| Objective:Tumor is one of the major global diseases that seriously endanger human life and health.Thyroid cancer is one of the most common endocrine system tumors.Although great progress has been made in its diagnosis and treatment,the incidence of thyroid cancer is still increasing year by year in recent years,and the incidence of patients has also been younger.Approximately 90% of thyroid cancers are well-differentiated papillary thyroid cancer(PTC).Although papillary thyroid cancer is usually indolent and has a good prognosis,the malignant progression of the disease and local recurrence are still the main reasons for its increased mortality.Therefore,exploration of the mechanism of the occurrence and development of papillary thyroid cancer to provide a theoretical basis for its early diagnosis and individualized treatment has become an urgent scientific problem in the field of thyroid cancer research.FOXK2,as one of the important transcription factors of the Forkhead Box(FOX)family,has been shown to control a grouping number of biological functions,such as cell proliferation,apoptosis,DNA repair,as well as regulating metabolism.More and more evidences show that FOXK2 is tightly related to the malignant process of tumors.FOXK2 can promote the malignant progression of colorectal cancer and liver cancer.On the contrary,FOXK2 can act as a tumor suppressor gene to inhibit the progression of breast cancer,non-small cell lung cancer(NSCLC)and glioma.These contradictory findings indicate that whether FOXK2 plays an oncogene or a tumor suppressor in tumor progression is tumor-specific.Autophagy is an evolutionarily highly conserved important process of degradation and recycling of intracellular substances in eukaryotes.During this process,some damaged proteins or organelles are encapsulated by autophagic vesicles with a double-layer membrane structure,and then sent to the lysosome for degradation and recycling.Autophagy plays an important role in maintaining cell homeostasis,promoting the circulation of substances and energy in cells,and helping cells resist external aggressions.It is an important mechanism for cells to protect themselves and maintain normal survival.Its dysfunction plays an important role in many diseases,especially in tumor formation.Autophagy eliminates damaged organelles and accumulated harmful substances to ensure the stability of the genome and inhibit the occurrence and development of tumors.The role of FOXK2 in papillary thyroid cancer remains to be clarified.In this study,we intend to explore the role of FOXK2 in papillary thyroid cancer and its possible mechanism of action to provide a theoretical basis for its early diagnosis and individualized treatment.Methods :This study is based on the genes screening and analysis that may play an important role in the occurrence and development of thyroid cancer through the TCGA database.FOXK2 is the candidate gene we selected.In order to further verify the expression of FOXK2 in papillary thyroid cancer,we collected 14 surgically resected papillary thyroid cancer patients with cancer and normal fresh thyroid tissue,and detected FOXK2 exprssion in papillary thyroid cancer by Western blot.At the same time,we collected 98 clinical cases of papillary thyroid cancer that were treated surgically and clinically and pathologically diagnosed in the Shengjing Hospital of China Medical University from 2011 to 2016,and further confirmed FOXK2 expression in the papillary thyroid through immunohistochemistry.The association between FOXK2 and clinicopathological characteristics of papillary thyroid cancer was analyzed by HSCORE score combined with statistical methods.At the same time,we also tested the expression of FOXK2 in human normal thyroid follicular cell lines(Nthy-ori-3-1)and papillary thyroid cancer cell lines(TPC-1,BCPAP,K1,BHT101)through Western blot experiments.Based on the relative high and low expression of FOXK2 in papillary thyroid cancer cell lines,we constructed RNAi-mediated knockdown PTC cells in BHT-101 and FOXK2-overexpressed cell lines in BCPAP.Transfection efficiency was examined by Western blot.We further explored the biological behavior of FOXK2 in papillary thyroid cancer cells through CCK8 cell proliferation experiments and cell colony formation experiments.The classic research methods of autophagy include:(1)Detection of p62 degradation and the conversion of LC3-I to LC3-II by Western blot experiment;(2)Observation of LC3 punctate aggregation by immunofluorescence experiment.(3)Observe the number of autophagosomes and autophagolysosomes by transmission electron microscope.We explored the regulation of FOXK2 on autophagy in papillary thyroid carcinoma.Furthermore,we use autophagy inhibitor 3-MA to explore whether FOXK2 regulates cell proliferation and cell colony formation due to its regulation of autophagy.Results:To explore whether FOXK2 is involved in the malignant process of thyroid cancer,we first analyzed the expression of FOXK2 in thyroid cancer through the TCGA database.We found that FOXK2 was significantly up-regulated in thyroid cancer tissues compared to normal tissues.Then Western blot analysis validated that FOXK2 expression was up-regulated in PTC tissues(n=14)compared to matched normal tissues.Consistent with TCGA database analysis and Western blot results,we further confirmed by immunohistochemical experiments that FOXK2 is highly expressed in 70.4% of PTC tissues(n=98).More importantly,through TCGA database analysis,we found that the expression of FOXK2 is closely related to the clinicopathological characteristics of thyroid cancer.The high expression of FOXK2 is associated with Tumor size(p=0.012),T stage(p=0.001)and AJCC stage(p=0.016).In summary,FOXK2 is highly expressed in papillary thyroid cancer and is closely related to its malignant progression.To characterize the biological functions of FOXK2 in PTC,we first detected the expression of FOXK2 in PTC cell lines.We found that the expression of FOXK2 was higher in most of PTC cell lines than that in human thyroid follicular epithelial cells Nthy-ori-3-1.Based on the relative high and low expression of FOXK2 in papillary thyroid cancer cell lines,we constructed RNAi-mediated knockdown PTC cells in BHT-101 and FOXK2-overexpressed cell lines in BCPAP.Transfection efficiency was examined by Western blot.Using CCK8 and colony formation experiments,we found that knockdown of FOXK2 can inhibit the proliferation and colony formation in BHT-101 cells.On the contrary,overexpression of FOXK2 can promote the proliferation and colony formation in BCPAP cells.Next,we want to explore the possible molecular mechanism of FOXK2 affecting cell proliferation.Given that autophagy is an important cell biological behavior affecting tumorigenesis,and there is also some relationship between FOXK2 and autophagy,we tested whether FOXK2 is involved in the regulation of autophagy levels in PTC cells.Using Western blotting,we found that silencing of FOXK2 promoted autophagy in BHT-101 cells,as demonstrated by the decrease of autophagy substrate p62 and increase of the conversion of the nonlipidated form(LC3-I)to the phosphatidyl ethanolamine-conjugated form(LC3-II)of LC3.Conversely,overexpression of FOXK2 suppressed autophagy levels in BCPAP cells.Next,we examined GFP-LC3 puncta to assess autophagosome formation in the nonspecific RNAi-treated or FOXK2 knockdown BHT-101 cells.We found that knockdown of FOXK2 promoted the number of autophagosomes.In addition,quantitative electron microscopic analysis showed that knockdown of FOXK2 increased autophagic structures compared with the sh RNA negative control cells.Furthermore,we found that silencing of FOXK2 can promote the expression of critical autophagy proteins ULK1,VPS34 and FOXO3.Conversely,overexpression of FOXK2 suppressed protein levels of ULK1,VPS34 and FOXO3 in BCPAP cells.Taken together,silencing of FOXK2 promotes autophagy by up-regulating the expression of ULK1,VPS34,and FOXO3 in PTC cells.Next,we wanted to explore whether FOXK2 regulates cell proliferation due to its regulation of autophagy.We found that the autophagy inhibitor 3-MA can reverse the autophagy caused by silencing FOXK2,as demonstrated by the increase of autophagy substrate p62 and decrease of the conversion of LC3-I to LC3-II in BHT-101 PTC cells.At the same time,3-MA can reverse the inhibition of cell proliferation and colony formation caused by silencing FOXK2.In summary,these findings suggested that silencing of FOXK2 inhibited cell proliferation and colony formation through up-regulating autophagy.Conclusion:1.FOXK2 is up-regulated in papillary thyroid carcinoma(PTC)tissue and cell lines.2.FOXK2 is correlated with the clinicopathological characteristics of PTC.3.FOXK2 knockdown inhibited the proliferation and colony formation of PTC cells through its regulating autophagy... |
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