Expression And Function Of FUNDC1 In Anaplastic Thyroid Carcinoma

Anaplastic Thyroid Carcinoma(ATC)is one of the most invasive malignant tumors with high degree of malignancy and poor prognosis.In general,the median survival time after diagnosis was only 5 to 6 months.Although the literature has documented long-term survivors,the diagnostic reliability of such cases is often questioned.Although the clinical incidence of anaplastic thyroid carcinoma is low,accounting for about 1%-9%of thyroid malignant tumors,due to rapid progress and high mortality,up to 14%-50%of thyroid cancer-related deaths are related to it every year.The main reason for patients to see a doctor is the rapid enlargement of the neck mass.Due to the rapid progress of the disease,patients often have local advanced stage when they see a doctor,and lose the opportunity of complete resection,or even if the tumor can be completely removed,due to the rapid postoperative recurrence and insensitivity to radiotherapy and chemotherapy,the mortality of patients is very high.Therefore,to understand the molecular pathogenesis of ATC tumor and its insensitivity to radiotherapy and chemotherapy may provide new hope for the treatment of the disease.Studies have showed that there is a big difference between cancer cells and non-cancer cells in their growth and metabolism,and the metabolic patterns of many cancer cells are reprogrammed by genes.Mitochondria are the energy supply places of cells,which supply energy for cells through aerobic metabolism,carry out tricarboxylic acid cycle and oxidative phosphorylation center,and provide ATP for cells.Besides supplying energy to cells,it is also the center of iron metabolism and lipid oxidation.Reactive oxygen species are produced in the process of oxidative phosphorylation,which participate in cell apoptosis,inflammatory reaction and senescence.Therefore,maintaining the stability of mitochondrial function can maintain the normal life activities of cells.Mitochondrial autophagy is to digest and decompose damaged and aging mitochondria into reusable substances,promote mitochondrial renewal,ensure the integrity of quantity and quality of mitochondria in cells,and is conducive to cell survival.Similarly,in tumor tissues,cells also rely on mitochondrial autophagy to update mitochondria and maintain the stability of the internal environment in tumor cells.It is correlated with the poor prognosis of tumor.The FUN 14 domain containing 1(FUNDC1)is a receptor protein of mitochondrial autophagy,which is composed of 155 amino acids.It is a three-time transmembrane protein.It is located in the outer membrane of mitochondria and is a highly conserved protein.FUNDC1 can induce mitochondrial autophagy in hypoxic environment.The study also found that the change of FUNDC1 expression and reversible phosphorylation after protein translation can regulate the change of mitochondrial autophagy level.FUNDC1 is highly expressed in a variety of tumor tissues and promotes the proliferation,invasion and migration of tumor cells by up regulating mitochondrial autophagy,but its role in ATC is not clear.Therefore,this study intends to explore the role of FUNDC1 in ATC from the following three aspects.This paper intends to study from the following three parts:In the first part,the clinical data and specimens of ATC patients were collected.The expression of FUNDC1 in cancer and para-cancerous tissues was detected by Immunohistochemical(IHC),quantitative real-time polymerase chain reaction(qRT-PCR)and Western Blot(WB).Combined with the clinical data and the expression of FUNDC1,the role of FUNDC1 in the occurrence and development of ATC was analyzed.In the second part,the ATC stable cell line with lentivirus knockdown of FUN DC1 expression was successfully constructed to study the effect of FUNDC1 knockdown on cell function in vitro.In the third part,the possible molecular mechanism of FUNDC1 in the occurrence and development of ATC was explored by gene chip technology.Finally,to demonstrate the role of FUNDC1 in the occurrence and development of ATC,as a potential target for targeted therapy of ATC,to provide a theoretical basis for the next step of basic and clinical treatment.Part 1.Expression of FUNDC1 in ATC and its correlation with clinical parameters and prognosisAimThe expression of FUNDC1 in ATC and paracancer tissues was detected,and its role in ATC was analyzed combined with clinical data.Methods1.Expression of FUNDC1 in 79 cases of ATC tissue and 40 cases of para-carcinoma thyroid tissue were detected by IHC method.2.The mRNA expression of FUNDC1 in cancer and para-carcinoma thyroid tissues of 26 patients with ATC was detected by qRT-PCR.Protein expression of FUNDC1 in cancer and para-carcinoma thyroid tissues of 3 patients with ATC was detected by WB.3.The relationship between the clinicicopathological data of patients and the expression of FUNDC1 was analyzed.The relationship between expression of FUNDC1 and the prognosis of patients was analyzed.Result1.IHC detection showed that the expression of FUNDC1 in ATC was significantly higher than in para-carcinoma tissues(p<0.05).2.qRT-PCR detection showed that the expression of FUNDC1 mRNA in ATC was significantly higher than that in para-carcinoma tissues(p<0.05).WB detection showed that the expression of FUNDC1 protein in ATC was higher than that in para-carcinoma tissues.3.The high expression of FUNDC1 was significantly associated with T staging,lymph node metastasis and TNM staging(p<0.05).4.In disease-free survival analysis and survival analysis,it was showed that the disease-free survival and overall survival of patients with high expression of FUNDC1 were shorter,suggesting that FUNDCl can be used as an independent prognostic factor for ATC and promote tumor progression(p<0.05).Part 2.The effects of knockdown of FUNDC1 on the function of ATC cells in vitroAimKnock down FUNDC1 expression with lentivirus in ATC cell line,explore its effect of cell function in vitro experiments.Methods1.The expression of FUNDC1 in ATC cell Lines 8305C and CAL-62 was detected.2.The ATC cell lines with higher expression of FUNDC1 were selected for subsequent experiments.3.To construct a stable transfected ATC cell line with lentivirus knockdown FUNDC1.4.WB and qRT-PCR were used to detect the effect of shRNA interference.5.Colony formation assay and MTT assay were used to detect the proliferation ability of ATC cells after shRNAi.The apoptosis of ATC cell was detected by flow cytometry.The invasion ability of ATC cells was detected by invasion assay.The migration ability of ATC cell was detected by cell scratch healing experiment.The migration and invasion ability of ATC cell was detected by Transwell assay.Result1.qRT-PCR showed that FUNDC1 was stably expressed in two ATC cell lines,and the higher expression was in CAL-62 cell lines.2.After lentiviral transfection,the expression levels of mRNA and protein of FUNDC1 decreased significantly(P<0.05).3.After inhibiting the expression of FUNDC1,the apoptosis rate of cells increased,the ability of cell proliferation decreased,and the ability of invasion and metastasis decreased significantly(P<0.05).Patr3 Molecular biology study on FUNDC1 in ATCAimUsing gene chip technology,to explore the possible molecular biological mechanism of FUNDC1 in the occurrence and development of ATC,and to explore the possibility of FUNDC1 protein as a potential target for ATC tumor therapy or adjuvant therapy for ATC,so as to provide theoretical basis for further basic and clinical research.Methods1.Using gene chip technology to detect the changes of gene expression profile after FUNDC1 shRNAi.2.IPA technology is used to analyze the signal pathway and functional mechanism of FUNDC1 promoting the occurrence and development of ATC.Result1.Gene chip results showed that FUNDC1 participates in the maintenance of cancer stem cell sternness and epithelial mesenchymal of tumor cells by activating the Sumoylation signaling pathway,which promoted the high invasion and metastasis of tumor cells,and participated in the recurrence of tumor and the resistance of tumor cells to chemotherapy(P<0.05).2.FUNDC1 promotes angiogenesis in tumor tissue by activating Sprouting,which is closely related to tumor invasion and metastasis.It inhibits the quantity of reactive oxygen species,avoids the accumulation of intracellular peroxides,inhibits cell apoptosis caused by oxidative stress injury,and participates in the regulation of cell metastasis and invasion(P<0.05).Conclusion1.The expression of FUNDC1 in ATC was significantly higher than that in para-carcinoma,and its high expression was significantly correlated with T staging,lymph node metastasis and TNM staging.In addition,patients with high FUNDC1 expression had shorter DFS and OS,which was associated with poor prognosis.FUNDC1 overexpression were independent risk factors for prognosis of ATC patients.2.After downregulating the expression of FUNDC1 in ATC cells,the proliferation,migration and invasion of ATC cells decreased,and the apoptosis rate increased.3.RNA gene chip detection showed that FUNDC1 was involved in the proliferation,invasion,metastasis and apoptosis of tumor cells,and involved in maintaining the stemness of cancer stem cells and Epithelial-Mesenchymal Transition of tumor cells,which may be related to tumor recurrence and chemotherapy resistance of tumor cells.These may be achieved by up-regulating Sumoylation,reducing intracellular Reactive Oxygen Species(ROS)accumulation and promoting the development of vascular system.