| BackgroundThe incidence rate of thyroid cancer,the most common endocrine malignancy,has increased rapidly over the past 10 years.Papillary thyroid carcinoma(PTC)is the pathological types of thyroid cancer,which is a kind of differentiated thyroid cancer.The occurrence and development of thyroid cancer is a complex process involving multiple factors,genes and signaling pathways.Therefore,it is of great significance to study the process of invasion and metastasis of thyroid cancer and explore the molecular mechanism of proliferation,apoptosis and metastasis of cancer cells for further improving the cure rate and survival rate of thyroid cancer patients.CITED1 is the first member of the CITED family of cofactors.Its overexpression has been observed in a variety of cancers including thyroid cancer,melanoma,hepatoblastoma,Wilms' tumor,and other cancer;moreover,high CITED1 levels are strongly correlated with a poor prognosis.Therefore,it is an oncogene in various cancers.ObjectiveWe investigated the effects of CITED1 on the biological behavior and tumor growth in in vitro and in vivo models of PTC,and determine whether the transcriptional regulator acts via the Wnt/?-catenin pathway.Our study provides a new idea for clinical diagnosis and molecular targeted therapy of PTC.Methods(1)CITED1 mRNA and protein levels in normal human thyroid cells(Nthy-ori 3–1)and human thyroid papillary cancer cell lines(K1,BCPAP,and TPC-1)detected by qRT-PCR and Western blotting analyses.The papillary thyroid cancer cells with the highest CITED1 expression were selected for the next experiment.(2)The recombinant lentivirus carrying CITED1-shRNA or thenegative control(NC)-shRNA was successfully infected into thyroid cancer K1 cells.The silencing efficiency of CITED 1 gene was detected by real-time fluorescent quantitative PCR and Western blotting.The cell proliferation was detected by CCK-8 method,the cell cycle distribution and apoptosis were detected by FCM,and the cell migration and invasion ability was detected by cell scratch healing assay and transwell experiments.Finally,the expression changes of ?-catenin,c-myc,cyclinD1 after slicing CITED1 were detected by Western blotting analyses.(3)Human thyroid papillary cancer K1 cells were inoculated under theskin to established the tumor model in nude mice.The length and width of transplanted tumor in nude mice were measured every 3 days,and the volume of transplanted tumor was calculated.After 27 days,the nude mice were sacrificed and the whole transplanted tumor tissue was collected and weighed,and the expression levels of?-catenin,c-myc,cyclinD1 in the transplanted tumor were detected by Western blotting analyses.Results(1)CITED1 was overexpressed in PTC cells compared to normal thyroid cells.Furthermore,CITED1 expression in the K1 cells was relatively high in the three PTC cell lines.Therefore,K1 cells were subjected to stable transfection with CITED1-shRNA.(2)After the recombinant lentivirus carrying CITED1-shRNA was successfully infected into papillary thyroid carcinoma K1 cells,the expression levels of CITED1 mRNA and protein were significantly decreased,which suggested that the K1 cells with CITED1 gene silencing was successfully obtained.After silencing CITED1 gene expression,the cell proliferation was significantly inhibited,the apoptosis rate was significantly increased,the proportion of G0 /G1 phase cells increased significanlty,the proportion of G2 /M and S phase cells decreased significanlty,and the cell migration abilities at 12 h and 24 h were significantly decreased.Transwell assay showed that the ability of cell migration and invasion were both reduced.And the expression of?-catenin,c-myc,cyclinD1 were increased after silencing CITED1.(3)The growth rate of transplanted tumor in the CITED1-shRNA group was slow,the volume and weight of transplanted tumor were also significantly reduced.The results of western blot were consistent with the results in vitro.The expression levels of?-catenin,c-myc,cyclinD1 in transplanted tumor in nude mice in the CITED1-shRNA group were increased.Conlcusion(1)The expression of CITED1 in PTC cells was significantly higher than that in normal thyroid tissue cells.(2)CITED1 can promote the proliferation,migration and invasion of PTC and inhibit apoptosis of tumor.(3)CITED1 contributes to progression of the malignant phenotype of PTC via the Wnt/?-catenin signal pathway. |
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