-environment-specific molecular interactions in cancer development and progression.AimsIn view of the inconsistency between clinical data conclusions and laboratory examination results,we speculated that:1.TSH was only a member of the PTC regulatory network,and other factors and pathways played a more important role in the occurrence and development of thyroid cancer.2.There may not be a simple positive correlation between TSH and papillary thyroid carcinoma,but there may be a double effect.In order to eliminate other potential interference and further explore the role of TSH in PTC,primary cultured thyroid cells and thyroid papillary caicinoma cells were used to observe a variety of biological changes after TSH treatment in vitro.It is important to identify other cell types in which DACH1 regulates hormone signaling.In addition to the breast,prostate,and ovary,DACH1 expressed in pituitary,thyroid,endocrine pancreas,and several other hormone-responsive cells;however,the function of DACH1 in these tissues remains uncertain.To explore DACH1 expression in thyroid papillary carcinoma and its effect,this study detected DACH1 expression in the pathological specimens of patients with thyroid papillary carcinoma,as well as by changing DACH1 expression level of the papillary thyroid cancer cell lines,to observe the effect of DACH1 on the biological activity of papillary thyroid cancer.Methods and results1.Primary thyroid cells of rats were extracted and cultured.After 72h treatment with different concentrations of TSH（0 m U/L,5 m U/L,10 m U/L,20 m U/L,40 m U/L,80 m U/L）,BRAF m RNA expression was down-regulated in all treatment groups compared with the control group.2.After 72 h treatment with different concentrations of TSH（0 m U/L,5 m U/L,20 m U/L）,the proliferation index of Nthy-ori-3-1 and TPC-1 cells decreased compared with the blank group,and there was no significant difference in the proliferation index among different concentrations.3.Flow cytometry showed that the proportion of G2/M+S phase decreased after 20 m U/L TSH treatment in Nthy-ori-3-1 and TPC-1 cells（P<0.05）,suggesting that cell proliferation was inhibited.4.The results of RNA seq analysis showed that in Nthy-ori-3-1 cells,the differential genes after TSH treatment were mainly enriched in ribosomal-related pathways,infectious diseases,virus and signal transduction.In TPC-1 cells,differential genes after TSH treatment were mainly concentrated in tumor-related pathways,cell transport and catabolism.The results of RNA seq showed that,in addition to the down-regulated expression of CXCL8,which was the inducer of thyroid cancer,the expression level of various chemokines such as CXCL1,CXCL2,CXCL3,CXCL5,CCL2,CCL20 and CCL28 were significantly reduced in TPC-1 cells after TSH treatment.5.ELISA and quantitative real-time PCR results showed that after TSH treatment,CXCL8 and CXCL12 of Nthy-ori-3-1 cells were downregulated,while CXCL10 was upregulated.In TPC-1 cells,CXCL8 was downregulated and CXCL10 was upregulated.6.Western blot results showed that after TSH treatment,BRAFV600E expression in Bcpap cells decreased,BRAF expression in Nthy-ori-3-1 and TPC-1 cells decreased,and DACH1 expression in Nthy-ori-3-1 and Bcpap cells increased.And TSH upregulated the expression of DACH1 in Bcpap and TPC-1 cells in a time-dependent manner.7.Immunohistochemical results of DACH1 in pathological tissues of papillary thyroid carcinoma showed that the expression level of DACH1 was significantly different between carcinoma and para-cancerous tissues（P=0.002）.In the analyses performed by different subgroups,there were significant differences in DACH1 expression between cancer and adjacent tissues in 45 year old,female and stage I-II subgroups（P=0.046,P=0.017,P=0.016,respectively）.When comparing different individuals,it was found that the expression level of DACH1 was significantly different in different samples,but the expression level of DACH1 in cancer tissues was higher than that in adjacent tissues.8.Western blot results showed that DACH1 expression in the three cell lines was Nthy-ori-3-1 cells>Bcpap cells>TPC-1 cells.According to the results,si RNA was transfected to Nthy-ori-3-1 cells,and the construct of p CMV-3x FLAG-DACH1 was transfected to TPC-1 cells.DACH1 plasmid and si RNA were respectively transfected to Bcpap cells to change the expression level of DACH1.9.After down-regulating the expression of DACH1 in Nthy-ori-3-1 cells with si RNA, the results showed that the cell proliferation increased,apoptosis and migration were significantly enhanced.According to ELISA and quantitative real-time PCR,the levels of CXCL10 in transfected cells were decreased,while the levels of CXCL8 andCXCL12 were increased.10.The expression of DACH1 in Bcpap cells was changed by overexpression DACH1 plasmid and si RNA fragments,and the results showed that the proliferation of Bcpap cells was inhibited after overexpression,but increased in silencing group.Apoptosis increased after overexpression and decreased after silencing.And the migration ability of the overexpression group was decreased while the migration ability of the silent group was enhanced.According to ELISA and quantitative real-time PCR results,CXCL8 and CXCL12 levels were decreased after overexpressing DACH1,while CXCL8 and CXCL12 levels were increased and CXCL10 levels were decreased after silencing DACH1.11.After the overexpressed plasmid was transfected into TPC-1 cells to upregulate the expression of DACH1,the results showed that the proliferation of TPC-1 cells decreased,the apoptosis increased,and the migration ability was decreased.According to ELISA and quantitative real-time PCR results,the levels of CXCL8 andCXCL12 in the overexpression group were decreased,while the changes of CXCL10 were not statistically significant.ConclusionThe results of this study suggested that TSH was not a key oncogenic factor in thyroidcancer progression.The carcinogenic effect of TSH on thyroid remained to be further explored.We hypothesized that TSH played a dual role in promoting cancer（more specifically,assisting other factors,such as IGF-1）and inhibiting the development of thyroid cancer.The effect of TSH on the fate of thyroid cancer cells was proposed to be due to the disruption of the balance between cancer induction and tumor suppression,which ultimately determined the fate of thyroid cells.We also found that the basic level of DACH1 in thyroid tissues of each patient varied greatly,but it was higher in cancer tissues than para-cancerous tissues of the same individual.For example,in some patients,DACH1 expression was"-"in para-cancerous tissues and"+"in cancer tissues;but in some other patients,the para-cancerous tissues were"++"and the cancer tissues were"+++".This explains why the expression of DACH1 in normal cell line is higher than that in the other two thyroid papillary cancer cell lines,it may be caused by the difference in the expression of DACH1 among the three individuals used to construct the cell lines.After the transfection of three thyroid cell lines to intervene the level of DACH1,we found that DACH1 can participate in the regulation of intracellular chemokines CXCL8,CXCL10 and CXCL12,and the up-regulation of DACH1 showed inhibition effects of cell proliferation and migration to a certain extent,and it could also facilitate cell apoptosis.These experimental results suggested that DACH1 may play a tumor-inhibiting role in thyroid papillary carcinoma,which was consistent with the role of DACH1 in breast cancer,lung cancer,kidney cancer,prostate cancer[14,15]. |
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