| Background and bjective:Thyroid cancer is the most common malignant tumor in the endocrine system.In the past few decades,the incidence of thyroid cancer has increased around the world.The rate of increase is highest among all malignant tumors,causing a serious economic and health burden.Papillary thyroid cancer is most common in patients with thyroid cancer,accounting for more than 70% of thyroid malignancies.It has a high degree of differentiation and low malignancy.If diagnosed and treated early,the prognosis is good,5-year survival rate can reach more than 90%.However,many patients with papillary thyroid cancer have reached the advanced stage or even metastasized when they are discovered.Mortality rate of patients with advanced stage is significantly increased and the prognosis is poor.Therefore,an in-depth understanding of the pathogenesis,early prevention and diagnosis of papillary thyroid cancer are important to reduce the incidence and improve patient survival and quality of life.Forkhead box E1(FOXE1),also known as thyroid transcription factor-2,is a member of the transcription factors forkhead / wing spiral family.FOXE1 is necessary for thyroid formation,development,and maintenance of adult thyroid differentiation.Studies have shown that FOXE1 is differentially expressed in papillary thyroid cancer and normal thyroid,but the internal mechanism is still not understood.MicroRNA(miRNA)is a short,non-coding endogenous RNA molecule that inhibits the expression of corresponding protein-coding genes by binding to the 3'-untranslated region(3'-UTR)of the target mRNA.More and more researches show that miRNA can be involved in tumorigenesis and development as regulators of oncogenes or tumor suppressor genes.Based on the above,we selected the FOXE1 gene as the target gene to study the role of miRNAs targeted to regulate FOXE1 in papillary thyroid cancer,and explored the possibility of target miRNAs as papillary thyroid cancer specific biomarkers.Provide evidence for effective prevention,early diagnosis,and early intervention of papillary thyroid cancer.Methods:1.We compared the expression of FOXE1 in papillary thyroid carcinoma and normal thyroid tissue based on TCGA database.We also selected papillary thyroid cancer cells TPC-1 cells and normal thyroid epithelial cells Nthy-ori 3-1 cells to detect mRNA and protein expression levels of FOXE1 by RT-qPCR and Western Blot.2.We predict miRNAs that may target FOXE1 through the Targetscan website,and RT-qPCR was used to detect the expression of candidate miRNAs in TPC-1 and Nthy-ori 3-1 cells.Tissue microarray was used to analyze the expression of target miRNA miR-138-5p in cancer tissues and adjacent tissues of 58 patients with papillary thyroid cancer,and further analysis was performed by combining the general and pathological data of the patients.3.The miR-138-5p inhibitor was transfected into TPC-1 cells and transfected with NC inhibitor as a control group.The mRNA and protein expression levels of FOXE1 were detected by RT-qPCR and Western Blot.The double luciferase reporter assay was used to verify the specific binding of miR-138-5p to FOXE1.4.The effects of transfection with miR-138-5p inhibitor on proliferation,migration,invasion,and apoptotic function of TPC-1 cells were examined by CCK-8 proliferation experiments,scratch experiments,transwell invasion experiments,and flow cytometry.5.RT-qPCR was used to detect the expression of miR-138-5p in serum samples from 41 case groups and 41 control groups.The case group was collected from patients with papillary thyroid cancer diagnosed in thyroid surgery at the China-Japan Union Hospital of Jilin University from January 2012 to December 2014 and the population from the chronic disease spectrum survey of Jilin Province from July to August 2012 as control group.Receiver operating characteristic(ROC)curves was conducted to evaluate the ability of biomarkers to distinguish between papillary thyroid cancer patients and normal controls.Results:1.The TCGA database analysis showed that compared to normal thyroid tissue,FOXE1 expression is lower in papillary thyroid cancer.In vitro cell experiments have shown that FOXE1 expression is lower in papillary thyroid cancer cells both at the mRNA level and protein level(P<0.05).2.According to the Targetscan website,miR-138-5p is predicted to be a miRNA that targets FOXE1.Through experiments,it was found that miR-138-5p is highly expressed in papillary thyroid cancer cell compared with thyroid epithelial cell.The difference is statistically significant(P<0.05).Tissue microarray results also proved that miR-138-5p expression is higher in papillary thyroid cancer tissues than adjacent tissues(P<0.05).Further comparison and analysis of miR-138-5p expression levels and the general situation and clinical pathological indicators of patients(sex,age,presence or absence of lymph node invasion,clinical stage,T stage,N stage)found that high expression of miR-138-5p was associated with early clinical stage(?-?)(P<0.05).3.After transfection with miR-138-5p inhibitor,FOXE1 mRNA and protein expression levels were significantly increased(P<0.05).The results of the double luciferase reporter assay confirmed that miR-138-5p specifically binds to FOXE1.4.The results of CCK-8 proliferation experiments showed that the proliferation ability of TPC-1 cells was inhibited after transfection with miR-138-5p inhibitor(P <0.05).The results of cell scratch experiments showed that the migration ability of TPC-1 cells were all reduced at 6h,12 h,and 24 h after scratching(P<0.05).The results of the transwell invasion experiment proved that compared with the control group,the invasion ability of TPC-1 was significantly reduced after transfection with miR-138-5p inhibitor,and the difference was statistically significant(P<0.05).Flow cytometry results showed that although there was no statistically significant difference in the level of apoptosis between the miR-138-5p inhibitor group and the NC inhibitor group(P>0.05),TPC-1 cells have a tendency to promote apoptosis after transfection with miR-138-5p inhibitor.5.Compared with serum samples from healthy controls,miR-138-5p was highly expressed in serum samples from patients with papillary thyroid cancer,and the difference was statistically significant(P <0.05).The evaluation results of the ROC curves showed that miR-138-5p expression was able to distinguish well papillary thyroid cancer patients and normal controls,suggesting that miR-138-5p could be used as a potential diagnostic biomarker of papillary thyroid cancer.Conclusion:1.Compared to normal thyroid tissue and thyroid epithelial cells,FOXE1 is low expressed in papillary thyroid cancer tissues and cells.2.MiR-138-5p is highly expressed in tissues,cells,and serum samples of papillary thyroid cancer compared with normal thyroid tissues,thyroid epithelial cells,and serum samples from healthy controls.3.MiR-138-5p promotes the proliferation,migration,and invasion of papillary thyroid cancer cells.4.MiR-138-5p may affect papillary thyroid cancer cell function through targeted regulation of FOXE1.5.MiR-138-5p can be used as a potential diagnosis biomarker of papillary thyroid cancer. |
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